Making walled‐, highly autofluorescent dinoflagellate algae cells accessible and amenable for immunofluorescence and application of fluorescent probes

Abstract

Immunofluorescence or fluorescent probe procedures with dinoflagellate algae have been hampered by their intrinsic pigment autofluorescence and rigid cell walls that cannot be digested by cellulolytic or pectinolytic enzymes. Highly autofluorescent, chlorophyll-containing cells from Symbiodinium were fixed and treated by a freeze-fracture procedure on microscope slides, intended to preserve their structure, break the cell wall, eliminate endogenous fluorescent pigments, and provide accessibility to antibodies. Formaldehyde fixation was carried out on intact cells in suspension, followed by freeze-fracture in liquid nitrogen. Finally, they were subjected through a series of tests for pigment extraction to eliminate the cell autofluorescence. Incubation in 70% isopropanol, followed by NaBH 4 after freeze-fracture on glass slides, proved to be the most effective treatment to remove the fluorescence from endogenous pigments and free aldehyde groups remaining after the fixation. The procedure was effective in preserving the cell structures during the lengthy incubations, and the cells were suitable for both, immunolocalization of internal antigens, and visualization of targets of fluorescent probes. The procedure was equally successful when applied to other Symbiodinium species. These results provide a powerful tool for performing immunofluorescence and fluorescent probe detection on these highly autofluorescent, chlorophyll-containing cells.

Carbonic anhydrase 2‐like in the giant clam, Tridacna squamosa : characterization, localization, response to light, and possible role in the transport of inorganic carbon from the host to its symbionts

“…All images were captured under predetermined optimal exposure settings. Of note, zooxanthellae are known to contain pigments that autofluoresce (Castillo-Medina et al 2011), therefore, they were visualized using a U-MWIG filter (Olympus) with excitation at 520-550 nm and 580 nm band-pass emission filter (red channel). CA2-like immunostaining was observed using the U-MNIBA filter (Olympus) with excitation at 470-490 nm and 515-550 nm band-pass emission filter (green channel).…”

Host and Symbiont Cell Cycle Coordination Is Mediated by Symbiotic State, Nutrition, and Partner Identity in a Model Cnidarian-Dinoflagellate Symbiosis

“…In addition to this challenge, Symbiodiniaceae cells in hospite possess a thick internal cell wall and a peripheral chloroplast with a wide photosynthetic absorption range that results in high autofluorescence during microscopy. Together, these algal characteristics make it difficult for the penetration and visualization of intracellular fluorescent probes even after fixation (50)(51)(52).…”

Cutting through host autofluorescence: fluorescence lifetime imaging microscopy for visualising intracellular bacteria in Symbiodiniaceae

“…Various sample preparation approaches such as exposure of cells to sodium borohydride ( Castillo-Medina et al ., 2011 ), high light ( Webster et al ., 2001 ; Maire et al ., 2021 ), combined hydrogen peroxide and light ( Yakubovskaya et al ., 2019 ), copper sulfate and ethanol ( Cohen et al , 2021 ), fluorescence lifetime gating ( Kodama, 2016 ), TrueVIEW quenching kit ( Astafyeva et al ., 2022b ) and post image processing using deep learning tools ( Jiang et al ., 2022 ) are used for reduction of background autofluorescence. Maire et al .…”