Flow Cytometers

Strictly speaking, a cytometer is a device used to measure individual cells. A flow cytometer is a cytometer that measures cells moving by the sensor in a continuous stream.

Devices that can measure cells can measure other objects too. The more general name of these objects is particles. A particle is an aggregate of smaller particles or molecules. A single molecule of the ambient or flow medium is not a particle.

Two examples of flow cytometers in operational use in oceanography are described in the following. In each case, the basis of the measurement is a captured image.

Flowcam
Figure 1. Montage of FlowCAM® images of microscopic planktonic organisms arranged about a graph showing changes in size distribution over a 20-hour period. Size is expressed as equivalent spherical diameter (ESD) in micrometers. The data were collected in Wells Harbor, Maine, on 16 August 1998. Credit: ©Fluid Imaging Technologies. Click to enlarge

Flow Cytometer And Microscope (FlowCAM®)

The Flow Cytometer And Microscope (FlowCAM®) is an operational instrument that measures particles in the size range 5-1000 µm at volumetric flow rates up to 10 ml per minute. The flow channel cross section is 0.3 x 3 mm. The device functions in stages. In the one model, surface fluorescence, or epifluorescence, of passing particles is monitored. If the epifluoresence of a single particle exceeds a threshold value, then the fluorescence is measured, that is, its value is recorded. At the same time, a signal is sent to the camera to take a picture of the same particle as seen through a microscope. A number of images are presented in Fig. 1.

In technical language, the signal sent to the camera is called a trigger signal. Similarly, taking a picture is technically called capturing an image. In FlowCAM®, this is done by means of an electronic camera with charge-coupled device (CCD). This registers scattered light from the illuminated particle on a discrete array of picture elements (pixels) positioned in an image plane of the magnifying, imaging optics.

In other FlowCAM® models, light scattered by the passing particles, in addition to or in place of excited epifluorescence, is monitored. This also triggers image-capturing, as in the original model.

Data collected with any optical device can easily be voluminous. Being able to process such data in a reasonable time is essential for the device to be used in a routine manner.

This issue of data processing is addressed in FlowCAM® by integration of a powerful digital computer. This is a Pentium processor, with 400-MHz clock rate. The computer serves as the platform for image-processing software. This analyzes images, displays these on a monitor, and stores the images, associated data, and other results of the image analysis.

FlowCAM® is being used in oceanographic research and in a number of monitoring applications. These include measurements from moorings and vessels, and in laboratories.

WHOI scientists
Figure 2. WHOI scientists A. Shalapyonok, H. Sosik, and R. Olson guide the FlowCytobot mounted inside a protective frame. Credit: T. Kleindinst ©Woods Hole Oceanographic Institution. Click to enlarge
Typical data from 1 hr of FlowCytobot
Figure 3. Typical data from 1 hr of FlowCytobot sampling; classification of cells considers all measured properties including orange fluorescence, critical for discrimination of Synechococcus and cryptophytes. Credit: H. Sosik ©Woods Hole Oceanographic Institution. Click to enlarge

Submersible flow cytometer (FlowCytobot)

FlowCytobot (Fig. 2) is a flow cytometer that can operate for extended periods while sitting on the seafloor, given the availability of shore power and communication links. It illuminates particles in a flow cell with laser light at 532 nm. Forward- and side-scattered light is detected at this wavelength. Light emitted at two longer wavelengths, 575 and 680 nm, due to fluorescence, is also detected. Photomultiplier tubes are located at each of the corresponding foci to ensure maximal sensitivity in light gathering before further signal amplification.

Signals from Synechococcus cells, nominal size 1 µm, and from phytoplankton up to 10 µm have been measured (Fig. 3).

FlowCytobot is capable of self-calibration by means of standard beads. It can also clean itself. Both operations are done routinely during submersion, ensuring high data quality.

In an early application, FlowCytobot was deployed for two months on the seafloor at 5-m depth near the Long-term Environmental Observatory (LEO-15) off New Jersey. Populations of Synechococcus and other phytoplankton, were observed during the period of operation. Quite large variations in these were observed, which in some cases have been attributed to changes in population growth rates.

Acknowledgments

We would like to thank K. Peterson and H. Sosik for images and comments.

Related Links

FlowCAM® / Fluid Imaging Technologies:
http://www.fluidimaging.com/
http://www.fluidimaging.com/pictures/flowCAM_brochure.pdf

FlowCytobot:
http://www.orionprogram.org/PDFs/AGU/OS41E-04Sosik.pdf
http://www.whoi.edu/science/B/sosiklab/067.pdf

References
Olson, R.J., A.A. Shalapyonok, and H.M. Sosik. 2003. An automated submersible flow cytometer for analyzing pico- and nanophytoplankton: FlowCytobot. Deep-Sea Research I 50: 301-315.
 
Sieracki, C.K., M.E. Sieracki, and C.S. Yentsch. 1998. An imaging-in-flow system for automated analysis of marine microplankton. Mar. Ecol. Prog. Ser. 168: 285-296.
 
Sosik, H.M., R.J. Olson, M.G. Neubert, and A.A. Shalapyonok. 2003. Growth rates of coastal phytoplankton from time-series measurements with a submersible flow cytometer. Limnol. Oceanogr. 48(5): 1756-1765.