Two-photon imaging with genetically encoded sensors is widely used to monitor neurophysiology. An additional fluorescent protein can provide anatomical landmarks for cell-type identification and motion detection. However, most red fluorescent proteins require a dedicated excitation laser. We made transgenic Drosophila with a long-Stokes-shift mScarlet variant (LSSmScarlet3) to image alongside green sensors with a single 920-nm laser. We describe excitation and emission spectra of the expressed protein and show that 920 nm elicits robust in vitro and in vivo fluorescence. Channel crosstalk is minimal. This approach can reduce equipment complexity and cost while placing functional calcium dynamics in their anatomical context.
","acknowledgements":"We thank Masahiro Fukuda, Hyunsoo Shawn Je (Programme in Neuroscience and Behavioural Disorders, Duke-NUS Medical School) for insightful and productive discussions, Danesha Suresh and Zhiyi Zhang for technical and administrative assistance, and Dénes Pálfi and Viktória Kiss (Femtonics HQ) for assistance with two-photon imaging on FEMTO3D Atlas Plug & Play. We additionally thank Dr. Michael Hausser (University of Hong Kong) and Dr. Jung Kim (University of Hong Kong) for providing temporary access to the FEMTO3D Atlas and lab space. The design of the custom chamber used for imaging was kindly provided by Dr. Anissa Kempf (University of Basel). We thank BDSC and Dr. Hiromu Tanimoto for providing fly stocks.
","authors":[{"affiliations":["Institute for Molecular and Cell Biology (IMCB), A*STAR, Singapore","Duke-NUS Medical School, Singapore"],"departments":["","Programme in Neuroscience and Behavioural Disorders"],"credit":["conceptualization","methodology","investigation","dataCuration","formalAnalysis","visualization","writing_originalDraft","writing_reviewEditing","fundingAcquisition"],"email":"xusangyu@gmail.com","firstName":"Sangyu","lastName":"Xu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4927-9204"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["investigation","writing_reviewEditing","methodology"],"email":"xy_zhang@duke-nus.edu.sg","firstName":"Xianyuan","lastName":"Zhang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0003-4483-750X"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["methodology","investigation","dataCuration","formalAnalysis","visualization","writing_reviewEditing"],"email":"kycheung@duke-nus.edu.sg","firstName":"King Yee","lastName":"Cheung","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-1098-4926"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["methodology","investigation","dataCuration","validation","writing_reviewEditing"],"email":"maiyishan@u.duke.nus.edu","firstName":"Yishan","lastName":"Mai","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7199-380X"},{"affiliations":["National University of Singapore, Singapore"],"departments":["Microscopy Core Facility — Multiphoton Microscopy Unit, Yong Loo Lin School of Medicine"],"credit":["methodology","investigation","validation","writing_reviewEditing"],"email":"wu.yue@nus.edu.sg","firstName":"Yue","lastName":"Wu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2484-4967"},{"affiliations":["Duke-NUS Medical School, Singapore","Institute of Molecular and Cell Biology, A*STAR, Singapore","National University of Singapore, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders","","Department of Physiology, Yong Loo Lin School of Medicine"],"credit":["conceptualization","supervision","fundingAcquisition","writing_reviewEditing"],"email":"claridge-chang.adam@duke-nus.edu.sg","firstName":"Adam","lastName":"Claridge-Chang","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4583-3650"}],"awards":[{"awardId":"MOH-OFYIRG20nov-0051","funderName":"National Medical Research Council (Singapore)","awardRecipient":"Sangyu Xu"},{"awardId":"MOH-001397-01","funderName":"National Medical Research Council (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"MOE-T2EP30222-0018","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"MOE-T2EP30223-0009","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"FY2022-MOET1-0001","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"S. X. was supported by an MOH-OFYIRG20nov-0051, and X. Z., K. Y. C., Y. M., and A. C.-C. were supported by the MOH-001397-01 grant from the National Medical Research Council, Singapore, by grants MOE-T2EP30222-0018 and MOE-T2EP30223-0009 from the Ministry of Education, Singapore, and by a Duke-NUS Medical School grant. X. Z. and A. C.-C. were supported by FY2022-MOET1-0001.
","image":{"url":"https://portal.micropublication.org/uploads/ce5a9aea0e4551e97f1410bf18dd2876.png"},"imageCaption":"A) An image of the mushroom body expressing LSSmScarlet3 in a fixed MB247-Gal4>UAS-LSSmScarlet3 fly collected with 940 nm excitation and 620 nm collection. White box: control ROI without LSSmScarlet3 expression. Orange box: test ROI with LSSmScarlet3 expression. Scale bar: 20 μm.
B) Excitation spectrum of LSSmScarlet3 in the sample shown in A. Three local maxima were further tested for emission: 860 nm, 940 nm, and 1040 nm.
C) Emission spectra of excitation at 860 nm, 940 nm, and 1040 nm.
D) LSSmScarlet3 expressed in live HEK293T cells under the control of a CMV promoter excited at various 920 nm laser power settings and images collected with a green and a red PMT. At laser power above 25 %, the red PMT was overloaded by the light emitted. Green signals began to emerge at above 50 % laser power.
E) Quantification of mean intensity of each image in D as a function of laser power.
F) Maximum intensity projection image of the mushroom body calyx and α-lobe in a live MB247>GCaMP8m;LSSmScarlet3Drosophila brain. Both fluorophores were simultaneously excited by the 920 nm laser at 20% power. (F) GCaMP8m signals detected with the green-filtered PMT at 80% gain, (F′) LSSmScarlet3 signals detected with the red-filtered PMT at 70% gain, and (F′′) merged signals. Scale bar = 20 μm.
","imageTitle":"Two-photon imaging of a Drosophila codon-optimized LSSmScarlet3
","methods":"Drosophila cultures were maintained on standard cornmeal-based medium (1.25 % w/v agar, 10.5 % w/v dextrose, 10.5 % w/v maize, and 2.1 % w/v yeast) (Temasek Life Sciences Laboratories 2018). Stocks were kept at room temperature unless otherwise specified. Crosses were performed at 25 ºC in a day-night cycling incubator (Mir-253, Sanyo). Adult male and female flies, aged 5–10 days, were used for experiments. UAS-GCamp8m was obtained from Bloomington Drosophila Stock Center (BDSC #92590). The MB247-Gal4 stock (Zars et al. 2000) was provided by Dr Hiromu Tanimoto (Tohoku University, Japan).
LSSmScarlet3 coding sequence was codon-optimized for Drosophila by GenScript (Piscataway, NJ, USA) and cloned into pcDNA3.4 (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. V880-01) to generate the mammalian expression construct pcDNA-DmLSSmScarlet3 and into pJFRC81-10XUAS-IVS-Syn21-GFP-p10 vector (Pfeiffer et al. 2012) between XhoI and XbaI restriction sites to generate Drosophila expression construct UAS-DmLSSmScarlet3, which was then injected into yw flies at the attp40 site balanced against Curly of Oster by BestGene (La Jolla, CA, USA). The pJFRC81-10XUAS-IVS-Syn21-GFP-p10 plasmid was a gift from Gerald Rubin (Addgene plasmid #36432; http://n2t.net/addgene:36432; RRID:Addgene_36432)
Fly brains expressing LSSmScarlet3 were dissected in cold PBS and fixed in 4 % PFA for 30 min and then mounted in PBS in 2 stacked reinforcement rings (Suremark) pasted on 1.0–1.2 mm-thick 25 × 75 mm glass slides (Biomedia), and then sealed with 170 µm-thick 18 × 18 mm glass cover slips with clear nail polish. Samples were imaged on a Leica TCS SP8 DIVE multiphoton microscope (Leica Microsystems, Wetzlar, Germany) as previously described (Cheung et al. 2025) with the exception that in the emission spectrum measurements the excitation wavelengths were fixed at 860 nm, 940 nm, or 1040 nm. Image acquisition and spectral detection were controlled using Leica LAS X software. Mean intensity of ROIs in images was calculated using Fiji (Schindelin et al. 2012). Excitation and emission spectra graphs were generated using matplotlib in Python.
HEK293T cells were seeded into small tissue culture dishes to ~ 70 % confluence in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA, USA) and transfected with pcDNA3.4-DmLSSmScarlet3 constructs using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) in Opti-MEM™ Medium (Thermo Fisher Scientific, Waltham, MA, USA) according to a standard protocol (Thermo Fisher Scientific 2016) in 35 mm cell culture dishes (Nunc, catalog #153066; Thermo Fisher Scientific, Waltham, MA, USA). The cells were imaged 48 hours after transfection.
Cell culture expressing LSSmScarlet3 was imaged with A-SCOPE (Thorlabs, NJ, USA) as previously described (Cheung et al. 2025). Laser power was set to 30 mW. Green PMT was bandpass filtered between 500 nm and 550 nm, and the red PMT was bandpass filtered between 570 nm and 640 nm. Detector gains were set to 5%.
Flies were mounted in a custom 3D-printed chamber using candle wax. Fly heads were opened using a pair of sharp Dumont No. 5 forceps (Fine Science Tools) and microdissection scissors (Fine Science Tools). A drop of PBS was then placed on the open fly head to keep the brain moist and to act as the objective immersion medium.
Z-stack images were acquired on a FEMTO3D Atlas Plug & Play 2-photon microscope (Femtonics, Budapest, Hungary) equipped with a 16× objective lens. Laser power of the 920 nm laser was set to 20% (112 mW). GCaMP8 emission light was collected by a green-filtered PMT at 80% gain, while LSSmScarlet3 emission light was collected by a red-filtered PMT at 70% gain.
The Drosophila mushroom body 2-photon image stack was subjected to Gaussian blur 3D processing (X: 0, Y: 0, Z: 2) and maximum intensity projection processing in Fiji (Schindelin et al. 2012). For the purpose of visualizing dual-channel images, signal collected by the green-filtered PMT was pseudo-colored to green, while signal collected by the red-filtered PMT was pseudo-colored to magenta.
Preprocessed data from the Fiji workflow were analyzed and visualized using custom Jupyter Notebooks running Python (version 3.10.16) with pandas (version 2.1.4), NumPy (version 1.26.4), and seaborn (version 0.12.2).
Data and code used to generate the figure in this paper, as well as construct sequences and maps, are available as a Zenodo repository (DOI: 10.5281/zenodo.19493335).
","reagents":"","patternDescription":"Understanding neural circuits requires monitoring both neural activity and anatomical relationships. Genetically encoded calcium indicators based on blue-excitable green fluorescent protein (GFP) are widely used, for example, the GFP–Calmodulin–M13-peptide (GCaMP) series of calcium sensors. Such sensors enable cellular-resolution functional imaging, but calcium signals are transient and can afford limited structural context. Alongside sensors, researchers sometimes co-express red fluorescent proteins (RFPs) as structural markers (e.g., tdTomato or mCherry) to provide landmarks for cellular identification and sample-motion detection. However, this strategy requires separate two-photon (2P) excitation wavelengths—typically 920 nm for GCaMP and ~1040 nm for some RFPs; this necessitates complex multi-laser setups that increase equipment cost, system complexity, and phototoxicity.
Long-Stokes-shift (LSS) fluorescent dyes exhibit large separations between excitation and emission peaks (Shcherbakova et al. 2012). Meanwhile, there has been considerable effort in developing LSS fluorescent proteins through diverse approaches, including excited-state proton transfer, chromophore modifications, and protein engineering (Piatkevich et al. 2010; Santos et al. 2021). Originally developed to improve FLIM-FRET (fluorescence lifetime imaging microscopy-Förster resonance energy transfer) sensors by reducing spectral overlap between donor and acceptor fluorophores, LSS variants have found broader applications in multicolor imaging (Shcherbakova et al. 2012; Piatkevich et al. 2010). A recently developed LSS variant of mScarlet, LSSmScarlet3, exhibits a 128 nm Stokes shift (ex 466 → em 598 nm) and, compared to previous versions, has improved pH stability and photostability (Subach et al. 2022). Similarly, monomeric cyan-excitable RFP 3, mCyRFP3 (Kim et al. 2022; Laviv et al. 2016), has been deposited as a Drosophila transgenic line (FlyBase 2025); however, to our knowledge, its performance in any species remains to be reported. Here, we describe a genetically encoded LSSmScarlet3 as a structural marker for single-laser two-color 2P imaging, demonstrating efficient excitation at 920 nm. Alongside GFP-based sensors, LSSmScarlet3 provides a simplified approach for dual-channel functional–structural imaging.
To verify whether a long-Stokes-shift RFP can be imaged with a 920 nm laser in Drosophila brains, we synthesized a Drosophila codon-optimized version of LSSmScarlet3. In fixed Drosophila tissue, LSSmScarlet3 was well expressed and readily distributed to the lobes of the mushroom body, indicating good neuronal expression and distribution (Figure 1A). On a spectrally tunable multiphoton microscope, LSSmScarlet3 had a peak excitation at 940 nm, with two smaller local maxima at 860 nm and 1040 nm (Figure 1B). At a frequently used 2P excitation wavelength of 920 nm, the mean ROI intensity measured was 99 arbitrary units (arb. u.), which was around 75% of the 940-nm peak intensity (132 arb. u.). Emission curves for the three excitation wavelengths tested (Figure 1C) show a peak emission wavelength around 620 nm.
