Citation:

Zucker, R., N. Chernoff, Jim Lazorchak, AND N. Dugan. Flow cytometry microscopy and hyperspectral imaging of microcystis, cyanobacteria and algae. CYTO2017, Boston, MA, June 10 - 14, 2017.

Impact/Purpose:

This data derived from a fluorescent microscope, PARISS hyperspectral imagier and a flow cytometer equipment show that cyanobacteria and algae can be identified. This research presents a new methodology to study photosynthetic organisms collected derived from lakes & streams which could be useful to identify and distinguish cyanobacteria from algae, understand their growth dynamics, cell viability and their potential toxicity in water.

Description:

The detection of algae and cyanobacteria is an important step in assessing water quality. Studies were initiated using microscopy, flow cytometry and hyperspectral imaging with two fresh water species that could be grown in the laboratory: Microcystis Aeruginosa (cyanobacteria), and Selenastrum (algae). Cyanobacteria was distinguished rapidly from algae using a fluorescent microscope that selectively excited different photosynthetic pigments in the two species with blue or green of light. Algae and cyanobacteria can both wavelengths. Generally, the cyanobacteria will be brighter with green light excitation while algae will be brighter with blue light excitation. Cyanobacteria emit mean autofluorescence light at 645-665 nm while algae emit mean autofluorescence light at 680-690 nm. By combining images obtained using these two excitation wavelengths, different organisms can be quickly classified as either an algae or a cyanobacteria. The identification of each organisms was then confirmed using the Prism and Reflector Imaging Spectrometer System (PARISS, hyperspectral imaging system).. Lake samples were obtained in the summer during a cyanobacteria lake bloom of microcystis and studied by microscopy and PARISS equipment. The following was observed: 1. Algae and cyanobacteria were identified based on their specific fluorescence emission of each species after excitation with blue and green light. 2. A library of wavelengths derived from algae and cyanobacteria could be subsequently used to identify the algae and cyanobacteria using PARISS system 3. Healthy live cyanobacteria could be distinguished from dying cyanobacteria based on the lack of fluorescence intensity using flow cytometry and microscopy. The Stratedigm flow cytometer could distinguish laboratory algae and cyanobacteria based on their size and emissions from a blue (488nm) or yellow (550nm) laser. The yellow laser emission was detected in the 676/29 range while the blue laser emission was detected in the 690/40 range. The cyanobacteria showed a very strong emission with yellow laser excitation while the algae showed a strong emission with blue laser excitation. These flow cytometry techniques derived on the laboratory species microcystis aeruginosa, were then applied to study lake picoplankton (1-2um in size) after filtration using 37 nylon mesh. 4. Picoplankton is defined as an organism between 1-2 um, which could be detected as two distinct clusters using yellow and blue laser excitation with two far red emission wavelengths detectors. Summary: This data show that cyanobacteria and algae can be identified using cytometric and microscope equipment. This research presents a new methodology to study photosynthetic organisms collected derived from lakes & streams which could be useful to identify and distinguish cyanobacteria from algae, understand their growth dynamics, cell viability and their potential toxicity in water. This abstract does not represent USEPA policy.

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