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In living butterflies, their UV-VIS absorption spectra, respectively with 11-cis and all-trans 3-hydroxy-retinal (A3) chromophore, can be recorded from the eyeshine, the light reflected from the compound eye after passing twice through the rhabdom waveguides. Here, a comprehensive pipeline for the analysis of eyeshine reflection spectra is presented. A microscope coupled with a broadband LED and a microspectrometer was used to record the photoconversions. Fitting temporal exponential relaxations to log-reflectance arrays yielded transient and baseline spectra, analogous to absorbance difference and sum, respectively. These spectra were subjected to singular value decomposition and visual pigment template fitting. The compound eye of the fritillary\n                    <jats:italic toggle=\"yes\">Fabriciana adippe<\/jats:italic>\n                    was exposed to a series of second-long light pulses, causing photorelaxations to 80% metahodopsin equilibrium with time constants 40-120 ms. The alpha peak wavelength was estimated to 547-552 nm for rhodopsin and 496-501 nm for metarhodopsin. The alpha peak absorbance ratio 1.25-1.35 is consistent with the isosbestic wavelength 530 nm. Below 405 nm, rhodopsin absorbs more than metarhodopsin. Baseline spectra enable concatenation of exposures, monitor pigment concentrations, and enhance template parameter estimation. The method can be used in many butterflies and could be adapted to fruitflies, facilitating studies of the relation between the pigment photophysical properties and the opsin amino acid sequence. Spectroscopic results can be complemented with physiological spectral sensitivity datasets analysed with the same fitting procedure.\n                  <\/jats:p>","DOI":"10.1177\/00037028261481892","type":"journal-article","created":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T19:30:52Z","timestamp":1787340652000},"update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":0,"title":["Reflection Spectroscopy of Bistable Visual Pigments in Living Butterflies"],"prefix":"10.1177","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1710-444X","authenticated-orcid":false,"given":"Primo\u017e","family":"Pirih","sequence":"first","affiliation":[{"name":"University of Salzburg"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"179","published-online":{"date-parts":[[2026,8,21]]},"reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1515\/znc-1977-7-833"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1007\/BF00312143"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1002\/cne.10582"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1113\/JP273917"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1242\/jeb.217703"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1098\/rspb.2021.1560"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-ento-061720-071644"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ydbio.2022.10.011"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1007\/s002390010071"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1523\/jneurosci.23-11-04527.2003"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msac067"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00359-025-01761-6"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-5320(68)90065-8"},{"key":"e_1_3_3_15_2","doi-asserted-by":"publisher","DOI":"10.1515\/znc-1979-3-421"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1007\/s001140100268"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1242\/jeb.205.8.1077"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00359-002-0307-9"},{"key":"e_1_3_3_19_2","doi-asserted-by":"publisher","DOI":"10.2108\/zsj.16.17"},{"key":"e_1_3_3_20_2","doi-asserted-by":"publisher","DOI":"10.1242\/jeb.030692"},{"key":"e_1_3_3_21_2","doi-asserted-by":"publisher","DOI":"10.1242\/jeb.083485"},{"key":"e_1_3_3_22_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00359-019-01330-8"},{"key":"e_1_3_3_23_2","doi-asserted-by":"publisher","DOI":"10.1098\/rstb.2021.0276"},{"key":"e_1_3_3_24_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.88.7.2783"},{"key":"e_1_3_3_25_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00359-005-0608-x"},{"key":"e_1_3_3_26_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.203.4385.1125"},{"key":"e_1_3_3_27_2","doi-asserted-by":"publisher","DOI":"10.1039\/d0fd00075b"},{"key":"e_1_3_3_28_2","doi-asserted-by":"publisher","DOI":"10.1098\/rstb.2021.0275"},{"key":"e_1_3_3_29_2","doi-asserted-by":"publisher","DOI":"10.1007\/BF00535663"},{"key":"e_1_3_3_30_2","doi-asserted-by":"crossref","unstructured":"Stavenga DG Schwemer J. 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