Having established the spectral properties of the fluorophore, we wanted to measure its performance on a standard 2P microscope equipped with one 920 nm laser and two photomultiplier tubes (PMTs) for detection: one green, one red (Figure 1D). When expressed in human embryonic kidney 293T (HEK293T) cells under the control of a CMV promoter, and with 920 nm excitation, LSSmScarlet3 was detected with the red PMT. As we varied the laser power input, we observed robust detection of LSSmScarlet3 by the red PMT at low gain, whereas the green PMT did not detect any signal below 50 % laser power. Above 25 % laser power, the red PMT was overloaded. Only at around 60 % laser power did we observe substantial cross-talk signal on the green PMT (Figure 1D, E). This separation between RFP and GFP-channel detection indicates that, with appropriate filtering, LSSmScarlet3 is suitable for marking cells without interference with the green channel.
Co-expression of LSSmScarlet3 and GCaMP8m in the mushroom body under UAS control, driven by MB247-Gal4, showed robust co-detection (Figure 1F) in vivo.
We demonstrate the utility of LSSmScarlet3 as a structural marker alongside GCaMP8m using a single laser for excitation. This arrangement promises substantial advantages over existing approaches, including simplicity in setup, elimination of sequential scanning for potential reduction of phototoxicity, and better temporal registration. The excitation maximum near 920 nm is ideal for most existing 2P setups, and we validated that the long Stokes shift minimizes crosstalk between green and red channels. Compared to other long Stokes shift FPs currently available, our transgenic fly is validated and ready to use.
LSSmScarlet3 is quite bright in our hands. Given the low threshold for PMT saturation from the highly expressed LSSmScarlet3 in HEK293T cells (Figure 1D), careful titration of expression levels may be needed. On the other hand, this also reassures users of highly detectable signals when expression levels are low, for example, in a fusion protein. This opens up many possibilities, such as co-imaging neuronal activity with synaptic markers, tagging proteins with subcellular localization signals, or monitoring reference transcription levels.
Transgenic LSSmScarlet3 provides a practical solution for dual-channel 2P imaging in Drosophila. Single laser, two-color imaging opens up avenues for developing LSSmScarlet3 variants for specific biological markers.
","references":[{"reference":"Thermo Fisher Scientific. 2016. Lipofectamine 3000 Reagent Protocol.","pubmedId":"","doi":""},{"reference":"Piatkevich KD, Hulit J, Subach OM, Wu B, Abdulla A, Segall JE, Verkhusha VV. 2010. Monomeric red fluorescent proteins with a large Stokes shift. Proc. Natl. Acad. Sci. U. S. A. 107: 5369-5374.","pubmedId":"","doi":"10.1073/pnas.0914365107"},{"reference":"Lssmscarlet L. . Chemically Stable Genetically Encoded Red Fluorescent Proteins with a.","pubmedId":"","doi":""},{"reference":"Pfeiffer BD, Truman JW, Rubin GM. 2012. Using translational enhancers to increase transgene expression in. Proc. Natl. Acad. Sci. U. S. A. 109: 6626-6631.","pubmedId":"","doi":"10.1073/pnas.1204520109"},{"reference":"Schindelin J, Arganda Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al., Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 9: 676-682.","pubmedId":"","doi":"10.1038/nmeth.2019"},{"reference":"Fly Base. 2025. FlyBase Reference Report: Wilson and Holtz, 2025.2.27, Physiology.","pubmedId":"","doi":""},{"reference":"Thermo Fisher Scientific. 2016. Lipofectamine 3000 Reagent Protocol.","pubmedId":"","doi":""},{"reference":"Temasek Life Sciences Laboratories. 2018. Recipe For Drosophila Media.","pubmedId":"","doi":"10.5281/zenodo.1185451"},{"reference":"Laviv T, Kim BB, Chu J, Lam AJ, Lin MZ, Yasuda R. 2016. Simultaneous dual-color fluorescence lifetime imaging with novel. Nat. Methods. 13: 989-992.","pubmedId":"","doi":"10.1038/nmeth.4046"},{"reference":"Cheung KY, Wu Y, Lee SY, Zhang X, Fukuda M, Suresh DD, Claridge Chang A. 2025. Two-photon characterisation of long-Stokes-shift dye ATTO 490LS for. Neuroscience","pubmedId":"","doi":""},{"reference":"Shcherbakova DM, Hink MA, Joosen L, Gadella TWJ, Verkhusha VV. 2012. An orange fluorescent protein with a large Stokes shift for. J. Am. Chem. Soc. 134: 7913-7923.","pubmedId":"","doi":"10.1021/ja3018972"},{"reference":"Schmid B, Schindelin J, Cardona A, Longair M, Heisenberg M. 2010. A high-level 3D visualization API for Java and ImageJ. BMC Bioinformatics. 11: 274-274.","pubmedId":"","doi":"10.1186/1471-2105-11-274"},{"reference":"Subach OM, Vlaskina AV, Agapova YK, Piatkevich KD, Patrushev MV, Samygina VR, Subach FV. 2022. LSSmScarlet2 and LSSmScarlet3, chemically stable genetically encoded red. Int. J. Mol. Sci. 23: 11051.","pubmedId":"","doi":"10.3390/ijms231911051"},{"reference":"Zars T, Fischer M, Schulz R, Heisenberg M. 2000. Localization of a short-term memory in Drosophila. Science. 288: 672-675.","pubmedId":"","doi":"10.1126/science.288.5466.672"},{"reference":"Kim BB, Wu H, Hao YA, Pan M, Chavarha M, Zhao Y, et al., Lin MZ. 2022. A red fluorescent protein with improved monomericity enables ratiometric. Sci. Rep. 12: 3678.","pubmedId":"","doi":"10.1038/s41598-022-07313-1"},{"reference":"Subach OM, Cranfill PJ, Davidson MW, Verkhusha VV. 2011. An enhanced monomeric blue fluorescent protein with the high chemical. PLoS One. 6: e28674.","pubmedId":"","doi":"10.1371/journal.pone.0028674"},{"reference":"Santos EM, Sheng W, Esmatpour Salmani R, Tahmasebi Nick S, Ghanbarpour A, Gholami H, et al., Borhan B. 2021. Design of Large Stokes Shift fluorescent proteins based on Excited State. J. Am. Chem. Soc. 143: 15091-15102.","pubmedId":"","doi":"10.1021/jacs.1c05039"}],"title":"Long-Stokes-Shift mScarlet3 as a Structural Marker for Two-Photon Imaging
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Steven Marygold"},"openAcknowledgement":false,"submitted":null}]},{"id":"f79dc76b-aafa-49b4-a4ca-3bcfcf39b531","decision":"revise","abstract":"Two-photon imaging with genetically encoded sensors is widely used to monitor neurophysiology. An additional fluorescent protein can provide anatomical landmarks for cell-type identification and motion detection. However, most red fluorescent proteins require a dedicated excitation laser. We made transgenic Drosophila with a long-Stokes-shift mScarlet variant (LSSmScarlet3) to image alongside green sensors with a single 920-nm laser. We describe excitation and emission spectra of the expressed protein and show that 920 nm elicits robust in vitro and in vivo fluorescence. Channel crosstalk is minimal. This approach can reduce equipment complexity and cost while placing functional calcium dynamics in their anatomical context.
","acknowledgements":"We thank Masahiro Fukuda, Hyunsoo Shawn Je (Programme in Neuroscience and Behavioural Disorders, Duke-NUS Medical School) for insightful and productive discussions, Danesha Suresh and Zhiyi Zhang for technical and administrative assistance, and Dénes Pálfi and Viktória Kiss (Femtonics HQ) for assistance with two-photon imaging on FEMTO3D Atlas Plug & Play. We additionally thank Dr. Michael Hausser (University of Hong Kong) and Dr. Jung Kim (University of Hong Kong) for providing temporary access to the FEMTO3D Atlas and lab space. The design of the custom chamber used for imaging was kindly provided by Dr. Anissa Kempf (University of Basel). We thank BDSC and Dr. Hiromu Tanimoto for providing fly stocks.
","authors":[{"affiliations":["Institute for Molecular and Cell Biology (IMCB), A*STAR, Singapore","Duke-NUS Medical School, Singapore"],"departments":["","Programme in Neuroscience and Behavioural Disorders"],"credit":["conceptualization","methodology","investigation","dataCuration","formalAnalysis","visualization","writing_originalDraft","writing_reviewEditing","fundingAcquisition"],"email":"xusangyu@gmail.com","firstName":"Sangyu","lastName":"Xu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4927-9204"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["investigation","writing_reviewEditing","methodology"],"email":"xy_zhang@duke-nus.edu.sg","firstName":"Xianyuan","lastName":"Zhang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0003-4483-750X"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["methodology","investigation","dataCuration","formalAnalysis","visualization","writing_reviewEditing"],"email":"kycheung@duke-nus.edu.sg","firstName":"King Yee","lastName":"Cheung","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-1098-4926"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["methodology","investigation","dataCuration","validation","writing_reviewEditing"],"email":"maiyishan@u.duke.nus.edu","firstName":"Yishan","lastName":"Mai","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7199-380X"},{"affiliations":["National University of Singapore, Singapore"],"departments":["Microscopy Core Facility — Multiphoton Microscopy Unit, Yong Loo Lin School of Medicine"],"credit":["methodology","investigation","validation","writing_reviewEditing"],"email":"wu.yue@nus.edu.sg","firstName":"Yue","lastName":"Wu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2484-4967"},{"affiliations":["Duke-NUS Medical School, Singapore","Institute of Molecular and Cell Biology, A*STAR, Singapore","National University of Singapore, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders","","Department of Physiology, Yong Loo Lin School of Medicine"],"credit":["conceptualization","supervision","fundingAcquisition","writing_reviewEditing"],"email":"claridge-chang.adam@duke-nus.edu.sg","firstName":"Adam","lastName":"Claridge-Chang","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4583-3650"}],"awards":[{"awardId":"MOH-OFYIRG20nov-0051","funderName":"National Medical Research Council (Singapore)","awardRecipient":"Sangyu Xu"},{"awardId":"MOH-001397-01","funderName":"National Medical Research Council (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"MOE-T2EP30222-0018","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"MOE-T2EP30223-0009","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"FY2022-MOET1-0001","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"S. X. was supported by an MOH-OFYIRG20nov-0051, and X. Z., K. Y. C., Y. M., and A. C.-C. were supported by the MOH-001397-01 grant from the National Medical Research Council, Singapore, by grants MOE-T2EP30222-0018 and MOE-T2EP30223-0009 from the Ministry of Education, Singapore, and by a Duke-NUS Medical School grant. X. Z. and A. C.-C. were supported by FY2022-MOET1-0001.
","image":{"url":"https://portal.micropublication.org/uploads/ce5a9aea0e4551e97f1410bf18dd2876.png"},"imageCaption":"A) An image of the mushroom body expressing LSSmScarlet3 in a fixed MB247-Gal4>UAS-LSSmScarlet3 fly collected with 940 nm excitation and 620 nm collection. White box: control ROI without LSSmScarlet3 expression. Orange box: test ROI with LSSmScarlet3 expression. Scale bar: 20 μm.
B) Excitation spectrum of LSSmScarlet3 in the sample shown in A. Three local maxima were further tested for emission: 860 nm, 940 nm, and 1040 nm.
C) Emission spectra of excitation at 860 nm, 940 nm, and 1040 nm.
D) LSSmScarlet3 expressed in live HEK293T cells under the control of a CMV promoter excited at various 920 nm laser power settings and images collected with a green and a red PMT. At laser power above 25 %, the red PMT was overloaded by the light emitted. Green signals began to emerge at above 50 % laser power.
E) Quantification of mean intensity of each image in D as a function of laser power.
F) Maximum intensity projection image of the mushroom body calyx and α-lobe in a live MB247>GCaMP8m;LSSmScarlet3Drosophila brain. Both fluorophores were simultaneously excited by the 920 nm laser at 20% power. (F) GCaMP8m signals detected with the green-filtered PMT at 80% gain, (F′) LSSmScarlet3 signals detected with the red-filtered PMT at 70% gain, and (F′′) merged signals. Scale bar = 20 μm.
","imageTitle":"Two-photon imaging of a Drosophila codon-optimized LSSmScarlet3
","methods":"Fly stocks and husbandry
Drosophila cultures were maintained on standard cornmeal-based medium (1.25 % w/v agar, 10.5 % w/v dextrose, 10.5 % w/v maize, and 2.1 % w/v yeast) (Temasek Life Sciences Laboratories 2018). Stocks were kept at room temperature unless otherwise specified. Crosses were performed at 25 ºC in a day-night cycling incubator (Mir-253, Sanyo). Adult male and female flies, aged 5–10 days, were used for experiments. UAS-GCamp8m was obtained from Bloomington Drosophila Stock Center (BDSC #92590). The MB247-Gal4 stock (Zars et al. 2000) was provided by Dr Hiromu Tanimoto (Tohoku University, Japan).
