{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T23:58:10Z","timestamp":1768521490674,"version":"3.49.0"},"posted":{"date-parts":[[2025,3,30]]},"group-title":"Bioengineering","reference-count":80,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,3,30]],"date-time":"2025-03-30T00:00:00Z","timestamp":1743292800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,3,30]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Intrinsically photosensitive retinal ganglion cells are photoreceptors discovered in the last 20 years. These cells project to the suprachiasmatic nucleus of the brain to drive circadian rhythms, regulated by ambient light levels. The photopigment responsible for photoactivation in these cells, melanopsin, has been shown to exhibit many unique activation features among opsins. Notably, the photopigment can exist in three states dependent on the intensity and spectrum of ambient light, which affects its function. Despite increasing knowledge about these cells and melanopsin, tools that can manipulate their three states, and do so with single-cell precision, are limited. This reduces the extent to which circuit-level phenomena, and studying the implications of melanopsin tri-stability in living systems, can be pursued. In this report, we evoke and modulate calcium transients in live cells and intrinsically photosensitive retinal ganglion cells from isolated retinal tissues following two-photon excitation using near-infrared light pulses. We demonstrate that two-photon activation of melanopsin can successfully stimulate melanopsin-expressing cells with high spatio-temporal precision. Moreover, we demonstrate that the functional tri-stability of the photopigment can be interrogated by multiphoton excitation using spectral-temporal modulation of a broadband, ultrafast laser source.<\/jats:p>","DOI":"10.1101\/2025.03.26.645437","type":"posted-content","created":{"date-parts":[[2025,3,31]],"date-time":"2025-03-31T01:31:05Z","timestamp":1743384665000},"source":"Crossref","is-referenced-by-count":0,"title":["Two-photon activation, deactivation, and coherent control of melanopsin in live cells"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3596-5799","authenticated-orcid":false,"given":"Carlos A.","family":"Renteria","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiho","family":"Kahng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brian","family":"Tibble","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9126-9491","authenticated-orcid":false,"given":"Rishyashring R.","family":"Iyer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8906-1082","authenticated-orcid":false,"given":"Jindou","family":"Shi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haya","family":"Algrain","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eric J.","family":"Chaney","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5089-0596","authenticated-orcid":false,"given":"Edita","family":"Aksamitiene","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuan-Zhi","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Phyllis","family":"Robinson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4791-6775","authenticated-orcid":false,"given":"Tiffany","family":"Schmidt","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9386-5630","authenticated-orcid":false,"given":"Stephen A.","family":"Boppart","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025040203500666000_2025.03.26.645437v1.1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1069609"},{"key":"2025040203500666000_2025.03.26.645437v1.2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1067262"},{"key":"2025040203500666000_2025.03.26.645437v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.tins.2011.07.001"},{"key":"2025040203500666000_2025.03.26.645437v1.4","doi-asserted-by":"publisher","DOI":"10.1007\/s00018-020-03641-5"},{"key":"2025040203500666000_2025.03.26.645437v1.5","doi-asserted-by":"publisher","DOI":"10.1126\/science.1076848"},{"key":"2025040203500666000_2025.03.26.645437v1.6","doi-asserted-by":"publisher","DOI":"10.1126\/science.1076701"},{"key":"2025040203500666000_2025.03.26.645437v1.7","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.44358"},{"key":"2025040203500666000_2025.03.26.645437v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/nature01761"},{"key":"2025040203500666000_2025.03.26.645437v1.9","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-neuro-072116-031324"},{"key":"2025040203500666000_2025.03.26.645437v1.10","doi-asserted-by":"crossref","first-page":"e80749","DOI":"10.7554\/eLife.80749","article-title":"Melanopsin