Hello, everybody. Welcome to Week 5 of our course here on Experimental Methods in Systems Biology. This week we'll have a few lectures on flow and mass cytometry. So a little bit about what we're going to go over first. The first lecture slides will go over flow cytometry. Which is pretty well-established technique in biology we'll go over. And at the instrumentation, how does it work. What are the different parts of the machine that allow it to work. And then talk about what you can measure, the types of things that you can do the experiments that you can do, and how you might treat yourselves to get the most information out of a flow cytometry experiment. And then we'll talk about one of the later developments in the field of cytometry called mass cytometry. Which although relatively new, it, it seems to be catching a lot of steam and will become more and more relevant, I think in the coming years. So we'll also talk about the components of a mass cytometer. How does it work? What can you measure, and do a little bit of contrasting between flow cytometry and mass cytometry. Why would you use one versus the other? And then finally we'll go into a bit of analysis of flow cytometry data. it, it can be actually quite complex. And you know, there's a large degree of kind of expert knowledge one needs in order to analyze these data properly. There's something called gating which you need to know how to apply to these data. Something called compensation which we'll touch on very briefly, but just so you know a little bit about what to do when sometimes when you have fluorophores that overlap in their spectral mission you can still analyze them quantitatively by applying compensation properly. And also a little bit about what you can do when you really want to obtain good quantitative data out of the flow cytometry experiment. There's some particular considerations which should be taken into account. So what is flow cytometry? Well, essentially, it's just a technology which allows you to characterize and sort cells as they move through a laser in a fluid stream. and, you know, this can be applied in a variety of ways. What it allows is measurements of multiple parameters, typically florescence based. On a single cell level, but within a potentially highly heterogeneous cell population. So you can get, for example, you know it, it's a very widely used technique in, in immunology, or for a clinician studying whole blood sample as you can get a lot of information about the different types of cells and blood. Look at their different morphologies by the way they scatter light. Look at different fluorescence markers. If you have stains that mark particular things in cells or on the surface of cells. And you can do this quantitatively. So there is many different types of flow cytometers. I've pasted a few here that we have at Mount Sinai and also at many other institutions. These are a lot of Becton Dickinson models. One of the leaders in the flow cytometry industry. Going from some of the older machines here, the FACS Calibur has two lasers. You can do four color imaging on that. There's a variety of other machines which have more and more lasers. You can do more and more colors. You know, that some of these machines have the capability of doing 18 colors although people rarely will get up to that amount. But capabilities there in case you really want to design a complex experiment. So there's also this mass cytometer, which we'll see a little bit later in these lectures. And also in some of the lab-based video. It's not a whole lot bigger than the flow cytometer, just works in a different way. It doesn't have lasers, but it allows you to look at way more so-called colors. I use the term color loosely there because it's really not color, but many, many different parameters you can look at on a mass cytometer. Of course, you can couple a flow cytometer to something called a cell sorter and essentially that is we'll talk a little bit more about that later, but it's just something. Which allows you to because you're working with single cells in a fluid stream. Based on what you measure in those cells, you can potentially then route them into different wells of a 96 well plate or into different tubes et cetera. So you can potentially purify cell populations in that way. It's it's a pretty pretty robust and and useful technique in many in many experimental applications. So what are exactly the components of a flow cytometer? And, and what are the, what, what are the principles that make it up and allow such a machine to work? Well the first part are the fluidics. And typically what you have, as shown here, is your sample that contains cells coming through some central tubes surrounded by what's called sheath fluid flowing this way. And the way that this flow cell is arranged, it uses hydrodynamic focusing. To make sure that all the cells are essentially in a single file line so that you predominantly have single cells then flowing past a laser one or more lasers here. So the laser shines on the cell and the cell, as the cell goes past the laser. You can collect multiple multiple parameters of the way that cell interacts with the light. The first is so-called forward scatter. So laser comes, the light gets scattered in certain angles in the forward direction. You can collect information on that based on photomultiplier tubes typically and then interfacing with an electronic apparatus which we won't describe in too much detail, but just suffice it to say that there's pretty well established ways of taking that signal and then transferring it into a digital format in the computer very, very rapidly. And you also can measure what's happening on the so-called side scatter channel. So this not only measures how the excitation light is scattered, but then the light is also passed through a series of filters, fluorescence filters. We'll talk a little bit more about that later, but based on how you filter that light and then reflect it pass a series of mirrors. You can measure fluorescence emission of any fluorophores that were in that particular cell in a number of channels here that collect