Flow cytometers

Screen shot 2013-04-07 at 11.46.23 AMNatsuko Porcino

While others may have to deal with cumbersome, archaic machines like light microscopes, part of my job here is to use a Flow Cytometer (FCM for short) – essentially a giant machine that send cells single file through a laser and quantifies them. Sound like something out of the future? I thought so, too, until I learned about them today.

It is a really neat piece of technology, but FHL doesn’t have one at the moment. So as everyone was settling in to work on their analyses today after sampling, I had another task – hop on a ferry to Anacortes and trek across a tide-permitting, snail-y strip of beach to Western Washington University’s Shannon Point Marine Center to learn how these things worked.

The path was narrow in parts when the tide came in, making it difficult to progress without using tree branches as leverage to hop across the water…
The dangers of being a scientist!

The FCM is a machine that counts, sorts, and sizes cells using lasers and a fluorophore’s natural ability to absorb light and then emit it at a slightly different wavelength. When these particles are hit with light, they are sent from their resting state to an excited state with higher energy. As the fluorophore returns to its resting state, it emits energy in the form of a longer wavelength. The FCM captures and records this data as graphs that you can look at and analyze.

the internal mechanism of a FCM

the internal mechanism of a FCM

FCMs can use both autofluorescence and dyes – as long as your sample has something can act as a fluorophore (ie an indicator), the FCM can use it! This attribute makes these machines incredibly versatile in their uses. Using different fluorophores that attach to different things, FCMs can do anything from sort cells by ploidy (using a nucleotide indicator) to find out enzyme production (using a protein indicator).

In our experiment, we will mostly be using the FCM to measure chlorophyll and phycoerythrin (A pigment that takes in a slightly different wavelength than chlorophyll. It’s found in all cyanobacteria). By measuring these levels against cell size, we hope to determine the different populations of phytoplankton present in our mesocosm bags, and to see if these populations change over time.

FCM diagram courtesy of MUSC Department of Regenerative Medicine and Cell Biology

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