activates divergent phototransduction pathways in intrinsically photosensitive retinal ganglion cell subtypes","volume":"12","year":"2023","journal-title":"eLife"},{"key":"2025040203500666000_2025.03.26.645437v1.11","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.20-02-00600.2000"},{"key":"2025040203500666000_2025.03.26.645437v1.12","unstructured":"Suprachiasmatic nucleus: The mind\u2019s clock. (Oxford University Press, New York, 1991)."},{"key":"2025040203500666000_2025.03.26.645437v1.13","doi-asserted-by":"publisher","DOI":"10.1038\/s41583-018-0026-z"},{"key":"2025040203500666000_2025.03.26.645437v1.14","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.09-03-01073.1989"},{"key":"2025040203500666000_2025.03.26.645437v1.15","doi-asserted-by":"publisher","DOI":"10.1038\/nature07682"},{"key":"2025040203500666000_2025.03.26.645437v1.16","doi-asserted-by":"crossref","first-page":"3747","DOI":"10.1113\/JP279524","article-title":"A kinetic analysis of mouse rod and cone photoreceptor responses","volume":"598","year":"2020","journal-title":"J. Physiol"},{"key":"2025040203500666000_2025.03.26.645437v1.17","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0228121"},{"key":"2025040203500666000_2025.03.26.645437v1.18","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1167\/iovs.61.12.10","article-title":"Protein phosphatase 2A and clathrin-mediated endocytosis facilitate robust melanopsin light responses and resensitization","volume":"61","year":"2020","journal-title":"Investig. Opthalmology Vis. Sci"},{"key":"2025040203500666000_2025.03.26.645437v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2015.02.011"},{"key":"2025040203500666000_2025.03.26.645437v1.20","doi-asserted-by":"publisher","DOI":"10.1177\/0748730407306043"},{"key":"2025040203500666000_2025.03.26.645437v1.21","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2016.03.007"},{"key":"2025040203500666000_2025.03.26.645437v1.22","doi-asserted-by":"publisher","DOI":"10.1038\/nature10206"},{"key":"2025040203500666000_2025.03.26.645437v1.23","doi-asserted-by":"publisher","DOI":"10.1016\/S0896-6273(00)00093-3"},{"key":"2025040203500666000_2025.03.26.645437v1.24","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M111.325969"},{"key":"2025040203500666000_2025.03.26.645437v1.25","doi-asserted-by":"publisher","DOI":"10.1021\/bi3004999"},{"key":"2025040203500666000_2025.03.26.645437v1.26","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1523629113"},{"key":"2025040203500666000_2025.03.26.645437v1.27","doi-asserted-by":"publisher","DOI":"10.1152\/jn.00062.2008"},{"key":"2025040203500666000_2025.03.26.645437v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/nature02033"},{"key":"2025040203500666000_2025.03.26.645437v1.29","doi-asserted-by":"publisher","DOI":"10.1126\/science.1077293"},{"key":"2025040203500666000_2025.03.26.645437v1.30","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-444-59426-6.00007-0"},{"key":"2025040203500666000_2025.03.26.645437v1.31","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2249"},{"key":"2025040203500666000_2025.03.26.645437v1.32","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.32671"},{"key":"2025040203500666000_2025.03.26.645437v1.33","doi-asserted-by":"publisher","DOI":"10.1038\/s43586-022-00136-4"},{"key":"2025040203500666000_2025.03.26.645437v1.34","doi-asserted-by":"crossref","unstructured":"Liu, Y.