Coupling gel electrophoresis with photoluminescence imaging reveals biochrome complexes in modern and fossil shells

Thoury, Mathieu 1 ; Marin, Frédéric2 ; Polacchi, Luca1 ; Merle, Didier3; Basuyaux, Olivier4; Albéric, Marie5; Habermeyer, Benoît6; Gueriau, Pierre7; Thomas, Jérôme2; Pasco, Hélène1; Guilard, Roger8; Lutet-Toti, Camille2

Published Sep 25, 2025 on Dryad. https://doi.org/10.5061/dryad.rr4xgxdjc

Abstract

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.

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.

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.