Construct generation
LSSmScarlet3 coding sequence was codon-optimized for Drosophila by GenScript (Piscataway, NJ, USA) and cloned into pcDNA3.4 (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. V880-01) to generate the mammalian expression construct pcDNA-DmLSSmScarlet3 and into pJFRC81-10XUAS-IVS-Syn21-GFP-p10 vector (Pfeiffer et al. 2012) between XhoI and XbaI restriction sites to generate Drosophila expression construct UAS-DmLSSmScarlet3, which was then injected into yw flies at the attp40 site balanced against Curly of Oster by BestGene (La Jolla, CA, USA). The pJFRC81-10XUAS-IVS-Syn21-GFP-p10 plasmid was a gift from Gerald Rubin (Addgene plasmid #36432; http://n2t.net/addgene:36432; RRID:Addgene_36432)
Excitation and emission scanning of mushroom body tissue
Fly brains expressing LSSmScarlet3 were dissected in cold PBS and fixed in 4 % PFA for 30 min and then mounted in PBS in 2 stacked reinforcement rings (Suremark) pasted on 1.0–1.2 mm-thick 25 × 75 mm glass slides (Biomedia), and then sealed with 170 µm-thick 18 × 18 mm glass cover slips with clear nail polish. Samples were imaged on a Leica TCS SP8 DIVE multiphoton microscope (Leica Microsystems, Wetzlar, Germany) as previously described (Cheung et al. 2025) with the exception that in the emission spectrum measurements the excitation wavelengths were fixed at 860 nm, 940 nm, or 1040 nm. Image acquisition and spectral detection were controlled using Leica LAS X software. Mean intensity of ROIs in images was calculated using Fiji (Schindelin et al. 2012). Excitation and emission spectra graphs were generated using matplotlib in Python.
Cell culture and transfection
HEK293T cells were seeded into small tissue culture dishes to ~ 70 % confluence in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA, USA) and transfected with pcDNA3.4-DmLSSmScarlet3 constructs using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) in Opti-MEM™ Medium (Thermo Fisher Scientific, Waltham, MA, USA) according to a standard protocol (Thermo Fisher Scientific 2016) in 35 mm cell culture dishes (Nunc, catalog #153066; Thermo Fisher Scientific, Waltham, MA, USA). The cells were imaged 48 hours after transfection.
2-photon imaging of LSSmScarlet3 in HEK293T cells
Cell culture expressing LSSmScarlet3 was imaged with A-SCOPE (Thorlabs, NJ, USA) as previously described (Cheung et al. 2025). Laser power was set to 30 mW. Green PMT was bandpass filtered between 500 nm and 550 nm, and the red PMT was bandpass filtered between 570 nm and 640 nm. Detector gains were set to 5%.
Simultaneous 2-photon imaging of GCaMP8m and LSSmScarlet3 in Drosophila mushroom body
Flies were mounted in a custom 3D-printed chamber using candle wax. Fly heads were opened using a pair of sharp Dumont No. 5 forceps (Fine Science Tools) and microdissection scissors (Fine Science Tools). A drop of PBS was then placed on the open fly head to keep the brain moist and to act as the objective immersion medium.
Z-stack images were acquired on a FEMTO3D Atlas Plug & Play 2-photon microscope (Femtonics, Budapest, Hungary) equipped with a 16× objective lens. Laser power of the 920 nm laser was set to 20% (112 mW). GCaMP8 emission light was collected by a green-filtered PMT at 80% gain, while LSSmScarlet3 emission light was collected by a red-filtered PMT at 70% gain.
Image processing
The Drosophila mushroom body 2-photon image stack was subjected to Gaussian blur 3D processing (X: 0, Y: 0, Z: 2) and maximum intensity projection processing in Fiji (Schindelin et al. 2012). For the purpose of visualizing dual-channel images, signal collected by the green-filtered PMT was pseudo-colored to green, while signal collected by the red-filtered PMT was pseudo-colored to magenta.
Data analysis and visualization
Preprocessed data from the Fiji workflow were analyzed and visualized using custom Jupyter Notebooks running Python (version 3.10.16) with pandas (version 2.1.4), NumPy (version 1.26.4), and seaborn (version 0.12.2).
Data and Code Availability
Data and code used to generate the figure in this paper, as well as construct sequences and maps, are available as a Zenodo repository (DOI: 10.5281/zenodo.19493335).
","reagents":"","patternDescription":"Understanding neural circuits requires monitoring both neural activity and anatomical relationships. Genetically encoded calcium indicators based on blue-excitable green fluorescent protein (GFP) are widely used, for example, the GFP–Calmodulin–M13-peptide (GCaMP) series of calcium sensors. Such sensors enable cellular-resolution functional imaging, but calcium signals are transient and can afford limited structural context. Alongside sensors, researchers sometimes co-express red fluorescent proteins (RFPs) as structural markers (e.g., tdTomato or mCherry) to provide landmarks for cellular identification and sample-motion detection. However, this strategy requires separate two-photon (2P) excitation wavelengths—typically 920 nm for GCaMP and ~1040 nm for some RFPs; this necessitates complex multi-laser setups that increase equipment cost, system complexity, and phototoxicity.
Long-Stokes-shift (LSS) fluorescent dyes exhibit large separations between excitation and emission peaks (Shcherbakova et al. 2012). Meanwhile, there has been considerable effort in developing LSS fluorescent proteins through diverse approaches, including excited-state proton transfer, chromophore modifications, and protein engineering (Piatkevich et al. 2010; Santos et al. 2021). Originally developed to improve FLIM-FRET (fluorescence lifetime imaging microscopy-Förster resonance energy transfer) sensors by reducing spectral overlap between donor and acceptor fluorophores, LSS variants have found broader applications in multicolor imaging (Shcherbakova et al. 2012; Piatkevich et al. 2010). A recently developed LSS variant of mScarlet, LSSmScarlet3, exhibits a 128 nm Stokes shift (ex 466 → em 598 nm) and, compared to previous versions, has improved pH stability and photostability (Subach et al. 2022). Similarly, monomeric cyan-excitable RFP 3, mCyRFP3 (Kim et al. 2022; Laviv et al. 2016), has been deposited as a Drosophila transgenic line (FlyBase 2025); however, to our knowledge, its performance in any species remains to be reported. Here, we describe a genetically encoded LSSmScarlet3 as a structural marker for single-laser two-color 2P imaging, demonstrating efficient excitation at 920 nm. Alongside GFP-based sensors, LSSmScarlet3 provides a simplified approach for dual-channel functional–structural imaging.
To verify whether a long-Stokes-shift RFP can be imaged with a 920 nm laser in Drosophila brains, we synthesized a Drosophila codon-optimized version of LSSmScarlet3. In fixed Drosophila tissue, LSSmScarlet3 was well expressed and readily distributed to the lobes of the mushroom body, indicating good neuronal expression and distribution (Figure 1A). On a spectrally tunable multiphoton microscope, LSSmScarlet3 had a peak excitation at 940 nm, with two smaller local maxima at 860 nm and 1040 nm (Figure 1B). At a frequently used 2P excitation wavelength of 920 nm, the mean ROI intensity measured was 99 arbitrary units (arb. u.), which was around 75% of the 940-nm peak intensity (132 arb. u.). Emission curves for the three excitation wavelengths tested (Figure 1C) show a peak emission wavelength around 620 nm.
Having established the spectral properties of the fluorophore, we wanted to measure its performance on a standard 2P microscope equipped with one 920 nm laser and two photomultiplier tubes (PMTs) for detection: one green, one red (Figure 1D). When expressed in human embryonic kidney 293T (HEK293T) cells under the control of a CMV promoter, and with 920 nm excitation, LSSmScarlet3 was detected with the red PMT. As we varied the laser power input, we observed robust detection of LSSmScarlet3 by the red PMT at low gain, whereas the green PMT did not detect any signal below 50 % laser power. Above 25 % laser power, the red PMT was overloaded. Only at around 60 % laser power did we observe substantial cross-talk signal on the green PMT (Figure 1D, E). This separation between RFP and GFP-channel detection indicates that, with appropriate filtering, LSSmScarlet3 is suitable for marking cells without interference with the green channel.
Co-expression of LSSmScarlet3 and GCaMP8m in the mushroom body under UAS control, driven by MB247-Gal4, showed robust co-detection (Figure 1F) in vivo.
We demonstrate the utility of LSSmScarlet3 as a structural marker alongside GCaMP8m using a single laser for excitation. This arrangement promises substantial advantages over existing approaches, including simplicity in setup, elimination of sequential scanning for potential reduction of phototoxicity, and better temporal registration. The excitation maximum near 920 nm is ideal for most existing 2P setups, and we validated that the long Stokes shift minimizes crosstalk between green and red channels. Compared to other long Stokes shift FPs currently available, our transgenic fly is validated and ready to use.
LSSmScarlet3 is quite bright in our hands. Given the low threshold for PMT saturation from the highly expressed LSSmScarlet3 in HEK293T cells (Figure 1D), careful titration of expression levels may be needed. On the other hand, this also reassures users of highly detectable signals when expression levels are low, for example, in a fusion protein. This opens up many possibilities, such as co-imaging neuronal activity with synaptic markers, tagging proteins with subcellular localization signals, or monitoring reference transcription levels.
Transgenic LSSmScarlet3 provides a practical solution for dual-channel 2P imaging in Drosophila. Single laser, two-color imaging opens up avenues for developing LSSmScarlet3 variants for specific biological markers.
","references":[{"reference":"Cheung KY, Wu Y, Lee SY, Zhang X, Fukuda M, Suresh DD, Claridge Chang A. 2025. Two-photon characterisation of long-Stokes-shift dye ATTO 490LS for. Neuroscience","pubmedId":"","doi":""},{"reference":"Fly Base. 2025. FlyBase Reference Report: Wilson and Holtz, 2025.2.27, Physiology.","pubmedId":"","doi":""},{"reference":"Kim BB, Wu H, Hao YA, Pan M, Chavarha M, Zhao Y, et al., Lin MZ. 2022. A red fluorescent protein with improved monomericity enables ratiometric. Sci. Rep. 12: 3678.","pubmedId":"","doi":"10.1038/s41598-022-07313-1"},{"reference":"Laviv T, Kim BB, Chu J, Lam AJ, Lin MZ, Yasuda R. 2016. Simultaneous dual-color fluorescence lifetime imaging with novel. Nat. Methods. 13: 989-992.","pubmedId":"","doi":"10.1038/nmeth.4046"},{"reference":"Lssmscarlet L. . Chemically Stable Genetically Encoded Red Fluorescent Proteins with a.","pubmedId":"","doi":""},{"reference":"Pfeiffer BD, Truman JW, Rubin GM. 2012. Using translational enhancers to increase transgene expression in. Proc. Natl. Acad. Sci. U. S. A. 109: 6626-6631.","pubmedId":"","doi":"10.1073/pnas.1204520109"},{"reference":"Piatkevich KD, Hulit J, Subach OM, Wu B, Abdulla A, Segall JE, Verkhusha VV. 2010. Monomeric red fluorescent proteins with a large Stokes shift. Proc. Natl. Acad. Sci. U. S. A. 107: 5369-5374.","pubmedId":"","doi":"10.1073/pnas.0914365107"},{"reference":"Santos EM, Sheng W, Esmatpour Salmani R, Tahmasebi Nick S, Ghanbarpour A, Gholami H, et al., Borhan B. 2021. Design of Large Stokes Shift fluorescent proteins based on Excited State. J. Am. Chem. Soc. 143: 15091-15102.","pubmedId":"","doi":"10.1021/jacs.1c05039"},{"reference":"Schindelin J, Arganda Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al., Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 9: 676-682.","pubmedId":"","doi":"10.1038/nmeth.2019"},{"reference":"Schmid B, Schindelin J, Cardona A, Longair M, Heisenberg M. 2010. A high-level 3D visualization API for Java and ImageJ. BMC Bioinformatics. 11: 274-274.","pubmedId":"","doi":"10.1186/1471-2105-11-274"},{"reference":"Shcherbakova DM, Hink MA, Joosen L, Gadella TWJ, Verkhusha VV. 2012. An orange fluorescent protein with a large Stokes shift for. J. Am. Chem. Soc. 134: 7913-7923.","pubmedId":"","doi":"10.1021/ja3018972"},{"reference":"Subach OM, Cranfill PJ, Davidson MW, Verkhusha VV. 2011. An enhanced monomeric blue fluorescent protein with the high chemical. PLoS One. 6: e28674.","pubmedId":"","doi":"10.1371/journal.pone.0028674"},{"reference":"Subach OM, Vlaskina AV, Agapova YK, Piatkevich KD, Patrushev MV, Samygina VR, Subach FV. 2022. LSSmScarlet2 and LSSmScarlet3, chemically stable genetically encoded red. Int. J. Mol. Sci. 23: 11051.","pubmedId":"","doi":"10.3390/ijms231911051"},{"reference":"Temasek Life Sciences Laboratories. 2018. Recipe For Drosophila Media.","pubmedId":"","doi":"10.5281/zenodo.1185451"},{"reference":"Thermo Fisher Scientific. 2016. Lipofectamine 3000 Reagent Protocol.","pubmedId":"","doi":""},{"reference":"Thermo Fisher Scientific. 2016. Lipofectamine 3000 Reagent Protocol.","pubmedId":"","doi":""},{"reference":"Zars T, Fischer M, Schulz R, Heisenberg M. 2000. Localization of a short-term memory in Drosophila. Science. 288: 672-675.","pubmedId":"","doi":"10.1126/science.288.5466.672"}],"title":"Long-Stokes-Shift mScarlet3 as a Structural Marker for Two-Photon Imaging","reviews":[{"reviewer":{"displayName":"Elizabeth Hong"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Steven Marygold"},"openAcknowledgement":false,"submitted":"1781161842034"}]},{"id":"be40a414-ccd7-4047-9fb2-eb8b037a9b94","decision":"accept","abstract":"Two-photon imaging with genetically encoded sensors is widely used to monitor neurophysiology. An additional fluorescent protein can provide anatomical landmarks for cell-type identification and motion detection. However, monomeric red fluorescent proteins often require a dedicated excitation laser. We made transgenic Drosophila with a long-Stokes-shift mScarlet variant (LSSmScarlet3) to image alongside green sensors with a single 920-nm laser. We describe excitation and emission spectra of the expressed protein and show robust fluorescence at 920 nm in vitro and in vivo with minimal channel crosstalk. This approach can reduce equipment complexity and cost while placing functional calcium dynamics in their anatomical context.
