How do biologists take a cellular census? Lasers.

This is the first in a series of posts describing techniques that scientists use regularly. Find more under the “Tools and Techniques” tab above. 

Tools and Techniques: FACS (Fluorescence Activated Cell Sorting)

It’s 9am, and I am getting ready to play with lasers. (Yeah, occasionally I have to pinch myself as a reminder that this is a real-life job).

This is the first time I’ve done this particular experiment, and I am a little nervous. Trying out a new technique is a bit like learning a combination in kickboxing class, or baking an especially complicated cake. Before any of the fun begins, there’s some legwork that needs to be done: hours researching protocols, procuring supplies, mentally running through the whole thing to try to anticipate roadblocks. The stakes are high! Failure to anticipate an experimental error might render all your hard work, along with expensive supplies, useless.

Today, I’m going to use lasers to count the number of cells within an organ. Organs are like cellular cities. They’re comprised of a hodgepodge of characters (individual cells), each belonging to a particular group responsible for a specific task. Some cells gobble up bacterial invaders, others pump out chemical signals in order to broadcast information across the tissue, and others store energy in the form of fat droplets. It is the exquisite cooperation between these “cell types” that allow an organ (and by extension, an organism) to survive.

14971
An image of the eclectic panoply of cells that comprise the brain of a mouse. Each color marks a particular cell type. Image courtesy of Margaret Davis and the Cell Image Library.

To fully understand how an organ functions, one might want to take a cellular census. However, historically, it’s been tricky to get a good sense of how many types of cells make up a given organ, and how many cells belong to each type. In the past, scientists relied on a kind of photography to take pictures of tissues, and then make guesses about who is doing what. This is a bit like trying to distinguish the job descriptions of “lawyer” and “investment banker” from an aerial shot of New York City – how do you know whether or not the small dot wearing a suit you’ve captured in your picture is on their way to sift through indictments or stock portfolios? Likewise, how do you know if the cells you’ve captured in your image are stretched long and thin because they’re reaching out to grab a pathogen, or in mid-race across a tissue to attend to a wound?

(No joke about the racing – check out this beautiful video of cells in a developing chicken migrating together, published in BioRxiv this week. They’ll go on to build the nervous system and head skeleton.)   

It would be better if we could meet our cells on the street and ask each one what they’re up to, and even better if we could keep track of what each cell answers in real time.

Enter the lasers.

The technique is called Fluorescence Activated Cell Sorting (or FACS, for short). In brief, it involves separating every cell within a tissue, lining them up neatly in a stream of liquid, and blasting them with lasers. The photons from the laser beam behave differently when they’re fired into a cell depending on the size of the cell, how wiggly its membrane is, and whether or not it has been stained with glowing dyes. As the assembly-line of cells marches through the machine, each one is illuminated by the laser, and the differences in laser light are recorded by a very powerful computer. It’s as if one stood at a turnstile during NYC rush hour and scribbled down the occupations of thousands and thousands of commuters.

FACS
Cells flow through a stream of liquid. Individually, they’re bombarded by light from a laser. The light is collected in a detector, and relayed to a powerful computer. Laser light reflects off of cells of particular sizes and shapes differently, allowing us to count individual populations.

My experiment begins with a morning of delicate dissections. I carefully excise the pad of tissue destined to become a fruit fly wing from hundreds of fruit fly larvae (I’ll spare you the goopy and gory details of this process). Once I have a collection of developing wings, I add a chemical called trypsin (the same chemical that we use to digest food in our stomachs!)that chews up the connective proteins between the cells, leaving each individual cell swimming alone in a sea of media. Then, I carefully bring my sample up four flights of stairs, to a brightly lit room with a machine the size of a small car humming in its center. I set the sample within the belly of this machine, and insert a long, thin straw into the liquid containing my cells. I turn on the computer, make sure the lasers are working well and that the machine is sucking up a healthy stream of cells in single-file, and ask the software to start counting.

Very little feels as wonderful as an experiment working the first time you try it, so when a reassuring stream of data materializes on my screen, I can’t help but rush to show off the success to the technician whose job it is to care for the machine and help newbies like me. After a career of experiments much more complicated than the one I’ve just pulled off, he gives me a very generous high-five, and then returns to more important tasks.

But I’m giddy. I will use this data to get a better picture of what’s going on as cells within the wing tissue divide and organize themselves into a massively complex organ. In the future, I will go beyond simply counting. I will sort and separate the cells based on size or shape or identifying chemical markers, and then perform even more illuminating experiments on particular populations – like sequencing their genomes or profiling the proteins displayed on their surfaces.

It’s worth a moment of appreciation for the thousands and thousands of hours that went into crafting these types of cell sorters. Getting human beings to behave in single file is challenging enough, and getting cells hundreds of times smaller than the period at the end of this sentence to do the same is a near-miraculous feat. That’s not to mention the precision with which all the lasers have to be aligned, and the clip at which the computer must be working. Tool building is a hugely important part of science, and thanks to the herculean effort of the engineers and biologists that have come before me, I’m able to ask some exciting questions and get some exciting data that would have been unthinkable in the recent past!

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s