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Flow Cytometry, Microscopy and Hyperspectral Imaging of microcystis, Cyanobacteria, and Algae- SETAC
Citation:
Zucker, R., N. Chernoff, Jim Lazorchak, AND N. Dugan. Flow Cytometry, Microscopy and Hyperspectral Imaging of microcystis, Cyanobacteria, and Algae- SETAC. SETAC, Minneapolis, MN, November 12 - 16, 2017.
Impact/Purpose:
Presentation at the Annual Meeting of the Society of Environmental Toxicology and Chemistry
Description:
The detection of cyanobacteria algae, and picoplankton, in water is an important step in assessing water quality. Studies were initiated using fluorescence microscopy, flow cytometry and hyperspectral imaging with two fresh water species that were cultured in the laboratory:Microcystis Aeruginosa (cyanobacteria), and Selenastrum capricornutum (algae). Cyanobacteria were distinguished rapidly from algae using a fluorescent microscope that selectively excited different photosynthetic pigments in the two species with blue or green fluorescent light. The cyanobacteria are brighter with green light excitation while algae are brighter with blue light excitation. By combining microscopic images sequentially obtained using these two excitation wavelengths, organisms can be quickly classified as either algae or cyanobacteria. The positive identification of each organism was then confirmed through its emission wavelengths using the Prism and Reflector Imaging Spectrometer System (PARISS, hyperspectral imaging system). The mean fluorescence emission from algae is about 25 nm longer than cyanobacteria. The Stratedigm flow cytometer can count 1000 organisms per second. This provides statistical accurate data om distinguishing large quantities of laboratory algae, cyanobacteria, and picoplankton. Their identification was based on their size and selective intensity emissions following excitation by a blue (488 nm), yellow (550 nm) or red (640 nm) lasers Fluorescence from algae and cyanobacteria are observed in all detection channels with these lasers. However, the intensity of cyanobacteria emission following yellow or red laser excitation was preferentially detected in different channels than the algae. Likewise, the emission intensity from algae was detected in different channels that cyanobacteria after excitation with the blue laser. Thus the organisms could be detected by size and selective fluorescence emission. Flow cytometry techniques developed with the laboratory species microcystis aeruginosa, and algae were then applied to study lake picoplankton which had a size between 1-2 um. New methodologies to study photosynthetic single cell organisms are described which could be useful to identify and distinguish cyanobacteria from algae. In the future this type of data may provide a useful technique to distinguish algae from cyanobacteria derived from streams and lakes. Upon excitation, cyanobacteria and algae fluoresce with a peak maximum at 645-665 nm and 680-690 nm, respectively. These data show that cyanobacteria and algae can be identified using the cytometric and microscope equipment (Pseudokirchneriella subcapitata) recently renamed as Pseudokirchneriella subcapitata algae).