Yokogawa Fluid Imaging Technologies, Inc.

- Automatically Differentiate Cyanobacteria from Other Algae

Based on proven FlowCam technology, the FlowCam Cyano automatically identifies cyanobacteria from other algae and particles in aquatic samples. Using a patent-pending combination of excitation wavelength, phycocyanin fluorescence measurement, and image recognition software, the system automates what was previously done using manual microscopy. After differentiating the cyanobacteria from the other algae in the sample, VisualSpreadsheet software can be used to further characterize specific types of algae found in the sample using 40+ physical parameters.

FLUORESCENCE DETECTION 

The FlowCam® Cyano utilizes a red laser (633 nm) to excite phycocyanin, a pigment present in cyanobacteria, and chlorophyll, a pigment present in green algae. Excitation emissions are unique for each pigment, and their ratios of relative fluorescence can be used to  differentiate between green algae and cyanobacteria.  

(Left): Relative fluorescence intensity wavelengths for photosynthetic pigments phycoerythrin, phycocyanin, and chlorophyll. The blue and green boxes on delineate the fluorescence range of phycocyanin (650±10 nm) and chlorophyll (700±10 nm), respectively, detected by the FlowCam Cyano. Image modified from French and Young, 1951.

FLOWCAM CYANO CONFIGURATION

A schematic diagram of the FlowCam Cyano is depicted to the left. The following describes the fluorescence of chlorophyll and phycocyanin from the point of excitation to its use in pigment analysis:

  1. A red laser (633 nm) excites microalgae fluorescence as a sample passes through the flow cell.
  2. Emitted fluorescence is sent through a longpass beamsplitter followed by selective notch filters. The notch filters restrict emissions received by the photomultiplier tubes (PMT). PMT 1 receives fluorescence representative of chlorophyll (700±10nm). PMT 2 receives fluorescence representative of phycocyanin (650±10nm).

DIFFERENTIATION USING FLUORESCENCE RATIO: VISUALSPREADSHEET ANALYSIS

3.  VisualSpreadsheet® , the analysis software associated with the FlowCam, calculates the Channel 2/Channel 1 ratio for each imaged cell, a ratio calculated from relative fluorescence intensities received by PMT 2. 

Imaged cells can be sorted in VisualSpreadsheet by Ch2/Ch1 ratio. In the figure above, the fluorescence ratio of each cell is plotted on the lower right graph, with Ch 2 peak as the y-axis and Ch 1 peak as the x-axis. Ch2/Ch1 ratios >1 are indicative of green algae (left) and Ch2/Ch1 ratios <1 are indicative of cyanobacteria (right).

  • High-sensitivity fluorescence detection (2 channels, phycocyanin and chlorophyll)
  • Ratiometric fluorescence to discriminate cyanobacteria from other algae
  • Classification using 40+ physical parameters measured from each cell
  • Integral biovolume calculations
  • Our intelligent image recognition and analysis software VisualSpreadsheet® 
  • Faster & easier analysis than manual microscopy