","acknowledgements":"We thank Masahiro Fukuda, Hyunsoo Shawn Je (Programme in Neuroscience and Behavioural Disorders, Duke-NUS Medical School) for insightful and productive discussions, Danesha Suresh and Zhiyi Zhang for technical and administrative assistance, and Dénes Pálfi and Viktória Kiss (Femtonics HQ) for assistance with two-photon imaging on FEMTO3D Atlas Plug & Play. We additionally thank Dr. Michael Hausser (University of Hong Kong) and Dr. Jung Kim (University of Hong Kong) for providing temporary access to the FEMTO3D Atlas and lab space. The design of the custom chamber used for imaging was kindly provided by Dr. Anissa Kempf (University of Basel). We thank BDSC and Dr. Hiromu Tanimoto for providing fly stocks.
","authors":[{"affiliations":["Institute of Molecular and Cell Biology (IMCB), A*STAR, Singapore","Duke-NUS Medical School, Singapore"],"departments":["","Programme in Neuroscience and Behavioural Disorders"],"credit":["conceptualization","methodology","investigation","dataCuration","formalAnalysis","visualization","writing_originalDraft","writing_reviewEditing","fundingAcquisition"],"email":"xusangyu@gmail.com","firstName":"Sangyu","lastName":"Xu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4927-9204"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["investigation","writing_reviewEditing","methodology"],"email":"xy_zhang@duke-nus.edu.sg","firstName":"Xianyuan","lastName":"Zhang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0003-4483-750X"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["methodology","investigation","dataCuration","formalAnalysis","visualization","writing_reviewEditing"],"email":"kycheung@duke-nus.edu.sg","firstName":"King Yee","lastName":"Cheung","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-1098-4926"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["methodology","investigation","dataCuration","validation","writing_reviewEditing"],"email":"maiyishan@u.duke.nus.edu","firstName":"Yishan","lastName":"Mai","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7199-380X"},{"affiliations":["National University of Singapore, Singapore"],"departments":["Microscopy Core Facility — Multiphoton Microscopy Unit, Yong Loo Lin School of Medicine"],"credit":["methodology","investigation","validation","writing_reviewEditing"],"email":"wu.yue@nus.edu.sg","firstName":"Yue","lastName":"Wu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2484-4967"},{"affiliations":["Duke-NUS Medical School, Singapore","Institute of Molecular and Cell Biology, A*STAR, Singapore","National University of Singapore, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders","","Department of Physiology, Yong Loo Lin School of Medicine"],"credit":["conceptualization","supervision","fundingAcquisition","writing_reviewEditing"],"email":"claridge-chang.adam@duke-nus.edu.sg","firstName":"Adam","lastName":"Claridge-Chang","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4583-3650"}],"awards":[{"awardId":"MOH-OFYIRG20nov-0051","funderName":"National Medical Research Council (Singapore)","awardRecipient":"Sangyu Xu"},{"awardId":"MOH-001397-01","funderName":"National Medical Research Council (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"MOE-T2EP30222-0018","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"MOE-T2EP30223-0009","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"FY2022-MOET1-0001","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"S. X. was supported by an MOH-OFYIRG20nov-0051, and X. Z., K. Y. C., Y. M., and A. C.-C. were supported by the MOH-001397-01 grant from the National Medical Research Council, Singapore, by grants MOE-T2EP30222-0018 and MOE-T2EP30223-0009 from the Ministry of Education, Singapore, and by a Duke-NUS Medical School grant. X. Z. and A. C.-C. were supported by FY2022-MOET1-0001.
","image":{"url":"https://portal.micropublication.org/uploads/ce5a9aea0e4551e97f1410bf18dd2876.png"},"imageCaption":"A) An image of the mushroom body expressing LSSmScarlet3 in a fixed MB247-Gal4>UAS-LSSmScarlet3 fly collected with 940 nm excitation and 620 nm collection. White box: control ROI without LSSmScarlet3 expression. Orange box: test ROI with LSSmScarlet3 expression. Scale bar: 20 μm.
B) Excitation spectrum of LSSmScarlet3 in the sample shown in A. Three local maxima were further tested for emission: 860 nm, 940 nm, and 1040 nm.
C) Emission spectra of excitation at 860 nm, 940 nm, and 1040 nm.
D) LSSmScarlet3 expressed in live HEK293T cells under the control of a CMV promoter excited at various 920 nm laser power settings and images collected with a green and a red PMT. At laser power above 25 %, the red PMT was overloaded by the light emitted. Green signals began to emerge at above 50 % laser power.
E) Quantification of mean intensity of each image in D as a function of laser power.
F) Maximum intensity projection image of the mushroom body calyx and α-lobe in a live MB247>GCaMP8m;LSSmScarlet3 Drosophila brain. Both fluorophores were simultaneously excited by the 920 nm laser at 20% power. (F) GCaMP8m signals detected with the green-filtered PMT at 80% gain, (F′) LSSmScarlet3 signals detected with the red-filtered PMT at 70% gain, and (F′′) merged signals. Scale bar = 20 μm.
","imageTitle":"Two-photon imaging of a Drosophila codon-optimized LSSmScarlet3
","methods":"Fly stocks and husbandry
Drosophila cultures were maintained on standard cornmeal-based medium (1.25 % w/v agar, 10.5 % w/v dextrose, 10.5 % w/v maize, and 2.1 % w/v yeast) (Temasek Life Sciences Laboratories 2018). Stocks were kept at room temperature unless otherwise specified. Crosses were performed at 25 ºC in a day-night cycling incubator (Mir-253, Sanyo). Adult male and female flies, aged 5–10 days, were used for experiments. UAS-jGCaMP8m (Zhang et al. 2023) was obtained from Bloomington Drosophila Stock Center (BDSC #92590). The MB247-Gal4 stock (Zars et al. 2000) was provided by Dr Hiromu Tanimoto (Tohoku University, Japan).
Construct generation
LSSmScarlet3 coding sequence was codon-optimized for Drosophila by GenScript (Piscataway, NJ, USA) and cloned into pcDNA3.4 (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. V880-01) to generate the mammalian expression construct pcDNA-DmLSSmScarlet3 and into pJFRC81-10XUAS-IVS-Syn21-GFP-p10 vector (Pfeiffer et al. 2012) between XhoI and XbaI restriction sites to generate Drosophila expression construct UAS-DmLSSmScarlet3, which was then injected into yw flies at the attp40 site balanced against Curly of Oster by BestGene (La Jolla, CA, USA). The pJFRC81-10XUAS-IVS-Syn21-GFP-p10 plasmid was a gift from Gerald Rubin (Addgene plasmid #36432; http://n2t.net/addgene:36432; RRID:Addgene_36432)
Excitation and emission scanning of mushroom body tissue
Fly brains expressing LSSmScarlet3 were dissected in cold PBS and fixed in 4 % PFA for 30 min and then mounted in PBS in 2 stacked reinforcement rings (Suremark) pasted on 1.0–1.2 mm-thick 25 × 75 mm glass slides (Biomedia), and then sealed with 170 µm-thick 18 × 18 mm glass cover slips with clear nail polish. Samples were imaged on a Leica TCS SP8 DIVE multiphoton microscope (Leica Microsystems, Wetzlar, Germany) as previously described (Cheung et al. 2026) with the exception that in the emission spectrum measurements the excitation wavelengths were fixed at 860 nm, 940 nm, or 1040 nm. Image acquisition and spectral detection were controlled using Leica LAS X software. Mean intensity of ROIs in images was calculated using Fiji (Schindelin et al. 2012). Excitation and emission spectra graphs were generated using matplotlib in Python.
Cell culture and transfection
HEK293T cells were seeded into small tissue culture dishes to ~ 70 % confluence in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA, USA) and transfected with pcDNA3.4-DmLSSmScarlet3 constructs using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) in Opti-MEM™ Medium (Thermo Fisher Scientific, Waltham, MA, USA) according to a standard protocol (Thermo Fisher Scientific 2016) in 35 mm cell culture dishes (Nunc, catalog #153066; Thermo Fisher Scientific, Waltham, MA, USA). The cells were imaged 48 hours after transfection.
2-photon imaging of LSSmScarlet3 in HEK293T cells
Cell culture expressing LSSmScarlet3 was imaged with A-SCOPE (Thorlabs, NJ, USA) as previously described (Cheung et al. 2026). Laser power was set to 30 mW. Green PMT was bandpass filtered between 500 nm and 550 nm, and the red PMT was bandpass filtered between 570 nm and 640 nm. Detector gains were set to 5%.
Simultaneous 2-photon imaging of GCaMP8m and LSSmScarlet3 in Drosophila mushroom body
Flies were mounted in a custom 3D-printed chamber using candle wax. Fly heads were opened using a pair of sharp Dumont No. 5 forceps (Fine Science Tools) and microdissection scissors (Fine Science Tools). A drop of PBS was then placed on the open fly head to keep the brain moist and to act as the objective immersion medium.
Z-stack images were acquired on a FEMTO3D Atlas Plug & Play 2-photon microscope (Femtonics, Budapest, Hungary) equipped with a 16× objective lens. Laser power of the 920 nm laser was set to 20% (112 mW). GCaMP8 emission light was collected by a green-filtered PMT at 80% gain, while LSSmScarlet3 emission light was collected by a red-filtered PMT at 70% gain.
Image processing
The Drosophila mushroom body 2-photon image stack was subjected to Gaussian blur 3D processing (X: 0, Y: 0, Z: 2) and maximum intensity projection processing in Fiji (Schindelin et al. 2012). For the purpose of visualizing dual-channel images, signal collected by the green-filtered PMT was pseudo-colored to green, while signal collected by the red-filtered PMT was pseudo-colored to magenta.
Data analysis and visualization
Preprocessed data from the Fiji workflow were analyzed and visualized using custom Jupyter Notebooks running Python (version 3.10.16) with pandas (version 2.1.4), NumPy (version 1.26.4), and seaborn (version 0.12.2).
Data and Code Availability
Data and code used to generate the figure in this paper, as well as construct sequences and maps, are available as a Zenodo repository (DOI: 10.5281/zenodo.19493335).
","reagents":"","patternDescription":"Understanding neural circuits requires monitoring both neural activity and anatomical relationships. Genetically encoded calcium indicators based on blue-excitable green fluorescent protein (GFP) are widely used, for example, the GFP–Calmodulin–M13-peptide (GCaMP) series of calcium sensors. Such sensors enable cellular-resolution functional imaging, but calcium signals are transient and can afford limited structural context. Alongside sensors, researchers sometimes co-express red fluorescent proteins (RFPs) as structural markers (e.g., tdTomato or mCherry) to provide landmarks for cellular identification and sample-motion detection. Single-laser excitation of red fluorescent proteins away from their excitation maxima (~920 nm rather than at their ~1040 nm peak) has been used for dual-color structural-functional imaging (Tillo et al. 2010; Ozbay et al. 2018), but relies on the tandem dimer tdTomato, whose two-photon cross-section at this wavelength is an order of magnitude higher than that of monomeric RFPs such as mCherry (~35–40 GM vs. ~5 GM) (Drobizhev et al. 2011). The resulting doubling of molecular weight complicates protein-fusion functionality (Shaner et al. 2005). Alternatively, a dedicated 1040 nm laser can be added to excite monomeric RFPs at their peak (Gee et al. 2014; Bugeon et al. 2022), resulting in multi-laser setups that increase equipment cost, system complexity, and phototoxicity. A monomeric red fluorophore with robust excitation at ~920 nm could therefore retain the simplicity of single-wavelength imaging while avoiding a tandem dimer, improving signal levels at low expression, and broadening applications to include fusion proteins and subcellular markers.
