Flow cytometry?

Question

Can someone please explain in detail how flow cytometry works? Can someone please explain how the machine detects the antibodies and fluoresence?

How does it count the cells? Why is it used? How does it work?

Answers from experts in this area would be appreciated...Thanks!

Answer 1

The above explanation mentions a number of the basics quite well. I'll see if I can add anything, without writing a book. I have done flow cytometry for about 20 years, and currently teach the flow cytometry and histology segments of a medical school course entitled "Methods in Pathobiology".

A flow cytometer is really a combination of three distinct systems - a fluidics system with tubes, valves, pressurized containers, etc; an optics system with prisms, lenses, filters, mirrors, lasers and photodetectors; and a computer system to collect, analyse and visualize the data. Essentially, a flow cytometer analyses particles moving single file in a fluid stream, by the extent and direction of reflected light, and the emission of fluorescent light, when the particles pass through a laser beam that intersects the fluid stream at a perpendicular angle. The speed of the fluid stream is quite fast, about 15 meters per second, which enables several thousand particles per second to be analysed. (From here on I'll say "cells" rather than "particles", since cells are the usual "particles" being analysed. However, the cytometer can also analyse other particles, like bacteria, isolated cell nuclei, isolated organelles like mitochondria; pollen grains, etc.) The analysis includes several parameters, typically cell size, degree of complexity (either internal or surface complexity), and fluorescence emission. Various cytometers can analyse 2, 3, 4 or more different wavebands of fluorescence simultaneously.

After the laser beam passes through the fluid stream, it approaches a photoreceptor diode, but it is blocked from entering that diode by a thin bar across the center of the collection lens. When a cell passes through the laser beam, light is deflected off the surface of the cell sufficiently to bypass the blocking bar and enter the lens. Larger cells deflect light at a greater angle, so the light deflected from larger cells hits the lens closer to its periphery. A photoreceptor converts these light impulses to electronic signals and sends them to the computer, which analyses the signals and creates a display on the monitor. The display can be viewed several ways, such as dot plots, histograms, contour plots. etc.

Another light detector is situated behind the fluid stream, at a position 90 degrees to both the stream and the laser beam. The only light that enters this lens is therefore that which is either reflected or emitted from the cell in a direction perpendicular to its direction of travel. This light is from two sources.

First, if the interior of the cell has many organelles or intracytoplasmic granules, these small objects will scatter the laser light in all directions, and some of that light will enter the perpendicular detector. Also, if a cell is complex on its surface, with many microvilli or other irregularities, this too will scatter the laser light. This light which enters the receptor lens is directed by additional lenses and prisms into a photomultiplier tube or PMT, which not only converts the light pulse into an electronic signal but also amplifies its intensity. This signal is ssent to the computer, is analysed, and is displayed as an indication of relative cell complexity.

If the cell contains any fluorescent compounds, the laser will stimulate these compounds and cause them to fluoresce. This fluorescent emission likewise emanates from the cell in all directions, so some of it enters the perpendicular detector lens. The light entering this lens is subdivided by prisms and various types of short-pass and long-pass filters and dichroic mirrors, eventually arriving at several different PMT's. The light is separated into its respective colors, so that only red light reaches one PMT, only green light reaches another PMT, etc. These PMT's treat the light the same way as the other PMT's, converting to an electronic signal, amplifying that signal, and sending it to the computer.

Suppose I have a culture containing three different linds of cells, and I wish to know whether any of these cells contain either of two different proteins. Any given cell may contain either protein, or neither one, or both. So I make or purchase an antibody made against one of the proteins, and label that antibody with a green-fluorescing compound. Then I get an antibody against the other protein, and label it with a red-fluorescing compound. I treat the cell culture with both antibodies, which bind to their respective proteins, taking with them their respective fluorescent markers. As the cells pass through the cytometer, the laser beam intercepts each cell individually, and within a couple of nanoseconds, the computer receives all the data regarding that cell's relative size, relative complexity, red fluorescence, and green fluorescence, and updates the monitor display accordingly. Cells which contain both proteins fluoresce red and green, while cells containing just one protein fluoresce one color, and cells without either protein do not fluoresce either red or green. Since this entire process can occur several thousand times per second, the monitor shows a continuously updated display of the data, in real time. Since we then have the size, complexity, red fluorescence and green fluorescent data for every cell, we can quickly do statistical analyses involving any combination of these parameters. For example, we can ask "what percentage of the larger cells with minimum complexity demonstrate green fluorescence but not red fluorescence?".

Flow cytometry enables us to acquire huge amounts of data very quickly (compared to the older method of sitting at a fluorescence microscope, visually counting red and green cells), which provides must faster and more accurate analysis of cellular characteristics.

Hope that helps. I know this account is pretty sketchy, but this is really quite a complex subject, and large volumes have been written about it.

Answer 2

In detail, no. As an expert, no. But, as someone that only on friday used flow cytometry I'll give it a go.

Flow cytometry passes particles through a tube specialised so that the particles go through in an order- one after another; this way only one particle can pass through a cross sectional area of the tube at one time. A fine laser is aimed at this thin cross sectional area of the tube. When the cross sectional area has just water passing through, the laser light passes through unhindered and is recorded. However, if a particle (such as a cell) passes through, it disrupts the laser light. The laser light is scattered either at a small angle (forward scattering), or at a perpendicular angle (side scattering). Different receptors detect forward and side scattering, and from this information detail on the size of the cell, and how granular it is can be deduced.

Counting the number of cells in a sample is easy: all the sample is passed through the tube, and the number of times the laser light is interupted, by a cell, indicates the number of cells in a sample.

Sometimes, such as for detecting antibodies, flourescent labels are used. These flourescent labels absorb light at a specific frequency (the frequency of the penetrating laser light), then moments later re-emmit it at a lower frequency. Detectors are used, which destinguish between scattared light and re-emmited lower frequency light. Labelling antibodies with such a flourescent molecule, allows you to detect cells displaying the antibody. It is then possible to find the frequency of cells, from the whole population, that express the antibody.

Answer 3

In normal English (versus the incomprehensible scientificese of the previous person) flow cytometry is the ablity to add fluorescent dyes that will stain some cells and not others. As they go past a laser the machine will detect the amount of fluorescence and direct that cell into a separate container. Thus you can take a solution of two or more cells and isolate a single type of cell (if you have to abilty to specifically label the cell of interest).