-Z. et al. Simultaneous two-photon activation and imaging of neural activity based on spectral\u2013temporal modulation of supercontinuum light. Neurophotonics 7, (2020).","DOI":"10.1117\/1.NPh.7.4.045007"},{"key":"2025040203500666000_2025.03.26.645437v1.35","doi-asserted-by":"publisher","DOI":"10.1126\/science.2321027"},{"key":"2025040203500666000_2025.03.26.645437v1.36","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-04470-8"},{"key":"2025040203500666000_2025.03.26.645437v1.37","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.1600675"},{"key":"2025040203500666000_2025.03.26.645437v1.38","doi-asserted-by":"publisher","DOI":"10.1038\/nbt899"},{"key":"2025040203500666000_2025.03.26.645437v1.39","doi-asserted-by":"publisher","DOI":"10.1364\/JOSAB.13.000481"},{"key":"2025040203500666000_2025.03.26.645437v1.40","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0907084106"},{"key":"2025040203500666000_2025.03.26.645437v1.41","doi-asserted-by":"publisher","DOI":"10.1529\/biophysj.108.130187"},{"key":"2025040203500666000_2025.03.26.645437v1.42","doi-asserted-by":"crossref","first-page":"1111","DOI":"10.1038\/nphys4257","article-title":"Coherent control of an opsin in living brain tissue","volume":"13","year":"2017","journal-title":"Nat. Phys"},{"key":"2025040203500666000_2025.03.26.645437v1.43","doi-asserted-by":"crossref","first-page":"4144","DOI":"10.1364\/OPEX.12.004144","article-title":"Multiphoton intrapulse interference Coherent control through scattering tissue","volume":"12","year":"2004","journal-title":"Opt. Express"},{"key":"2025040203500666000_2025.03.26.645437v1.44","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0407733101"},{"key":"2025040203500666000_2025.03.26.645437v1.45","doi-asserted-by":"crossref","first-page":"1484","DOI":"10.1364\/OL.547032","article-title":"Label-free hyperspectral multiphoton microscopy","volume":"50","year":"2025","journal-title":"Opt. Lett"},{"key":"2025040203500666000_2025.03.26.645437v1.46","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1002\/lpor.201200014","article-title":"Coherent fiber supercontinuum for biophotonics","volume":"7","year":"2013","journal-title":"Laser Photonics Rev"},{"key":"2025040203500666000_2025.03.26.645437v1.47","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1514209112"},{"key":"2025040203500666000_2025.03.26.645437v1.48","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2019.07.016"},{"key":"2025040203500666000_2025.03.26.645437v1.49","doi-asserted-by":"publisher","DOI":"10.1126\/science.ade2024"},{"key":"2025040203500666000_2025.03.26.645437v1.50","doi-asserted-by":"crossref","first-page":"3187","DOI":"10.1063\/1.1531620","article-title":"Multiphoton intrapulse interference. II. Control of two- and three-photon laser induced fluorescence with shaped pulses","volume":"118","year":"2003","journal-title":"J. Chem. Phys"},{"key":"2025040203500666000_2025.03.26.645437v1.51","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1021\/jp036150o","article-title":"Multiphoton intrapulse interference 3: Probing microscopic chemical environments","volume":"108","year":"2004","journal-title":"J. Phys. Chem. A"},{"key":"2025040203500666000_2025.03.26.645437v1.52","doi-asserted-by":"crossref","first-page":"1813","DOI":"10.1021\/cr020668r","article-title":"Experimental coherent laser control of physicochemical processes","volume":"104","year":"2004","journal-title":"Chem. Rev"},{"key":"2025040203500666000_2025.03.26.645437v1.53","doi-asserted-by":"crossref","first-page":"759","DOI":"10.1364\/OPEX.14.000759","article-title":"Use of coherent control for selective two-photon fluorescence microscopy in live organisms","volume":"14","year":"2006","journal-title":"Opt. Express"},{"key":"2025040203500666000_2025.03.26.645437v1.54","doi-asserted-by":"crossref","first-page":"17256","DOI":"10.1364\/OE.21.017256","article-title":"Two-photon imaging of multiple fluorescent proteins by phase-shaping and linear unmixing with a single broadband laser","volume":"21","year":"2013","journal-title":"Opt. Express"},{"key":"2025040203500666000_2025.03.26.645437v1.55","doi-asserted-by":"crossref","first-page":"3444","DOI":"10.1364\/OL.35.003444","article-title":"Dispersion-based pulse shaping for multiplexed two-photon fluorescence microscopy","volume":"35","year":"2010","journal-title":"Opt. Lett"},{"key":"2025040203500666000_2025.03.26.645437v1.56","doi-asserted-by":"publisher","DOI":"10.1126\/science.1130747"},{"key":"2025040203500666000_2025.03.26.645437v1.57","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0904589106"},{"key":"2025040203500666000_2025.03.26.645437v1.58","doi-asserted-by":"crossref","first-page":"12741","DOI":"10.1364\/OE.17.012741","article-title":"Multiplexed two-photon