Long-Stokes-shift (LSS) fluorescent dyes exhibit large separations between excitation and emission peaks (Shcherbakova et al. 2012). Meanwhile, there has been considerable effort in developing LSS fluorescent proteins through diverse approaches, including excited-state proton transfer, chromophore modifications, and protein engineering (Piatkevich et al. 2010; Santos et al. 2021). Originally developed to improve FLIM-FRET (fluorescence lifetime imaging microscopy-Förster resonance energy transfer) sensors by reducing spectral overlap between donor and acceptor fluorophores, LSS variants have found broader applications in multicolor imaging (Shcherbakova et al. 2012; Piatkevich et al. 2010). A recently developed LSS variant of mScarlet, LSSmScarlet3, exhibits a 128 nm Stokes shift (ex 466 → em 598 nm) and, compared to previous versions, has improved pH stability and photostability (Subach et al. 2022). Similarly, monomeric cyan-excitable RFP 3, mCyRFP3 (Kim et al. 2022), has been reported as a functional fusion tag in cell-based imaging and has recently been incorporated into Drosophila transgenic reagents (FlyBase 2025). Here, as a complementary contribution, we describe a genetically encoded LSSmScarlet3 as a structural marker for single-laser two-color 2P imaging, demonstrating efficient excitation at 920 nm. Alongside GFP-based sensors, LSSmScarlet3 provides a simplified approach for dual-channel functional–structural imaging.
To verify whether a long-Stokes-shift RFP can be imaged with a 920 nm laser in Drosophila brains, we synthesized a Drosophila codon-optimized version of LSSmScarlet3. In fixed Drosophila tissue, LSSmScarlet3 was well expressed and readily distributed to the lobes of the mushroom body, indicating good neuronal expression and distribution (Figure 1A). On a spectrally tunable multiphoton microscope, LSSmScarlet3 had a peak excitation at 940 nm, with two smaller local maxima at 860 nm and 1040 nm (Figure 1B). At a frequently used 2P excitation wavelength of 920 nm, the mean ROI intensity measured was 99 arbitrary units (arb. u.), which was around 75% of the 940-nm peak intensity (132 arb. u.). Emission curves for the three excitation wavelengths tested (Figure 1C) show a peak emission wavelength around 620 nm.
Having established the spectral properties of the fluorophore, we wanted to measure its performance on a standard 2P microscope equipped with one 920 nm laser and two photomultiplier tubes (PMTs) for detection: one green, one red (Figure 1D). When expressed in human embryonic kidney 293T (HEK293T) cells under the control of a CMV promoter, and with 920 nm excitation, LSSmScarlet3 was detected with the red PMT. As we varied the laser power input, we observed robust detection of LSSmScarlet3 by the red PMT at low gain, whereas the green PMT did not detect any signal below 50 % laser power. Above 25 % laser power, the red PMT was overloaded. Only at around 60 % laser power did we observe substantial cross-talk signal on the green PMT (Figure 1D, E). This separation between RFP and GFP-channel detection indicates that, with appropriate filtering, LSSmScarlet3 is suitable for marking cells without interference with the green channel.
Co-expression of LSSmScarlet3 and GCaMP8m in the mushroom body under UAS control, driven by MB247-Gal4, showed robust co-detection (Figure 1F) in vivo.
We demonstrate the utility of LSSmScarlet3 as a structural marker alongside GCaMP8m using a single laser for excitation. This arrangement promises substantial advantages over existing approaches, including simplicity in setup, elimination of sequential scanning for potential reduction of phototoxicity, and better temporal registration. Strong excitation at 920 nm is ideal for most existing 2P setups, and we validated that the long Stokes shift minimizes crosstalk between green and red channels. Compared to other long Stokes shift FPs currently available, our transgenic fly is validated and ready to use.
LSSmScarlet3 is quite bright in our hands. Given the low threshold for PMT saturation from the highly expressed LSSmScarlet3 in HEK293T cells (Figure 1D), careful titration of expression levels may be needed. On the other hand, this also reassures users of highly detectable signals when expression levels are low, for example, in a fusion protein. This opens up many possibilities, such as co-imaging neuronal activity with synaptic markers, tagging proteins with subcellular localization signals, or monitoring reference transcription levels.
Transgenic LSSmScarlet3 provides a practical solution for dual-channel 2P imaging in Drosophila. Single-laser, two-color imaging opens up avenues for developing LSSmScarlet3 variants for specific biological markers. For applications with strong promoters where structural bulk is not a concern, using tdTomato excited with ~920 nm remains a viable option. LSSmScarlet3 offers particular advantages when a monomeric tag is needed, such as with fusion proteins, weak promoters, or subcellular localization signals where expression levels may be limiting.
","references":[{"reference":"Zhang Y, Rozsa M, Liang Y, Bushey D, Wei Z, Zheng J, et al., Looger LL. 2023. Fast and sensitive GCaMP calcium indicators for imaging neural populations. Nature. 615: 884-891.","pubmedId":"","doi":"10.1038/s41586-023-05828-9"},{"reference":"Piatkevich KD, Hulit J, Subach OM, Wu B, Abdulla A, Segall JE, Verkhusha VV. 2010. Monomeric red fluorescent proteins with a large Stokes shift. Proc. Natl. Acad. Sci. U. S. A. 107: 5369-5374.","pubmedId":"","doi":"10.1073/pnas.0914365107"},{"reference":"Pfeiffer BD, Truman JW, Rubin GM. 2012. Using translational enhancers to increase transgene expression in. Proc. Natl. Acad. Sci. U. S. A. 109: 6626-6631.","pubmedId":"","doi":"10.1073/pnas.1204520109"},{"reference":"Schindelin J, Arganda Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al., Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 9: 676-682.","pubmedId":"","doi":"10.1038/nmeth.2019"},{"reference":"Shaner NC, Steinbach PA, Tsien RY. 2005. A guide to choosing fluorescent proteins. Nat. Methods. 2: 905-909.","pubmedId":"","doi":"10.1038/nmeth819"},{"reference":"Fly Base. 2025. FlyBase Reference Report: Wilson and Holtz, 2025.2.27, Physiology.","pubmedId":"","doi":""},{"reference":"Tillo SE, Hughes TE, Makarov NS, Rebane A, Drobizhev M. 2010. A new approach to dual-color two-photon microscopy with fluorescent. BMC Biotechnol. 10: 6.","pubmedId":"","doi":"10.1186/1472-6750-10-6"},{"reference":"Cheung KY, Wu Y, Lee SY, Zhang X, Fukuda M, Suresh DD, Claridge Chang A. 2026. Two-photon characterisation of long-Stokes-shift dye ATTO 490LS for. MicroPubl. Biol. 2026","pubmedId":"","doi":"10.17912/micropub.biology.001955"},{"reference":"Thermo Fisher Scientific. 2016. Lipofectamine 3000 Reagent Protocol.","pubmedId":"","doi":""},{"reference":"Temasek Life Sciences Laboratories. 2018. Recipe For Drosophila Media.","pubmedId":"","doi":"10.5281/zenodo.1185451"},{"reference":"Bugeon S, Duffield J, Dipoppa M, Ritoux A, Prankerd I, Nicoloutsopoulos D, et al., Harris KD. 2022. A transcriptomic axis predicts state modulation of cortical interneurons. Nature. 607: 330-338.","pubmedId":"","doi":"10.1038/s41586-022-04915-7"},{"reference":"Gee JM, Smith NA, Fernandez FR, Economo MN, Brunert D, Rothermel M, et al., Tvrdik P. 2014. Imaging activity in neurons and glia with a Polr2a-based and cre-dependent. Neuron. 83: 1058-1072.","pubmedId":"","doi":"10.1016/j.neuron.2014.07.024"},{"reference":"Drobizhev M, Makarov NS, Tillo SE, Hughes TE, Rebane A. 2011. Two-photon absorption properties of fluorescent proteins. Nat. Methods. 8: 393-399.","pubmedId":"","doi":"10.1038/nmeth.1596"},{"reference":"Laviv T, Kim BB, Chu J, Lam AJ, Lin MZ, Yasuda R. 2016. Simultaneous dual-color fluorescence lifetime imaging with novel. Nat. Methods. 13: 989-992.","pubmedId":"","doi":"10.1038/nmeth.4046"},{"reference":"Shcherbakova DM, Hink MA, Joosen L, Gadella TWJ, Verkhusha VV. 2012. An orange fluorescent protein with a large Stokes shift for. J. Am. Chem. Soc. 134: 7913-7923.","pubmedId":"","doi":"10.1021/ja3018972"},{"reference":"Subach OM, Vlaskina AV, Agapova YK, Piatkevich KD, Patrushev MV, Samygina VR, Subach FV. 2022. LSSmScarlet2 and LSSmScarlet3, chemically stable genetically encoded red. Int. J. Mol. Sci. 23: 11051.","pubmedId":"","doi":"10.3390/ijms231911051"},{"reference":"Zars T, Fischer M, Schulz R, Heisenberg M. 2000. Localization of a short-term memory in Drosophila. Science. 288: 672-675.","pubmedId":"","doi":"10.1126/science.288.5466.672"},{"reference":"Kim BB, Wu H, Hao YA, Pan M, Chavarha M, Zhao Y, et al., Lin MZ. 2022. A red fluorescent protein with improved monomericity enables ratiometric. Sci. Rep. 12: 3678.","pubmedId":"","doi":"10.1038/s41598-022-07313-1"},{"reference":"Santos EM, Sheng W, Esmatpour Salmani R, Tahmasebi Nick S, Ghanbarpour A, Gholami H, et al., Borhan B. 2021. Design of Large Stokes Shift fluorescent proteins based on Excited State. J. Am. Chem. Soc. 143: 15091-15102.","pubmedId":"","doi":"10.1021/jacs.1c05039"},{"reference":"Ozbay BN, Futia GL, Ma M, Bright VM, Gopinath JT, Hughes EG, Restrepo D, Gibson EA. 2018. Three dimensional two-photon brain imaging in freely moving mice using a. Sci. Rep. 8: 8108.","pubmedId":"","doi":"10.1038/s41598-018-26326-3"}],"title":"Long-Stokes-Shift mScarlet3 as a Structural Marker for Two-Photon Imaging","reviews":[{"reviewer":{"displayName":"Elizabeth Hong"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Steven Marygold"},"openAcknowledgement":false,"submitted":null}]},{"id":"4989150b-a013-4e2f-befe-2583281563a5","decision":"publish","abstract":"Two-photon imaging with genetically encoded sensors is widely used to monitor neurophysiology. An additional fluorescent protein can provide anatomical landmarks for cell-type identification and motion detection. However, monomeric red fluorescent proteins often require a dedicated excitation laser. We made transgenic Drosophila with a long-Stokes-shift mScarlet variant (LSSmScarlet3) to image alongside green sensors with a single 920-nm laser. We describe excitation and emission spectra of the expressed protein and show robust fluorescence at 920 nm in vitro and in vivo with minimal channel crosstalk. This approach can reduce equipment complexity and cost while placing functional calcium dynamics in their anatomical context.
","acknowledgements":"We thank Masahiro Fukuda, Hyunsoo Shawn Je (Programme in Neuroscience and Behavioural Disorders, Duke-NUS Medical School) for insightful and productive discussions, Danesha Suresh and Zhiyi Zhang for technical and administrative assistance, and Dénes Pálfi and Viktória Kiss (Femtonics HQ) for assistance with two-photon imaging on FEMTO3D Atlas Plug & Play. We additionally thank Dr. Michael Hausser (University of Hong Kong) and Dr. Jung Kim (University of Hong Kong) for providing temporary access to the FEMTO3D Atlas and lab space. The design of the custom chamber used for imaging was kindly provided by Dr. Anissa Kempf (University of Basel). We thank BDSC and Dr. Hiromu Tanimoto for providing fly stocks.
