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

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. Methods 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.