microscopy of dynamic biological samples with shaped broadband pulses","volume":"17","year":"2009","journal-title":"Opt. Express"},{"key":"2025040203500666000_2025.03.26.645437v1.59","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2019.10.025"},{"key":"2025040203500666000_2025.03.26.645437v1.60","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.0151-22.2022"},{"key":"2025040203500666000_2025.03.26.645437v1.61","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3217"},{"key":"2025040203500666000_2025.03.26.645437v1.62","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-018-0183-z"},{"key":"2025040203500666000_2025.03.26.645437v1.63","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-020-0795-y"},{"key":"2025040203500666000_2025.03.26.645437v1.64","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.0572-16.2016"},{"key":"2025040203500666000_2025.03.26.645437v1.65","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.81983"},{"key":"2025040203500666000_2025.03.26.645437v1.66","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1186\/s13064-019-0132-2","article-title":"Cellular properties of intrinsically photosensitive retinal ganglion cells during postnatal development","volume":"14","year":"2019","journal-title":"Neural Develop"},{"key":"2025040203500666000_2025.03.26.645437v1.67","doi-asserted-by":"publisher","DOI":"10.1126\/science.aay3152"},{"key":"2025040203500666000_2025.03.26.645437v1.68","doi-asserted-by":"publisher","DOI":"10.1364\/OL.29.000775"},{"key":"2025040203500666000_2025.03.26.645437v1.69","doi-asserted-by":"publisher","DOI":"10.1038\/24329"},{"key":"2025040203500666000_2025.03.26.645437v1.70","doi-asserted-by":"publisher","DOI":"10.1016\/j.tins.2013.10.004"},{"key":"2025040203500666000_2025.03.26.645437v1.71","doi-asserted-by":"publisher","DOI":"10.1038\/emm.2014.121"},{"key":"2025040203500666000_2025.03.26.645437v1.72","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2023.09.023"},{"key":"2025040203500666000_2025.03.26.645437v1.73","doi-asserted-by":"crossref","first-page":"2172","DOI":"10.1364\/OL.37.002172","article-title":"Wave-breaking-extended fiber supercontinuum generation for high compression ratio transform-limited pulse compression","volume":"37","year":"2012","journal-title":"Opt. Lett"},{"key":"2025040203500666000_2025.03.26.645437v1.74","doi-asserted-by":"crossref","first-page":"1113","DOI":"10.1364\/OE.20.001113","article-title":"Nonlinear polarization dynamics in a weakly birefringent all-normal dispersion photonic crystal fiber: toward a practical coherent fiber supercontinuum laser","volume":"20","year":"2012","journal-title":"Opt. Express"},{"key":"2025040203500666000_2025.03.26.645437v1.75","doi-asserted-by":"crossref","first-page":"2206","DOI":"10.1364\/JOSAB.11.002206","article-title":"Frequency-resolved optical gating with the use of second-harmonic generation","volume":"11","year":"1994","journal-title":"J. Opt. Soc. Am. B"},{"key":"2025040203500666000_2025.03.26.645437v1.76","doi-asserted-by":"publisher","DOI":"10.3791\/55396"},{"key":"2025040203500666000_2025.03.26.645437v1.77","doi-asserted-by":"publisher","DOI":"10.3791\/2367"},{"key":"2025040203500666000_2025.03.26.645437v1.78","doi-asserted-by":"crossref","first-page":"2540","DOI":"10.1038\/s41598-020-59227-5","article-title":"Dynamic tracking algorithm for time-varying neuronal network connectivity using wide-field optical image video sequences","volume":"10","year":"2020","journal-title":"Sci. Rep"},{"key":"2025040203500666000_2025.03.26.645437v1.79","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-022-01450-8"},{"key":"2025040203500666000_2025.03.26.645437v1.80","first-page":"424","volume-title":"in Medical Image Computing and Computer-Assisted Intervention \u2013 MICCAI 2016","volume":"9901","year":"2016"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.03.26.645437","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T08:22:36Z","timestamp":1768465356000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.03.26.645437"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,30]]},"references-count":80,"URL":"https:\/\/doi.org\/10.1101\/2025.03.26.645437","relation":{},"subject":[],"published":{"date-parts":[[2025,3,30]]},"subtype":"preprint"}}