","authors":[{"affiliations":["Institute of Molecular and Cell Biology (IMCB), A*STAR, Singapore","Duke-NUS Medical School, Singapore"],"departments":["","Programme in Neuroscience and Behavioural Disorders"],"credit":["conceptualization","methodology","investigation","dataCuration","formalAnalysis","visualization","writing_originalDraft","writing_reviewEditing","fundingAcquisition"],"email":"xusangyu@gmail.com","firstName":"Sangyu","lastName":"Xu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4927-9204"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["investigation","writing_reviewEditing","methodology"],"email":"xy_zhang@duke-nus.edu.sg","firstName":"Xianyuan","lastName":"Zhang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0003-4483-750X"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["methodology","investigation","dataCuration","formalAnalysis","visualization","writing_reviewEditing"],"email":"kycheung@duke-nus.edu.sg","firstName":"King Yee","lastName":"Cheung","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-1098-4926"},{"affiliations":["Duke-NUS Medical School, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders"],"credit":["methodology","investigation","dataCuration","validation","writing_reviewEditing"],"email":"maiyishan@u.duke.nus.edu","firstName":"Yishan","lastName":"Mai","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7199-380X"},{"affiliations":["National University of Singapore, Singapore"],"departments":["Microscopy Core Facility — Multiphoton Microscopy Unit, Yong Loo Lin School of Medicine"],"credit":["methodology","investigation","validation","writing_reviewEditing"],"email":"wu.yue@nus.edu.sg","firstName":"Yue","lastName":"Wu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2484-4967"},{"affiliations":["Duke-NUS Medical School, Singapore","Institute of Molecular and Cell Biology (IMCB), A*STAR, Singapore","National University of Singapore, Singapore"],"departments":["Programme in Neuroscience and Behavioural Disorders","","Department of Physiology, Yong Loo Lin School of Medicine"],"credit":["conceptualization","supervision","fundingAcquisition","writing_reviewEditing"],"email":"claridge-chang.adam@duke-nus.edu.sg","firstName":"Adam","lastName":"Claridge-Chang","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4583-3650"}],"awards":[{"awardId":"MOH-OFYIRG20nov-0051","funderName":"National Medical Research Council (Singapore)","awardRecipient":"Sangyu Xu"},{"awardId":"MOH-001397-01","funderName":"National Medical Research Council (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"MOE-T2EP30222-0018","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"MOE-T2EP30223-0009","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"},{"awardId":"FY2022-MOET1-0001","funderName":"Ministry of Education (Singapore)","awardRecipient":"Adam Claridge-Chang"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"S. X. was supported by an MOH-OFYIRG20nov-0051, and X. Z., K. Y. C., Y. M., and A. C.-C. were supported by the MOH-001397-01 grant from the National Medical Research Council, Singapore, by grants MOE-T2EP30222-0018 and MOE-T2EP30223-0009 from the Ministry of Education, Singapore, and by a Duke-NUS Medical School grant. X. Z. and A. C.-C. were supported by FY2022-MOET1-0001.
","image":{"url":"https://portal.micropublication.org/uploads/ce5a9aea0e4551e97f1410bf18dd2876.png"},"imageCaption":"A) An image of the mushroom body expressing LSSmScarlet3 in a fixed MB247-Gal4>UAS-LSSmScarlet3 fly collected with 940 nm excitation and 620 nm collection. White box: control ROI without LSSmScarlet3 expression. Orange box: test ROI with LSSmScarlet3 expression. Scale bar: 20 μm.
B) Excitation spectrum of LSSmScarlet3 in the sample shown in A. Three local maxima were further tested for emission: 860 nm, 940 nm, and 1040 nm.
C) Emission spectra of excitation at 860 nm, 940 nm, and 1040 nm.
D) LSSmScarlet3 expressed in live HEK293T cells under the control of a CMV promoter excited at various 920 nm laser power settings and images collected with a green and a red PMT. At laser power above 25 %, the red PMT was overloaded by the light emitted. Green signals began to emerge at above 50 % laser power.
E) Quantification of mean intensity of each image in D as a function of laser power.
F) Maximum intensity projection image of the mushroom body calyx and α-lobe in a live MB247>GCaMP8m;LSSmScarlet3 Drosophila brain. Both fluorophores were simultaneously excited by the 920 nm laser at 20% power. (F) GCaMP8m signals detected with the green-filtered PMT at 80% gain, (F′) LSSmScarlet3 signals detected with the red-filtered PMT at 70% gain, and (F′′) merged signals. Scale bar = 20 μm.
","imageTitle":"Two-photon imaging of a Drosophila codon-optimized LSSmScarlet3
","methods":"Fly stocks and husbandry
Drosophila cultures were maintained on standard cornmeal-based medium (1.25 % w/v agar, 10.5 % w/v dextrose, 10.5 % w/v maize, and 2.1 % w/v yeast) (Temasek Life Sciences Laboratories 2018). Stocks were kept at room temperature unless otherwise specified. Crosses were performed at 25 ºC in a day-night cycling incubator (Mir-253, Sanyo). Adult male and female flies, aged 5–10 days, were used for experiments. UAS-jGCaMP8m (Zhang et al. 2023) was obtained from Bloomington Drosophila Stock Center (BDSC #92590). The MB247-Gal4 stock (Zars et al. 2000) was provided by Dr Hiromu Tanimoto (Tohoku University, Japan).
Construct generation
LSSmScarlet3 coding sequence was codon-optimized for Drosophila by GenScript (Piscataway, NJ, USA) and cloned into pcDNA3.4 (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. V880-01) to generate the mammalian expression construct pcDNA-DmLSSmScarlet3 and into pJFRC81-10XUAS-IVS-Syn21-GFP-p10 vector (Pfeiffer et al. 2012) between XhoI and XbaI restriction sites to generate Drosophila expression construct UAS-DmLSSmScarlet3, which was then injected into yw flies at the attp40 site balanced against Curly of Oster by BestGene (La Jolla, CA, USA). The pJFRC81-10XUAS-IVS-Syn21-GFP-p10 plasmid was a gift from Gerald Rubin (Addgene plasmid #36432; http://n2t.net/addgene:36432; RRID:Addgene_36432)
Excitation and emission scanning of mushroom body tissue
Fly brains expressing LSSmScarlet3 were dissected in cold PBS and fixed in 4 % PFA for 30 min and then mounted in PBS in 2 stacked reinforcement rings (Suremark) pasted on 1.0–1.2 mm-thick 25 × 75 mm glass slides (Biomedia), and then sealed with 170 µm-thick 18 × 18 mm glass cover slips with clear nail polish. Samples were imaged on a Leica TCS SP8 DIVE multiphoton microscope (Leica Microsystems, Wetzlar, Germany) as previously described (Cheung et al. 2026) with the exception that in the emission spectrum measurements the excitation wavelengths were fixed at 860 nm, 940 nm, or 1040 nm. Excitation and emission scans were performed using the second multiphoton laser source, with the microscope intensity control set to 14.14%. The system reported a laser-source output of 1.78 W at 1,200 nm; power at the objective was not measured. Image acquisition and spectral detection were controlled using Leica LAS X software. Mean intensity of ROIs in images was calculated using Fiji (Schindelin et al. 2012). Excitation and emission spectra graphs were generated using matplotlib in Python.
Cell culture and transfection
HEK293T cells were seeded into small tissue culture dishes to ~ 70 % confluence in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA, USA) and transfected with pcDNA3.4-DmLSSmScarlet3 constructs using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) in Opti-MEM™ Medium (Thermo Fisher Scientific, Waltham, MA, USA) according to a standard protocol (Thermo Fisher Scientific 2016) in 35 mm cell culture dishes (Nunc, catalog #153066; Thermo Fisher Scientific, Waltham, MA, USA). The cells were imaged 48 hours after transfection.
2-photon imaging of LSSmScarlet3 in HEK293T cells
Cell culture expressing LSSmScarlet3 was imaged with A-SCOPE (Thorlabs, NJ, USA) as previously described (Cheung et al. 2026). Laser power was set to 30 mW at the objective. Green PMT was bandpass filtered between 500 nm and 550 nm, and the red PMT was bandpass filtered between 570 nm and 640 nm. Detector gains were set to 5%.
Simultaneous 2-photon imaging of GCaMP8m and LSSmScarlet3 in Drosophila mushroom body
Flies were mounted in a custom 3D-printed chamber using candle wax. Fly heads were opened using a pair of sharp Dumont No. 5 forceps (Fine Science Tools) and microdissection scissors (Fine Science Tools). A drop of PBS was then placed on the open fly head to keep the brain moist and to act as the objective immersion medium.
Z-stack images were acquired on a FEMTO3D Atlas Plug & Play 2-photon microscope (Femtonics, Budapest, Hungary) equipped with a 16× objective lens. Laser power of the 920 nm laser was set to 20% (112 mW at the objective). GCaMP8m emission light was collected by a green-filtered PMT at 80% gain, while LSSmScarlet3 emission light was collected by a red-filtered PMT at 70% gain.
Image processing
The Drosophila mushroom body 2-photon image stack was subjected to Gaussian blur 3D processing (X: 0, Y: 0, Z: 2) and maximum intensity projection processing in Fiji (Schindelin et al. 2012). For the purpose of visualizing dual-channel images, signal collected by the green-filtered PMT was pseudo-colored to green, while signal collected by the red-filtered PMT was pseudo-colored to magenta.
Data analysis and visualization
Preprocessed data from the Fiji workflow were analyzed and visualized using custom Jupyter Notebooks running Python (version 3.10.16) with pandas (version 2.1.4), NumPy (version 1.26.4), and seaborn (version 0.12.2).
Data and Code Availability
Data and code used to generate the figure in this paper, as well as construct sequences and maps, are available as a Zenodo repository (DOI: 10.5281/zenodo.19493335).
","reagents":"","patternDescription":"Understanding neural circuits requires monitoring both neural activity and anatomical relationships. Genetically encoded calcium indicators based on blue-excitable green fluorescent protein (GFP) are widely used, for example, the GFP–Calmodulin–M13-peptide (GCaMP) series of calcium sensors. Such sensors enable cellular-resolution functional imaging, but calcium signals are transient and can afford limited structural context. Alongside sensors, researchers sometimes co-express red fluorescent proteins (RFPs) as structural markers (e.g., tdTomato or mCherry) to provide landmarks for cellular identification and sample-motion detection. Single-laser excitation of red fluorescent proteins away from their excitation maxima (~920 nm rather than at their ~1040 nm peak) has been used for dual-color structural-functional imaging (Tillo et al. 2010; Ozbay et al. 2018), but relies on the tandem dimer tdTomato, whose two-photon cross-section at this wavelength is an order of magnitude higher than that of monomeric RFPs such as mCherry (~35–40 GM vs. ~5 GM) (Drobizhev et al. 2011). The resulting doubling of molecular weight complicates protein-fusion functionality (Shaner et al. 2005). Alternatively, a dedicated 1040 nm laser can be added to excite monomeric RFPs at their peak (Gee et al. 2014; Bugeon et al. 2022), resulting in multi-laser setups that increase equipment cost, system complexity, and phototoxicity. A monomeric red fluorophore with robust excitation at ~920 nm could therefore retain the simplicity of single-wavelength imaging while avoiding a tandem dimer, improving signal levels at low expression, and broadening applications to include fusion proteins and subcellular markers.
Long-Stokes-shift (LSS) fluorescent dyes exhibit large separations between excitation and emission peaks (Shcherbakova et al. 2012). Meanwhile, there has been considerable effort in developing LSS fluorescent proteins through diverse approaches, including excited-state proton transfer, chromophore modifications, and protein engineering (Piatkevich et al. 2010; Santos et al. 2021). Originally developed to improve FLIM-FRET (fluorescence lifetime imaging microscopy-Förster resonance energy transfer) sensors by reducing spectral overlap between donor and acceptor fluorophores, LSS variants have found broader applications in multicolor imaging (Shcherbakova et al. 2012; Piatkevich et al. 2010). A recently developed LSS variant of mScarlet, LSSmScarlet3, exhibits a 128 nm Stokes shift (ex 466 → em 598 nm) and, compared to previous versions, has improved pH stability and photostability (Subach et al. 2022). Similarly, monomeric cyan-excitable RFP 3, mCyRFP3 (Kim et al. 2022), has been reported as a functional fusion tag in cell-based imaging and has recently been incorporated into Drosophila transgenic reagents (FlyBase 2025). Here, as a complementary contribution, we describe a genetically encoded LSSmScarlet3 as a structural marker for single-laser two-color 2P imaging, demonstrating efficient excitation at 920 nm. Alongside GFP-based sensors, LSSmScarlet3 provides a simplified approach for dual-channel functional–structural imaging.
To verify whether a long-Stokes-shift RFP can be imaged with a 920 nm laser in Drosophila brains, we synthesized a Drosophila codon-optimized version of LSSmScarlet3. In fixed Drosophila tissue, LSSmScarlet3 was well expressed and readily distributed to the lobes of the mushroom body, indicating good neuronal expression and distribution (Figure 1A). On a spectrally tunable multiphoton microscope, LSSmScarlet3 had a peak excitation at 940 nm, with two smaller local maxima at 860 nm and 1040 nm (Figure 1B). At a frequently used 2P excitation wavelength of 920 nm, the mean ROI intensity measured was 99 arbitrary units (arb. u.), which was around 75% of the 940-nm peak intensity (132 arb. u.). Emission curves for the three excitation wavelengths tested (Figure 1C) show a peak emission wavelength around 620 nm.
Having established the spectral properties of the fluorophore, we wanted to measure its performance on a standard 2P microscope equipped with one 920 nm laser and two photomultiplier tubes (PMTs) for detection: one green, one red (Figure 1D). When expressed in human embryonic kidney 293T (HEK293T) cells under the control of a CMV promoter, and with 920 nm excitation, LSSmScarlet3 was detected with the red PMT. As we varied the laser power input, we observed robust detection of LSSmScarlet3 by the red PMT at low gain, whereas the green PMT did not detect any signal below 50 % laser power. Above 25 % laser power, the red PMT was overloaded. Only at around 60 % laser power did we observe substantial cross-talk signal on the green PMT (Figure 1D, E). This separation between RFP and GFP-channel detection indicates that, with appropriate filtering, LSSmScarlet3 is suitable for marking cells without interference with the green channel.
Co-expression of LSSmScarlet3 and GCaMP8m in the mushroom body under UAS control, driven by MB247-Gal4, showed robust co-detection (Figure 1F) in vivo.
