Spectral flow cytometry and imaging studies on cyanobacteria and microplastics using 64 narrow bandpass filters and a widefield fluorescence research microscope
Spectral flow cytometers yield increased resolution due in part to the 64 narrow bandpass detectors and unmixing software that are used to remove autofluorescence derived from metabolic fluorescence molecules. We have applied this flow technology to study the following applications without unmixing: 1) cyanobacteria metabolism, 2) effects of nutrient deficiency or chemical exposure on cyanobacteria, and 3) microplastic detection in cells and solution by spectral scatter. A research Nikon Ti2 microscope was used to confirm and add to the data derived from a Cytek Aurora spectral flow cytometer. Time measurements on cyanobacteria were made during specific wavelength illumination to monitor photosynthesis and metabolism. 3D localization of fluorescent polystyrene beads in mammalian cells was made using widefield confocal microscopy.
Microcystis aeruginosa is a species of cyanobacteria present in inland lakes, streams, and water supplies. These bacteria can form harmful algal blooms, contaminating water with toxic compounds. The relationship between cyanobacteria, nutrient availability, growth, and death was investigated using an in vitro Microcystis aeruginosa cell line. Changes in cell metabolism were measured in the green (542 nm mean) and red (660 nm mean) fluorescent ranges. There was an increase in green fluorescence (potentially due to a buildup of metabolic proteins) followed by a decrease in red fluorescence from photosynthetic pigments. A decrease in cell size measured by light scatter was also detected.
Different wavelengths of light and chemicals hydrogen peroxide (H2O2) and potassium permanganate (KMnO4), which are commonly used to treat water contaminated with cyanobacteria, affect cyanobacteria’s physiology. An initial increase in red fluorescence (660 nm) followed by a decrease was shown after prolonged exposure to different wavelengths of light, indicating decreased photosynthesis. H2O2 and KMnO4 similarly induced as much as a fivefold increase in green fluorescence emission (542 nm) and an increase in red fluorescence within 24 hours before a decrease over longer periods of time. Microscope observations of Microcystis were correlated to flow cytometry data showing decreased photosynthesis fluorescence, viability, and size after chemical treatment. This measurement approach detects a sensitive and early change in cyanobacteria functioning.
Fluorescent particles were accumulated into cells using an in vitro cellular model system. The 200 nm fluorescence polystyrene beads were detected by measuring cellular fluorescence intensity. Particles were incorporated into several mammalian cell lines in a dose dependent manner and confirmed by microscopy. Using widefield confocal microscopy with deconvolution, the particles were found to concentrate in the endoplasmic reticulum surrounding the nuclei and near the mitochondria. Mitochondria morphology was studied using transfection.
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