How Does the FlowCam Cyano Work?

An explanation of the configuration and fluorescence detection of the FlowCam® Cyano

Phycocyanin and Chlorophyll Fluoresence as detected by the FlowCam Cyano

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.

 

 

Visio-Optical Schematic FlowCam Cyano

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

FlowCam Cyano cyano and green algae visp

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). 

Download the poster presented at IAGLR 2017 on the FlowCam Cyano technology.