Identifying and Classifying Aquatic Microorganisms with an Imaging Particle Analysis System

By AZOM

Table of Content

Introductions
FlowCAM System
VisualSpreadsheet Software
Advantages of FlowCAM
Example
Conclusion
About Fluid Imaging Technologies

Introductions

FlowCAM® was first launched in the market in 1999 and since then is being widely used for freshwater and oceanographic research. To date, more than 150 instruments have been installed across the globe in renowned laboratories like Bigelow Laboratory for Oceanographic Sciences, Alfred Wegner Institute, Scripps Institution of Oceanography, the Chinese Academy of Sciences, and Laboratoire d'Oceanologie de Villefrance.

Figure 1. Typical FlowCAM algae images acquired from an ocean sample.

Besides differentiating zooplankton and phytoplankton, FlowCAM is also being employed for algal research, water quality monitoring of source drinking water, HAB monitoring, ballast water research and biofuel production. With ongoing product enhancements through customer feedback, the system continues to fulfill the expanding requirements of the researchers.

FlowCAM System

Figure 2. Benchtop FlowCAM

The FlowCAM combines the function of an automated microscope for in-depth morphological analysis of shape and size of algal cells and has the ability to add fluorescence values to additionally differentiate cell types that are identical to a flow cytometer. First, the sample is drawn via a rectangular flow cell by a peristaltic pump or an optional syringe. While the sample travels through the camera’s field-of-view, a flash LED located at the back of the flow cell offers back lighting. Then, the camera is triggered synchronously with the flash, thus effectively freezing the sample for the camera to obtain the flowing sample’s image.

Figure 3: FlowCAM architecture block diagram

Since the FlowCAM does not utilize sheath fluid, it can accept a broad range of particle sizes up to 2,000 µm when compared to other standard instruments.

VisualSpreadsheet Software

After each image of the field-of-view is obtained, the VisualSpreadsheet software thresholds each cell from the background, and records only those images of the cells as separate images within a "collage". All images have indexed to them of approximately 26 different measurements which are made from the cell image as it is obtained. These measurements fall under a number of categories and they include morphological measurements such as length, diameter, width, circularity and perimeter.

In addition, “gray-scale" measurements such as color information, transparency and intensity, and spectral measurements such as width, peak and area measurements from the signals obtained in the two channels of fluorescence. Using a digital camera on a microscope, morphological measurements can be made except for the difference being that the measurements are being perfumed on a moving stream of particles rather than on a static microscope slide.

This means that the FlowCAM can attain and calculate countless number of particles each minute, thus obtaining much more data automatically and producing higher statistical significance.

Advantages of FlowCAM

The FlowCAM offers a number of benefits. For instance, it depends mainly on morphological and gray-scale discrimination of algal cells rather than on spectral discrimination of a flow cytometer. On the other hand, owing to FlowCAM’s fluorescence capabilities, spectral data can be utilized in addition to the morphological data for additional discrimination of algal cells.

This overcomes the limitation of flow cytometers, which have just a single morphological measurement, relative size often expressed as Equivalent Spherical Diameter obtained from the forward-scatter signal. Besides this easy morphological measurement, the flow cytometer can only differentiate cells depending on their spectral signature, by gathering fluorescence signals in varied narrow wavelength bands to estimate a steady electromagnetic spectrum as might be gathered by a spectrometer.

Figure 4. FlowCAM run results

Figure 5. Staurastrum cells isolated by VisualSpreadsheet statistical classification

Example

The FlowCAM was used to run a sample of mixed algae cultures to show how both the instrument and software utilize morphological and spectral processing to differentiate algae. Following this, “Fluorescence Trigger" mode was selected and the system was activated, whereby the camera was simply triggered to capture an image when a fluorescence event takes place upon laser excitation.

In a sparse sample, this ensures that all preferred particles i.e. living algae are captured, while sediment, detritus and other non-fluorescing particles travel without being captured. The FlowCAM also uses "Scatter Trigger" mode simultaneously with Fluorescence Trigger for non-fluorescing organisms and other particles.

Figure 4 illustrates the result of a FlowCAM run with this sample. The left portion of the window displays summary statistics, scatterplots and histograms, while the right portion of the window displays cell images in a collage window, which can be seen interactively, filtered, and categorized by the VisualSpreadsheet software.

The statistical pattern recognition capability of the VisualSpreadsheet is then utilized to automatically categorize various types of microorganisms identified n the sample. This is performed by producing libraries of cell images of a specific taxa and storing them.

Conclusion

The FlowCAM is an ideal tool for identification and classification of aquatic microorganisms. With advanced statistical pattern recognition software, the system can characterize algal species whose difference might be very subtle for other automated analyses. Moreover, FlowCAM can assess a wide range of particle sizes of approximately 2,000 µm when compared to other standard systems.

About Fluid Imaging Technologies

Fluid Imaging manufactures the FlowCAM®, a continuous-imaging particle analysis instrument. FlowCAM combines the speed of data acquisition of typical particle analyzers with the detailed individual particle information typical of microscopy. Intuitive VisualSpreadsheet software facilitates rapid analysis of particle data and sophisticated pattern recognition operations for individual particle characterization.

This information has been sourced, reviewed and adapted from materials provided by Fluid Imaging Technologies.

For more information on this source, please visit Fluid Imaging Technologies.

Date Added: May 15, 2013 | Updated: Jun 11, 2013
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