DOI: 10.5061/dryad.rr4xgxdjc
title: Coupling gel electrophoresis with photoluminescence imaging reveals biochrome complexes in modern and fossil shells

description: Electrophoresis is commonly used to visualize mixtures of proteins, such
 as those occluded in calcareous biominerals. However, it is ineffective
 for the detection of biochromes, a major class of low molecular weight
 organic compounds commonly associated with calcified exoskeletons. We
 describe a novel approach based on the coupling between electrophoresis
 and luminescence spectral imaging to reveal invisible biochromes, identify
 them chemically, evidence their putative interaction with exoskeletal
 macromolecules, and purify them in large amounts. Our protocol relies on
 three key steps: a mild extraction of all organics from bleached skeletal
 powder (step1); an optimized electrophoretic fractionation immediately
 followed by direct on-gel spectral image acquisition performed before
 classical gel staining (step 2); a large-scale purification via
 preparative electrophoresis coupled to luminescence spectral imaging, to
 obtain significant amounts of biochromes of interest (step 3). Steps 1 to
 3 were successfully applied to recent gastropod shell extracts, while
 steps 1 and 2 were applied to their fossil equivalents. Our protocol
 enabled direct, non-invasive on‐gel identification of porphyrin molecules,
 even in trace amounts. It opens new avenues for the study of a wide range
 of biological composites, mineralized or not, that contain luminescent
 biochromes. It is particularly well-suited to ancient specimens and
 fossils to trace the origin and evolution of biochrome complexes in the
 geological record.
description: Acquisition : The detection part consisted of
 a 4 megapixel CMOS camera (ORCA -Flash 4.0 LT Plus, Hamamatsu) with a
 sensitivity ranging from 350 to 1100 nm. The camera was fitted with a
 UV-VIS-IR 60 mm 1:4 Apo Macro lens (CoastalOptics) in front of which was
 positioned a filter wheel fitted with 8 Interference band-pass filters
 (Semrock) to perform multi-spectral acquisitions. The illumination part
 was composed of 16 LED lights ranging from 365 to 700 nm (CoolLED
 pE-4000), coupled to a liquid light-guide fiber fitted with a fiber-optic
 ring light-guide to allow homogeneous illumination. Emission and
 excitation spectra were collected using a modified spectrofluorometer
 (Fluorolog 3–22, HORIBA Jobin Yvon), which allows spectra to be collected
 directly on the gels using an optical bundle connected to a focusing lens.
 The entrance and exit slits' widths of the monochromators were set at
 10 nm to collect spectra with good signal-to-noise and a spectral
 resolution compatible with the detection of Q-bands. Emission spectra were
 collected using an excitation at 400 nm. Excitation spectra were collected
 using a 732 ± 34 nm bandpass filter (from Semrock) at the entrance of the
 emission monochromator to eliminate any stray light. The spectra in the
 tube were collected under a UV excitation generated by a Jaxman U1C torch
 and using a JETI 1211 portable spectrometer sensitive between 300 and 1000
 nm, fitted with a high-pass transmission filter with a cut-off wavelength
 at 409 nm to eliminate the reflectance of the UV excitation.
 Data processing : The black and white images
 were combined using ImageJ software to produce the three-channel false
 colour phtoluminescence images. The intensity levels of the image
 corresponding to Figure 2b were linearly stretched for each channel to
 optimise visualisation. For the image corresponding to Figure 3b, a square
 root scale was used to stretch image intensity levels to enhance the
 visibility of lower intensity values while compressing higher intensity
 values. The spectra collected with the
 spectrofluorometer were normalized according to the intensity maximum. The
 spectra shown were not modified.
description: # Coupling gel electrophoresis with photoluminescence imaging reveals
 biochrome complexes in modern and fossil shells
 [https://doi.org/10.5061/dryad.rr4xgxdjc](https://doi.org/10.5061/dryad.rr4xgxdjc) ## Description of the data and file structure The experimental method aimed at coupling in an innovative way *sodium dodecyl sulfate polyacrylamide gel electrophoresis (*SDS-PAGE) with luminescence spectral imaging in order to detect and chemically characterize the presence of invisible biochromes eluted on gel. ## 1. Dataset Overview This dataset contains multispectral imaging data and corresponding spectral measurements associated with **Figures 2 and 3** of the related publication. Data include raw grayscale images at selected excitation/emission wavelengths, composite false-colour images generated from these channels, and tabulated spectral measurements. --- ## 2. File Inventory and Description ### Composite Images * **`composite_image_figure2.png`** (4.39 MB) * False-colour composite generated from multispectral channels corresponding to Figure 2. * **`composite_image_Tubes_ref_R708_G571_B472_figure3.png`** (3.27 MB) * False-colour composite generated from reference tube images using three wavelengths mapped to RGB (R = 708 nm, G = 571 nm, B = 472 nm), shown in Figure 3. ### Raw Multispectral Images * **`image__x405-472-30-5s_figure3.tif`** (8.39 MB) * Raw grayscale image acquired with 405 nm excitation, 472/30 nm emission filter, 5 s exposure (Figure 3). * **`image_gel_x405_472-30_2s_figure2.tif`** (8.39 MB) * Gel sample imaged with 405 nm excitation, 472/30 nm emission filter, 2 s exposure (Figure 2). * **`image_gel_x405_514-30_2s_figure2.tif`** (8.39 MB) * Gel sample imaged with 405 nm excitation, 514/30 nm emission filter, 2 s exposure (Figure 2). * **`image_gel_x405_685-40_2s_figure2.tif`** (8.39 MB) * Gel sample imaged with 405 nm excitation, 685/40 nm emission filter, 2 s exposure (Figure 2). * **`image_Tubes_ref_G66-40.tif`** (4.21 MB) * Reference tube imaged using a 660/40 nm bandpass filter (green channel). * **`image-tubeEP_x405-514-30-5s_figure3.tif`** (8.39 MB) * Tube sample imaged with 405 nm excitation, 514/30 nm emission filter, 5 s exposure (Figure 3). * **`image-tubeEP_x405-708-75-2s_figure3.tif`** (8.39 MB) * Tube sample imaged with 405 nm excitation, 708/75 nm emission filter, 2 s exposure (Figure 3). ### Spectral Data * **`spectra_figure2.csv`** (31.27 KB) * Note: Numeric values use a comma as the decimal separator (e.g. `0,34`). * Tabulated excitation and emission spectra relevant to samples shown in Figure 2. * Columns: Wavelength (nm), Intensity (a.u.). * **`spectra_figure3.csv`** (38.48 KB) * Note: Numeric values use a comma as the decimal separator (e.g. `0,34`). * Tabulated excitation and emission spectra relevant to samples shown in Figure 3. * Columns: Wavelength (nm), Intensity (a.u.). --- ## 3. Notes on Data Use * Composite images were generated by mapping raw grayscale channels to the RGB colour space. * `.tif` files are uncompressed grayscale images representing individual spectral acquisitions. * `.csv` files can be opened in any text editor or imported into analysis environments such as R, Python, or MATLAB.