We demonstrate the utility of LSSmScarlet3 as a structural marker alongside GCaMP8m using a single laser for excitation. This arrangement promises substantial advantages over existing approaches, including simplicity in setup, elimination of sequential scanning for potential reduction of phototoxicity, and better temporal registration. Strong excitation at 920 nm is ideal for most existing 2P setups, and we validated that the long Stokes shift minimizes crosstalk between green and red channels. Compared to other long Stokes shift FPs currently available, our transgenic fly is validated and ready to use.
LSSmScarlet3 is quite bright in our hands. Given the low threshold for PMT saturation from the highly expressed LSSmScarlet3 in HEK293T cells (Figure 1D), careful titration of expression levels may be needed. On the other hand, this also reassures users of highly detectable signals when expression levels are low, for example, in a fusion protein. This opens up many possibilities, such as co-imaging neuronal activity with synaptic markers, tagging proteins with subcellular localization signals, or monitoring reference transcription levels.
Transgenic LSSmScarlet3 provides a practical solution for dual-channel 2P imaging in Drosophila. Single-laser, two-color imaging opens up avenues for developing LSSmScarlet3 variants for specific biological markers. For applications with strong promoters where structural bulk is not a concern, using tdTomato excited with ~920 nm remains a viable option. LSSmScarlet3 offers particular advantages when a monomeric tag is needed, such as with fusion proteins, weak promoters, or subcellular localization signals where expression levels may be limiting.
","references":[{"reference":"Bugeon S, Duffield J, Dipoppa M, Ritoux A, Prankerd I, Nicoloutsopoulos D, et al., Harris KD. 2022. A transcriptomic axis predicts state modulation of cortical interneurons. Nature. 607: 330-338.","pubmedId":"","doi":"10.1038/s41586-022-04915-7"},{"reference":"Cheung KY, Wu Y, Lee SY, Zhang X, Fukuda M, Suresh DD, Claridge-Chang A. 2026. Two-photon characterisation of long-Stokes-shift dye ATTO 490LS for single-laser multicolour imaging. MicroPubl. Biol. 2026
","pubmedId":"","doi":"10.17912/micropub.biology.001955"},{"reference":"Drobizhev M, Makarov NS, Tillo SE, Hughes TE, Rebane A. 2011. Two-photon absorption properties of fluorescent proteins. Nat. Methods. 8: 393-399.","pubmedId":"","doi":"10.1038/nmeth.1596"},{"reference":"FlyBase. 2025. FlyBase Reference Report: Wilson and Holtz, 2025.2.27, Physiology.
","pubmedId":"","doi":""},{"reference":"Gee JM, Smith NA, Fernandez FR, Economo MN, Brunert D, Rothermel M, et al., Tvrdik P. 2014. Imaging activity in neurons and glia with a Polr2a-based and Cre-dependent GCaMP5G-IRES-tdTomato reporter mouse. Neuron. 83: 1058-1072.
","pubmedId":"","doi":"10.1016/j.neuron.2014.07.024"},{"reference":"Kim BB, Wu H, Hao YA, Pan M, Chavarha M, Zhao Y, et al., Lin MZ. 2022. A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3. Sci. Rep. 12: 3678.
","pubmedId":"","doi":"10.1038/s41598-022-07313-1"},{"reference":"Laviv T, Kim BB, Chu J, Lam AJ, Lin MZ, Yasuda R. 2016. Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins. Nat. Methods. 13: 989-992.
","pubmedId":"","doi":"10.1038/nmeth.4046"},{"reference":"Ozbay BN, Futia GL, Ma M, Bright VM, Gopinath JT, Hughes EG, Restrepo D, Gibson EA. 2018. Three dimensional two-photon brain imaging in freely moving mice using a miniature fiber coupled microscope with active axial-scanning. Sci. Rep. 8: 8108.
","pubmedId":"","doi":"10.1038/s41598-018-26326-3"},{"reference":"Pfeiffer BD, Truman JW, Rubin GM. 2012. Using translational enhancers to increase transgene expression in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 109: 6626-6631.
","pubmedId":"","doi":"10.1073/pnas.1204520109"},{"reference":"Piatkevich KD, Hulit J, Subach OM, Wu B, Abdulla A, Segall JE, Verkhusha VV. 2010. Monomeric red fluorescent proteins with a large Stokes shift. Proc. Natl. Acad. Sci. U. S. A. 107: 5369-5374.","pubmedId":"","doi":"10.1073/pnas.0914365107"},{"reference":"Santos EM, Sheng W, Esmatpour Salmani R, Tahmasebi Nick S, Ghanbarpour A, Gholami H, et al., Borhan B. 2021. Design of large Stokes shift fluorescent proteins based on excited state proton transfer of an engineered photobase. J. Am. Chem. Soc. 143: 15091-15102.
","pubmedId":"","doi":"10.1021/jacs.1c05039"},{"reference":"Schindelin J, Arganda Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al., Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 9: 676-682.","pubmedId":"","doi":"10.1038/nmeth.2019"},{"reference":"Shaner NC, Steinbach PA, Tsien RY. 2005. A guide to choosing fluorescent proteins. Nat. Methods. 2: 905-909.","pubmedId":"","doi":"10.1038/nmeth819"},{"reference":"Shcherbakova DM, Hink MA, Joosen L, Gadella TWJ, Verkhusha VV. 2012. An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. Am. Chem. Soc. 134: 7913-7923.
","pubmedId":"","doi":"10.1021/ja3018972"},{"reference":"Subach OM, Vlaskina AV, Agapova YK, Piatkevich KD, Patrushev MV, Samygina VR, Subach FV. 2022. LSSmScarlet2 and LSSmScarlet3, chemically stable genetically encoded red fluorescent proteins with a large Stokes’ shift. Int. J. Mol. Sci. 23: 11051.
","pubmedId":"","doi":"10.3390/ijms231911051"},{"reference":"Temasek Life Sciences Laboratories. 2018. Recipe for Drosophila media.
","pubmedId":"","doi":"10.5281/zenodo.1185451"},{"reference":"Thermo Fisher Scientific. 2016. Lipofectamine 3000 Reagent Protocol.","pubmedId":"","doi":""},{"reference":"Tillo SE, Hughes TE, Makarov NS, Rebane A, Drobizhev M. 2010. A new approach to dual-color two-photon microscopy with fluorescent proteins. BMC Biotechnol. 10: 6.
","pubmedId":"","doi":"10.1186/1472-6750-10-6"},{"reference":"Zars T, Fischer M, Schulz R, Heisenberg M. 2000. Localization of a short-term memory in Drosophila. Science. 288: 672-675.","pubmedId":"","doi":"10.1126/science.288.5466.672"},{"reference":"Zhang Y, Rozsa M, Liang Y, Bushey D, Wei Z, Zheng J, et al., Looger LL. 2023. Fast and sensitive GCaMP calcium indicators for imaging neural populations. Nature. 615: 884-891.","pubmedId":"","doi":"10.1038/s41586-023-05828-9"}],"title":"Long-Stokes-Shift mScarlet3 as a Structural Marker for Two-Photon Imaging","reviews":[],"curatorReviews":[{"curator":{"displayName":"Steven Marygold"},"openAcknowledgement":false,"submitted":null}]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon chilense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aedes japonicus","label":"Aedes japonicus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aegorhinus vitulus","label":"Aegorhinus vitulus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alaimidae","label":"Alaimidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"allobates femoralis","label":"Allobates femoralis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alnus glutinosa","label":"Alnus glutinosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alosa aestivalis","label":"Alosa aestivalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alosa pseudoharengus","label":"Alosa pseudoharengus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alternaria alternata","label":"Alternaria alternata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"amynthas agrestis","label":"Amynthas Agrestis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ancylostoma caninum","label":"Ancylostoma caninum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ancylostoma ceylanicum","label":"Ancylostoma ceylanicum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anemone multifida","label":"Anemone multifida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anguilla rostrata","label":"Anguilla rostrata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anisakis simplex","label":"Anisakis simplex","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anomala albopilosa","label":"Anomala albopilosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anthomyiidae sp","label":"Anthomyiidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anthomyiidae sp","label":"Anthomyiidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arabidopsis","label":"Arabidopsis","imageSrc":"arabidopsis.png","imageAlt":"Arabidopsis graphic by Zoe Zorn CC BY 4.0","mod":"TAIR","modLink":"https://arabidopsis.org","linkVariable":""},{"value":"architeuthis dux","label":"Architeuthis dux","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arion vulgaris","label":"Arion vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"armeria","label":"Armeria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"artemia","label":"Artemia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arthrobacter sp.","label":"Arthrobacter sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ascaridia","label":"Ascaridia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ascaridia galli","label":"Ascaridia galli","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"asparagopsis taxiformis","label":"Asparagopsis taxiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"astatotilapia burtoni","label":"Astatotilapia burtoni","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"avena sativa","label":"Avena sativa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aves","label":"Aves","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus","label":"Bacillus (firmicutes)","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus cereus","label":"Bacillus cereus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus mycoides","label":"Bacillus mycoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus subtilis","label":"Bacillus subtilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus thuringiensis","label":"Bacillus thuringiensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus toyonensis","label":"Bacillus toyonensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus wiedmannii","label":"Bacillus wiedmannii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacteria","label":"Bacteria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacteriophage","label":"Bacteriophage","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bactrocera","label":"Bactrocera sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"batrachospermum gelatinosum","label":"Batrachospermum gelatinosum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"betula lenta","label":"Betula lenta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"betula nigra","label":"Betula nigra","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombus dahlbohmii","label":"Bombus dahlbohmii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombus terrestris","label":"Bombus terrestris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombyx mori","label":"Bombyx mori","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bos taurus","label":"Bos Taurus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brachygobius doriae","label":"Brachygobius doriae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brassica oleracea","label":"Brassica oleracea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brassica rapa","label":"Brassica rapa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brugia malayi","label":"Brugia malayi","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"burkholderia thailandensis","label":"Burkholderia thailandensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"buttiauxella","label":"Buttiauxella","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caenorhabditis brenneri","label":"Caenorhabditis brenneri","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis briggsae","label":"Caenorhabditis briggsae","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"c. elegans","label":"Caenorhabditis elegans","imageSrc":"c-elegans.jpg","imageAlt":"C. elegans graphic by Zoe Zorn CC BY 4.0","mod":"WormBase","modLink":"https://wormbase.org","linkVariable":""},{"value":"caenorhabditis inopinata","label":"Caenorhabditis inopinata","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis japonica","label":"Caenorhabditis japonica","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis nigoni","label":"Caenorhabditis nigoni","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caenorhabditis remanei","label":"Caenorhabditis remanei","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis tropicalis","label":"Caenorhabditis tropicalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calidifontibacillus","label":"Calidifontibacillus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calidifontibacillus erzuremensis","label":"Calidifontibacillus erzuremensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calliphora sp","label":"Calliphora sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caltha sagittata","label":"Caltha sagittata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cambarus latimanus","label":"Cambarus latimanus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"candida albicans","label":"Candida albicans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"canis familiaris","label":"Canis familiaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cannabis sativa","label":"Cannabis sativa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caretta caretta","label":"Caretta caretta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cassiopea xamachana","label":"Cassiopea xamachana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caulobacter vibrioides","label":"Caulobacter vibrioides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cephalopods","label":"Cephalopoda","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cerastium arvense","label":"Cerastium arvense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ceriodaphnia","label":"Ceriodaphnia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ceroglossus suturalis","label":"Ceroglossus suturalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chaetoceros","label":"Chaetoceros","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chamaecrista fasciculata","label":"Chamaecrista fasciculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chilicola chalcidiformis","label":"Chilicola chalcidiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chitinimonas","label":"Chitinimonas","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chlamydomonas reinhardtii","label":"Chlamydomonas reinhardtii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chromobacterium","label":"Chromobacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chrysemys picta","label":"Chrysemys picta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chrysoperla rufilabris","label":"Chrysoperla rufilabris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"citrus","label":"Citrus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"clavibacter sp.","label":"Clavibacter sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"colinus virginianus","label":"Colinus virginianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"crassostrea virginica","label":"Crassostrea virginica","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"crithidia fasciculata","label":"Crithidia fasciculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cutibacterium acnes","label":"Cutibacterium acnes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cyanobacteria","label":"Cyanobacteria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"daphnia","label":"Daphnia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"daphnia pulex","label":"Daphnia pulex","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"diabrotica virgifera","label":"Diabrotica virgifera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"diabrotica virgifera virgifera virus 1","label":"Diabrotica virgifera virgifera virus 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"d. discoideum","label":"Dictyostelium discoideum","imageSrc":"dicty.png","imageAlt":"D. discoideum","mod":"dictyBase","modLink":"http://dictybase.org","linkVariable":""},{"value":"diptera","label":"Diptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dotocryptus bellicosus","label":"Dotocryptus bellicosus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"drechmeria coniospora","label":"Drechmeria coniospora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"drosophila","label":"Drosophila","imageSrc":"drosophila.png","imageAlt":"Drosophila graphic by Zoe Zorn CC BY 4.0","mod":"FlyBase","modLink":"https://flybase.org/doi/","linkVariable":"doi"},{"value":"dryopteris campyloptera","label":"Dryopteris campyloptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dryopteris expansa","label":"Dryopteris expansa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dryopteris intermedia","label":"Dryopteris intermedia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dugesia dorotocephala","label":"Dugesia dorotocephala","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"elasmobranchii","label":"Elasmobranchii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"embryophyta","label":"Embryophyta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enoploteuthis chunii","label":"Enoploteuthis chunii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enterobacter aerogenes","label":"Enterobacter aerogenes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enterococcus raffinosus","label":"Enterococcus raffinosus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"epichloë coenophiala","label":"Epichloë coenophiala","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"equus caballus","label":"Equus caballus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erigeron sp","label":"Erigeron sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eristalis","label":"Eristalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eruca vesicaria","label":"Eruca vesicaria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erwinia carotovora","label":"Erwinia carotovora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erythronium americanum","label":"Erythronium