Exploiting the autofluorescent properties of photosynthetic pigments for analysis of pigmentation and morphology in live Fremyella diplosiphon cells

Abstract

Fremyella diplosiphon is a freshwater, filamentous cyanobacterium that exhibits light-dependent regulation of photosynthetic pigment accumulation and cellular and filament morphologies in a well-known process known as complementary chromatic adaptation (CCA). One of the techniques used to investigate the molecular bases of distinct aspects of CCA is confocal laser scanning microscopy (CLSM). CLSM capitalizes on the autofluorescent properties of cyanobacterial phycobiliproteins and chlorophyll a. We employed CLSM to perform spectral scanning analyses of F. diplosiphon strains grown under distinct light conditions. We report optimized utilization of CLSM to elucidate the molecular basis of the photoregulation of pigment accumulation and morphological responses in F. diplosiphon.

Keywords: autofluorescence; confocal laser scanning microscopy; cyanobacteria; fluorescence imaging; light; microscopy; morphology; phycobiliproteins.

Figure 1.

Slide preparation and imaging scheme. Population Z-series scans are obtained by scanning immobilized Fremyella diplosiphon cells in the x, y, and z axes for each sub-section of the cover-slip covered area (e.g., A, B, C, D, E, and F) on a slide.

Figure 2.

Differential Interference Contrast (DIC) images of filament populations of Fremyella diplosiphon strains grown in broad-band red and green light. Wild-type pigmentation SF33 (A, B) and ΔrcaE mutant (C, D) strains. Images at 20× dry objective. Bars, 50 μm.

Figure 3.

Spectral emission collection of SF33 GL-grown cells after excitation with 488-nm laser. An emission spectrum was gathered at 10.7-nm increments in the range from 500 nm to 750 nm. All images at 63× oil objective. Bars, 10 μm.

Figure 4.

Spectral emission collection of SF33 RL-grown cells after excitation with 488-nm laser. An emission spectrum was gathered at 10.7-nm increments in the range from 500 nm to 750 nm. All images at 63× oil objective. Bars, 10 μm.

Figure 5.

Spectral emission collection of SF33 GL-grown cells after excitation with 543-nm laser. An emission spectrum was gathered at 10.7-nm increments in the range from 550 nm to 750 nm. All images at 63× oil objective. Bars, 10 μm.

Figure 6.

Spectral emission collection of SF33 RL-grown cells after excitation with 543-nm laser. An emission spectrum was gathered at 10.7-nm increments in the range from 550 nm to 750 nm. All images at 63× oil objective. Bars, 10 μm.

Figure 7.

Spectral emission collection of SF33 GL-grown cells after excitation with 633-nm laser. An emission spectrum was gathered at 10.7-nm increments in the range from 640 nm to 750 nm all images at 63× oil objective. Bars, 10 μm.

Figure 8.

Spectral emission collection of SF33 RL-grown cells after excitation with 633-nm laser. An emission spectrum was gathered at 10.7-nm increments in the range from 640 nm to 750 nm. All images at 63× oil objective. Bars, 10 μm.

Figure 9.

Phycobiliprotein autofluorescence of Fremyella diplosiphon strains grown in broad-band red and green light. Wild-type pigmentation SF33 (A, B) and ΔrcaE mutant (C, D) strains. Maximum-projection images from a Z-series of GL- and RL-adapted filaments were acquired at 63× oil objective with a 2× zoom setting. False colors are scaled equally among images. Bars, 5 μm.