Monday, August 22, 2011

Flow Cytometry In A Nutshell

FACS was used in the trial treatments of leukemia last Saturday's post.
Fluorescence-activated cell sorting analysis, or FACS, uses a flow cytometer to separate individual cells in a heterogenous suspension based on epitope type. Fluorochrome-labeled antibodies are added to the cell sample. The antibodies bind to specific epitopes on or within the cell. The fluidics system delivers a stream of cells or particles one at a time through an interrogation point, where it passes through a laser. A cell traveling through the laser beam scatters the beam’s light forwards and sideways as a function of its size and granularity, respectively. When the laser beam strikes cells labeled with fluorescent-labeled antibodies, the fluorescent dye becomes excited and fluoresces at a unique wavelength. The intensity of the scattered and fluorescent light is collected and filtered by the optics system, recorded by the detector which translates the light into a quantifiable electrical impulse that can be represented graphically as a dot plot or a histogram.

We can use different fluorochromes at the same time, as long as their emission peaks are far enough apart for us to easily distinguish. A peripheral computer can instantaneously analyze the forward and side scatter light and fluorescence to identify the characteristics of individual cells and separate them into different subpopulations by charging each droplet with either a negative or positive charge, depending on the intensity and wavelength of fluorescence, as they leave the stream. The droplet is deflected either to the right or left by charged electrodes into one of three sample tubes. Intensity and wavelength of fluorescence and be plotted in a two dimensional box plot, where subpopulations in the sample can be distinguished by looking at two parameters. 

2D results with two different parameters makes visualizing cell populations so easy!
A histogram measuring the frequency of celled labeled with antibody A is plotted on the y-axis of the two-parameter box plot, and another histogram measuring the frequency of cells labeled with antibody B is plotted on the x-axis. The box plot in this example shows two subpopulations, distinguished by the intensity of fluorescence f protein expression.

28 scientific replies:

GMSoccerPicks said...

I should understand this better. I feel kinda bad being so ignorant regarding this stuff considering im directly related to a medicine nobel prize winner (hint #1, he won it for an antibodies related work. hint #2, he won it in 1984) hahaha little brag there, i know.

Natural One said...

The only thing I know about fluorescence is that they're great for plant growth. lol "Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength.[1] It is a form of luminescence."

Sub-Radar-Mike said...

So yet another post that caused me to have 32 google/wiki tabs open at once... knowledge avalanche!

Timothy Bowen said...

So much science!

Gareth Thomas said...

As with Sub-Radar-Mike, these articles always send me on a wiki hunt.

Inverse said...

I saw this (or something similar) on House the other day :D

Mekkor said...

i had to translate like half of the words to understand that but in the end i got it (at least i think i got it) ^^

Kid Shuffle said...

your posts blow me away every time i read them! this is neat

MUFCfollowers said...

Such detail and professionalism in your presentation. Is this your Doctorate work?!

That Bastard From Bellingham said...

Yay, I haven't a clue as to what you're saying heeere! :D

No, I understand it (inasmuch as a layman can), but what I'd like to know is what does this accomplish? Is this a way to test particular cells or graph 'em or somethin'?

Sketchar said...

It would be interesting to use light with wavelengths matching the resonant wavelength of cancer cells to move them to some where else, or destroy them entirely, that would also cause no tissue damage.

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