americanum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"escherichia coli","label":"Escherichia coli","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eukaryota","label":"Eukaryotes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"felis catus","label":"Felis catus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"francisella novicida","label":"Francisella novicida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"francisella tularensis","label":"Francisella tularensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fraxinus americana","label":"Fraxinus americana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fucus distichus","label":"Fucus distichus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fungi","label":"Fungi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gasteropelecus sp.","label":"Gasteropelecus sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"geranium sp","label":"Geranium sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"girardia","label":"Girardia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"glaucomys volans","label":"Glaucomys volans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"glycine max","label":"Glycine max","imageSrc":"","imageAlt":"","mod":"Soybase","modLink":"https://soybase.org","linkVariable":""},{"value":"glyptemys insculpta","label":"Glyptemys insculpta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gossypium hirsutum","label":"Gossypium hirsutum","imageSrc":"","imageAlt":"","mod":"CottonGen","modLink":"https://www.cottongen.org/","linkVariable":""},{"value":"gromphadorhina portentosa","label":"Gromphadorhina portentosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gryllodes sigillatus","label":"Gryllodes sigillatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"haliotis rufescens","label":"Haliotis rufescens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hepacivirus hominis","label":"Hepatitis C Virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"herpes simplex virus type 1","label":"Herpes simplex virus type 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"human","label":"Human","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"human coronavirus oc43","label":"Human coronavirus OC43","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hydra vulgaris","label":"Hydra vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hydropsyche sp","label":"Hydropsyche sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hymenoptera","label":"Hymenoptera","imageSrc":"","imageAlt":"","mod":"Hymenoptera Genome Database","modLink":"https://hymenoptera.elsiklab.missouri.edu/","linkVariable":""},{"value":"hypochaeris radicata","label":"Hypochaeris radicata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hypodynerus vespiformis","label":"Hypodynerus vespiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"iflaviridae","label":"Iflaviridae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"iflavuris","label":"Iflavirus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ipomoea hederacea","label":"Ipomoea hederacea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ischnomera","label":"Ischnomera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ischnomera ruficollis","label":"Ischnomera ruficollis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"julidochromis marlieri","label":"Julidochromis marlieri","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"juniperus virginiana","label":"Juniperus virginiana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"kluyveromyces marxianus","label":"Kluyveromyces marxianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"l. casei","label":"L. casei","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lacticaseibacillus casei","label":"Lacticaseibacillus casei","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"larentiinae sp","label":"Larentiinae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"laurus nobilis","label":"Laurus nobilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lepidoptera","label":"Lepidoptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"leucanthemum vulgare","label":"Leucanthemum vulgare","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"linepithema humile","label":"Linepithema humile","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"liometopum occidentale","label":"Liometopum occidentale","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lolium arundinaceum","label":"Lolium arundinaceum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lumbriculus variegatus","label":"Lumbriculus variegatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lumbricus terrestris","label":"Lumbricus terrestris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lupinus polyphyllus","label":"Lupinus polyphyllus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lycorma delicatula","label":"Lycorma delicatula","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lynx rufus","label":"Lynx rufus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"magnaporthe oryzae","label":"Magnaporthe oryzae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mammalia","label":"Mammalia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"manihot esculenta","label":"Manihot esculenta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"medicago lupulina","label":"Medicago lupulina","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"meloidogyne","label":"Meloidogyne","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mimus polyglottos","label":"Mimus polyglottos","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bryophyta","label":"Mosses","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mouse","label":"Mouse","imageSrc":"","imageAlt":"","mod":"MGI","modLink":"https://informatics.jax.org","linkVariable":""},{"value":"m. minutoides","label":"Mus minutoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mycobacterium smegmatis","label":"Mycobacterium smegmatis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nakaseomyces glabratus","label":"Nakaseomyces glabratus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nauphoeta cinerea","label":"Nauphoeta cinerea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"neurospora","label":"Neurospora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"n. benthamiana","label":"Nicotiana benthamiana","imageSrc":"","imageAlt":"","mod":"Solgenomics Network","modLink":"https://solgenomics.net/organism/Nicotiana_benthamiana/genome","linkVariable":""},{"value":"nicotiana tabacum","label":"Nicotiana tabacum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"noctuidae","label":"Noctuidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"noctuidae sp","label":"Noctuidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nothobranchius furzeri","label":"Nothobranchius furzeri","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"onchocerca volvulus","label":"Onchocerca volvulus","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"orconectes virilis","label":"Orconectes virilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ormia ochracea","label":"Ormia ochracea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"o. sativa","label":"Oryza sativa","imageSrc":"","imageAlt":"","mod":"Gramene","modLink":"https://www.gramene.org/","linkVariable":""},{"value":"other","label":"Other","imageSrc":"","imageAlt":"","mod":null,"modLink":null,"linkVariable":null},{"value":"oxalis enneaphylla","label":"Oxalis enneaphylla","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paenarthrobacter nicotinovorans","label":"Paenarthrobacter nicotinovorans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paenarthrobacter nicotinovorans","label":"Paenarthrobacter nicotinovorans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pantoea","label":"Pantoea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pantoea agglomerans","label":"Pantoea agglomerans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"papaver sp","label":"Papaver sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paramecium bursaria","label":"Paramecium bursaria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"partitiviridae","label":"Partitiviridae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pelodiscus sinensis","label":"Pelodiscus sinensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"perezia recurvata","label":"Perezia recurvata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"petromyzon marinus","label":"Petromyzon marinus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis","label":"Photinus pyralis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis associated partiti-like virus","label":"Photinus pyralis associated partiti-like virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis iflavirus 1","label":"Photinus pyralis iflavirus 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"physcomitrium patens","label":"Physcomitrium patens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pinus strobus","label":"Pinus strobus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pinus taeda","label":"Pinus taeda","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"platycheirus","label":"Platycheirus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"plectus sambesii","label":"Plectus sambesii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pogonomyrmex occidentalis","label":"Pogonomyrmex occidentalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"poncirus trifoliata","label":"Poncirus trifoliata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"populus deltoides","label":"Populus deltoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"potato virus y","label":"Potato virus Y","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"primula magellanica","label":"Primula magellanica","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pristionchus pacificus","label":"Pristionchus pacificus","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"prunus persica","label":"Prunus persica","imageSrc":"","imageAlt":"","mod":"Genome Database for Rosaceae","modLink":"https://www.rosaceae.org/","linkVariable":""},{"value":"psalmopoeus iriminia","label":"Psalmopoeus iriminia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudanabaena sp.","label":"Pseudanabaena sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas","label":"Pseudomonas","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas aeruginosa","label":"Pseudomonas aeruginosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas glycinae","label":"Pseudomonas glycinae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas putida","label":"Pseudomonas putida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas syringae","label":"Pseudomonas syringae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pterophyllum scalare","label":"Pterophyllum scalare","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"python regius","label":"Python regius","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"quercus macrocarpa","label":"Quercus macrocarpa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ralstonia solanacearum","label":"Ralstonia solanacearum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ranitomeya imitator","label":"Ranitomeya imitator","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ranunculus peduncularis","label":"Ranunculus peduncularis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"rat","label":"Rat","imageSrc":"","imageAlt":"","mod":"RGD","modLink":"https://rgd.mcw.edu","linkVariable":""},{"value":"rheinheimera","label":"Rheinheimera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ribes rubrum","label":"Ribes rubrum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"sars-cov-2","label":"SARS-CoV-2","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. cerevisiae","label":"Saccharomyces cerevisiae","imageSrc":"yeast.png","imageAlt":"Yeast graphic by Zoe Zorn CC BY 4.0","mod":"SGD","modLink":"https://yeastgenome.org","linkVariable":""},{"value":"saccharomyces paradoxus","label":"Saccharomyces paradoxus ","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. uvarum","label":"Saccharomyces uvarum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"schistosoma","label":"Schistosoma","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"schizosaccharomyces japonicus","label":"Schizosaccharomyces japonicus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. pombe","label":"Schizosaccharomyces pombe","imageSrc":"pombe.png","imageAlt":"Pombe graphic by Zoe Zorn © Caltech","mod":"PomBase","modLink":"https://www.pombase.org/reference/PMID:","linkVariable":"pmId"},{"value":"schmidtea mediterranea","label":"Schmidtea mediterranea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"senecio sp","label":"Senecio sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"simocephalus","label":"Simocephalus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"siraitia grosvenorii","label":"Siraitia grosvenorii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"solanum lycopersicum","label":"Solanum lycopersicum","imageSrc":"","imageAlt":"","mod":"Solgenomics Network","modLink":"https://solgenomics.net/organism/1/view/","linkVariable":""},{"value":"sorghum","label":"Sorghum","imageSrc":"","imageAlt":"","mod":"SorghumBase","modLink":"https://www.sorghumbase.org","linkVariable":""},{"value":"spiroplasma eriocheiris","label":"Spiroplasma eriocheiris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"staphylococcus aureus","label":"Staphylococcus aureus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"staphylococcus epidermidis","label":"Staphylococcus epidermidis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"steinernema carpocapsae","label":"Steinernema carpocapsae","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"https://wormbase.org","linkVariable":""},{"value":"steinernema hermaphroditum","label":"Steinernema hermaphroditum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"stenotrophomonas geniculata","label":"Stenotrophomonas geniculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"streptococcus gordonii ","label":"Streptococcus gordonii ","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"streptococcus mutans","label":"Streptococcus mutans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":" streptococcus pneumoniae","label":"Streptococcus pneumoniae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. purpuratus","label":"Strongylocentrotus purpuratus","imageSrc":"","imageAlt":"","mod":"Echinobase","modLink":"https://www.echinobase.org","linkVariable":""},{"value":"strongyloides ratti","label":"Strongyloides ratti","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"sulfolobus","label":"Sulfolobus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"symphoricarpos albus","label":"Symphoricarpos albus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"syncirsodes","label":"Syncirsodes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"synechococcus elongatus","label":"Synechococcus elongatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"syrphidae","label":"Syrphidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tarantobelus jeffdanielsi","label":"Tarantobelus jeffdanielsi","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"taraxacum officinale","label":"Taraxacum officinale","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tatochila theodice","label":"Tatochila theodice","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tetrahymena","label":"Tetrahymena","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tetramorium immigrans","label":"Tetramorium immigrans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tomato brown rugose fruit virus","label":"ToBRFV","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trachemys scripta","label":"Trachemys scripta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tribolium castaneum","label":"Tribolium castaneum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trichoptera","label":"Trichoptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trichuris muris","label":"Trichuris muris","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"trifolium repens","label":"Trifolium repens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trypoxylus dichotomus","label":"Trypoxylus dichotomus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tsuga canadensis","label":"Tsuga canadensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ulva expansa","label":"Ulva expansa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"universal","label":"Universal","imageSrc":"","imageAlt":"","mod":null,"modLink":null,"linkVariable":null},{"value":"vargula hilgendorfii","label":"Vargula hilgendorfii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"vespula vulgaris","label":"Vespula vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"virus","label":"Virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"watasenia scintillans","label":"Watasenia scintillans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"wolbachia pipientis","label":"Wolbachia pipientis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"xenopus","label":"Xenopus","imageSrc":"xenopus.png","imageAlt":"Xenopus graphic by Zoe Zorn CC BY 4.0","mod":"XenBase","modLink":"https://xenbase.org","linkVariable":""},{"value":"xenorhabdus griffiniae","label":"Xenorhabdus griffiniae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"yramea cytheris","label":"Yramea cytheris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"zaprionus indianus","label":"Zaprionus indianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"zea mays","label":"Zea mays","imageSrc":"","imageAlt":"","mod":"MaizeGDB","modLink":"https://www.maizegdb.org","linkVariable":""},{"value":"zebrafish","label":"Zebrafish","imageSrc":"zebrafish.png","imageAlt":"Zebrafish graphic by Zoe Zorn CC BY 4.0","mod":"ZFIN","modLink":"https://zfin.org","linkVariable":""}]}},"pageContext":{"id":"9df151ac-4349-47ad-832a-97a46d0457fc","citedBy":[],"parsedCsv":{"csvHeader":[],"csvData":[]}}}, "staticQueryHashes": ["2114697108"]}