Journal: bioRxiv

Article Title: Crystalline guanine packed within vacuoles serves as nitrogen store in Chromera velia

doi: 10.64898/2026.01.31.703024

Figure Lengend Snippet: Bright-field images and Raman spectra of reference guanine crystals (a, b) and guanine crystals isolated from Chromera velia cells (c, d). Raman spectra (b, d) were acquired using a 532 nm excitation laser at 50 mW. Yellow crosses in (a) and (c) indicate the positions from which the single-point spectra shown in (b) and (d) were collected. Extra Raman bands at 1124 and 1513 cm -1 , observed only in the spectra of C. velia guanine crystals, are due to an unidentified polyenic compound present in the cell walls. The band at 1004 cm -1 corresponds to phenylalanine vibrations of proteins.

Article Snippet: For in-situ determination of the chemical composition of intracellular structures, a confocal Raman microscope (alpha300 RSA; Oxford Instruments - WITec, Germany) was used as previously described ( ; ).

Techniques: Isolation

Journal: bioRxiv

Article Title: Crystalline guanine packed within vacuoles serves as nitrogen store in Chromera velia

doi: 10.64898/2026.01.31.703024

Figure Lengend Snippet: a) Cells grown in nitrogen replete medium; Diperential interference contrast (DIC) and polarised light (PM) microscopy images; guanine crystals are observed from day 0 to day 5 (compare to ). Arrows on the grayscale (GS) images mark individual crystals as they were counted. b) Cells kept in nitrogen-deplete medium from day 0 to day 5. Decrease in numbers of guanine crystals was observed from day 3, and on day 5, the total absence of crystals was noted. c) Reappearance of guanine crystals upon transfer of the cells to nitrogen replete medium was observed from day 1. d) Brightfield and polarisation micrographs, along with Raman chemical maps revealed the distribution of crystalline guanine, lipido-protein mass, an unknown polyene located in the cell wall, and water in a typical C. velia cell after 5 days of cultivation under nitrogen replete conditions Scale bar = 3 µm. e) Brightfield, polarisation micrographs and Raman chemical maps in nitrogen depleted conditions of showing the distribution of neutral lipids, starch grains, an unknown polyene located in the cell wall, and water in a typical C. velia cell cultured under nitrogen-depleted conditions for 5 days. Scale bar = 3 µm. f) Box plot showing average guanine crystals counts from day 0 to day 5 under nitrogen repleted and depleted conditions. X-axis represents days, and Y axis represents the number of guanine crystals per cell (n = 30 cells per condition and day) in C. velia cells under nitrogen repleted and depleted conditions. Color legend: Blue boxes represent nitrogen repleted and red boxes represented nitrogen depleted conditions. Horizontal lines represent the mean; whiskers indicate the variability outside of the upper and lower quartiles, and black dots represent outliners. g) Guanine content as measured by HPLC in C.velia cells under nitrogen repleted and depleted conditions, mean and standard deviations of the triplicate samples in both conditions are shown.

Article Snippet: For in-situ determination of the chemical composition of intracellular structures, a confocal Raman microscope (alpha300 RSA; Oxford Instruments - WITec, Germany) was used as previously described ( ; ).

Techniques: Microscopy, Starch, Cell Culture

Journal: bioRxiv

Article Title: Crystalline guanine packed within vacuoles serves as nitrogen store in Chromera velia

doi: 10.64898/2026.01.31.703024

Figure Lengend Snippet: a) Bright-field microscopy image of C. velia cells stained with Lysotracker TM Green DND-26, which accumulates in acidic vacuolar compartments. b) Polarized light micrograph highlighting the birefringence of guanine crystals, which appear as bright, refractile structures. c) Fluorescence microscopic image revealing the specific labelling of the vacuoles stained by DND-26. d) Autofluorescence of the plastids. e) Merged image of c) fluorescent staining and d) plastid autofluorescence, revealing the spatial distinction between guanine containing vacuoles and plastids. f) Merged image overlaying b) polarised light birefringence of guanine crystals and c) DND-26 staining of the vacuoles (magenta) indicating localisation of birefringent guanine crystals within the labelled vacuoles, Scale bar = 10 μm. g-o) Chromera velia zoospores isolated by the method described in Richtova et al; 2023 and fixed in 4% paraformaldehyde. Images g, j, m) Zoospores observed under diperential interference contrast. Images h, k, n) observed under polarized light microscopy highlighting the birefringence of guanine crystals and images (i, l, o) shows the autofluorescence of plastids in the same cells. Scale bar =5 µm. p) Isolated zoospores from Chromera velia cells under bright field, polarised and Raman microspectroscopy. q) Raman spectrum maps depicting the presence of lipid droplets, crystalline anhydrous guanine, plastid and polyene.

Article Snippet: For in-situ determination of the chemical composition of intracellular structures, a confocal Raman microscope (alpha300 RSA; Oxford Instruments - WITec, Germany) was used as previously described ( ; ).

Techniques: Microscopy, Staining, Fluorescence, Isolation, Light Microscopy