Flow cytometry in aquatic science

Julie Hall
Andrea Cumming

 

How many algal cells will mussels eat? What proportion of plankton in a sample will die if exposed to a toxic substance? To answer these kinds of questions, researchers have traditionally had to view tiny subsamples under a microscope and count individual cells. This is still the only practical option for some kinds of research. However, a high-tech method in use at NIWA – flow cytometry – is making the task much easier and more accurate in many projects.

What is flow cytometry?

Flow cytometry is the measurement of characteristics of single cells suspended in solution. A beam of laser light is focused on the moving cells and light is scattered by and emitted from the cells. The information is picked up on detectors and converted into a form suitable for electronic storage and analysis. Various features of cells can be used to identify different populations or types of cells. NIWA’s flow cytometer counts cells at a rate of 100 to 1800 per second and measures five different features.

The amount of laser light scattered forward gives us an estimate of the size of the cell.

The amount of laser light scattered sideways at 90 degrees gives us a measure of the internal complexity of the cell.

The other three features of cells measured by the flow cytometer depend on the fluorescence emitted when the laser light excites fluorescent material in the cells and causes emission of light of a certain wavelength. The fluorescence of a cell may come from natural pigments or from added stains. The instrument can detect fluorescence at three different wavelengths – 530, 585 and 670 nm – on its three fluorescence channels: FL1, FL2 and FL3.

Identifying, counting and sorting

We can make similar measurements on a wide range of cell types which have naturally fluorescing pigments, like chlorophyll a, or we can add fluorescent stains to identify cellular features and use them to identify different populations of cells. In the example above right, the presence of algae in the sample was first confirmed by the presence of chlorophyll a fluorescence. Then different algal groups were identified on the basis of other cell characteristics measured with the flow cytometer.

A common device used with the cytometer is also illustrated in the figure. To enable an accurate cell count we add a known number of fluorescent beads (the red population) to the sample. This allows us to calculate the volume of sample analysed and hence calculate the number of cells per ml of sample. The difference in the spread of the pink and blue populations compared to bead population is due to natural variation in the cells compared to the beads which are much more uniform in their characteristics.

The advantages of flow cytometry over traditional microscope counts of algae are that sample preparation is quicker, counting time is reduced from 10–20 minutes per sample to about 2 minutes per sample, and we are able to count many more cells (5000–10,000 by flow cytometry compared to 400 for microsope counts), so counts are much more accurate.

NIWA’s flow cytometer can also sort cells from identified populations. This allows us to identify a population of cells and then collect only this type of cell. Isolated cells can be checked by microscopy to make sure they are identified correctly by the flow cytometer.

Apart from using flow cytometry to count bacteria and algal cells (in both marine and freshwater samples), the instrument can be used to investigate the metabolic activity (or health) of cells to measure the impact of toxic substances. We have also used the flow cytometer to quantify the amount of DNA in cells.

Julie Hall and Andrea Cumming are based at NIWA in Hamilton.

NB: the PDF of this article contains additional illustrations and related information.