different ranges of wavelengths of light. So. Kind of a boring part of flow cytometry but a somewhat important part that I'll just note here is that one of the reasons that these machines are pretty big is that they just need to hold a lot of fluid and pump it around. So a lot of it, of good flow cytometry is making sure you have proper liquid management, that you have plenty of sheath fluid. This fluid that makes sure, make sure that the cells are actually being aligned in a in a nice line. And of course somewhere where all your waste goes. As the flow cell puts all of your, all of your cells from your sample into this single file line. It comes down to what's called this interrogation point where cells come in contact with a laser beam. And the first thing that is being interrogated is how the cells interact with excitation light in terms of forward scatter. So that is indicative often of cell size. How big are the cells that are passing by your laser? And also side scatter. So how is the excitation light hitting your cell and then being reflected at a 90 degree angle. The side scatter is often indicative of the granularity of the cells so how many organelles are in the cell? How complex is the intercellular environment? And of course then really one of the main things you want to collect in a flow cytometry experiment is fluorescence emission profiles of any fluorescent tags you might have put into the cell. and, and figuring out how much of that fluorescent probe actually go into each individual cell as they go by. That's also collected from light going through the side scatter channel as I mentioned briefly on the previous slide. So just to note here that based on all of these properties that one can measure as the cells go by the laser and the flow cytometer. You can do what's called Fluorescence Activated Cell Sorting often called FACS. And I'm not talking about you know, sending a document to somebody and you know, by a telephone line. [LAUGH] That hopefully will die soon I don't know why people still use fax machines, but anyways you'll hear a lot of people referring to flow cytometry as a fax machine and this is technically incorrect. FACS refers to the coupling of a flow cytometer to the sorting apparatus and one can do a lot of flow cytometry and never do FACS. So just you know, just so that you're, that you're being precise in the way that you talk about this instrumentation. You should, you should appreciate that point. So the way that, that works, okay, you have your flow cell you know, aligning your cells with the laser beam. And then based on what's going on in your different detectors, either forward scatter, side scatter or the different fluorescent signals that are coming out of different cells. You can decide whether you want to put a cell in different tubes for sorting later on. So you know, it, it's very useful, say for example if you have a a tag for green fluorescent protein and, but not all of the cells in your population are expressing the green fluorescent protein. But you're interested in getting a pure population of cells that only expressed a green fluorescent protein. So you can prepare those for flow cytometry, put them in and then based on whether each cell is GFP positive or negative an electro, an electric charge can be applied to that cell. And then be deflected into your GFP positive or GFP negative two. And there's a lot of different things you can do with that really. You can use your imagination to explore all sorts of things and you're not only limited to two way sorting. There's machines that can do four way sorting or perhaps even more you know, different, sorting into different bins. So a little bit more details about what these light scattering properties actually mean. I mentioned in the previous sli, slide, there's two types of light scattering that are typically considered in flow cytometry. One is forward scatter. As I mentioned, that's typically indicative of cell size. So the laser beam comes, hits the cell, and the degree of scattering in the forward direction tell you a little bit about how big that cell is. The laser beam also comes and gets reflected at all angles the angle at, at 90 degrees is typically referred to as side scatter, and is more indicative of the complexity of the cellular environment in terms of. You know, say number of mitochondria or you know, basically how many organelles this cell might have etc. And it can be used to differentiate different cell types. For example, in whole blood. And it's also used to differentiate cells that are going by your laser beam as opposed to other debris that might have gotten into your sample as it's been prepared. And so that light scattering is important for many reasons, but the real power of the flow spectrometer is to be able to look at fluorescence on the single cell level quite rapidly. So, as your cells go by the laser they go through this side scatter chamber side scatter optical path it can go past a series of mirrors and filters. Okay, so what do I mean by filters? So there's, there's a couple of different types of filters that are typically used in flow cytometry as well as fluorescence microscopy. We'll talk a little bit more next week about it. So their so called long pass filters which will only let wavelengths of light through which are past it's particular filter wavelength. So in this case I'm showing a, a long pass filter which is centered around 500 nanometer light. And this is the, this is a typical plot that such a filter would have if you plotted the percentage of, of of light intensity that gets transmitted through that filter as a function of wavelength. It's centered at 500 here, and of course, there's, it's not extremely sharp. Some are sharper than others. But you can see here, here that primarily only light greater than 500 nanometers is getting through that filter. So you can also have band pass filters. So like a long pass filter, it only lets certain wavelengths of light through. But in this case it cuts off at a low end and at a high end. So, in this case you have a, a center of your filter. In this case, the same 500. But you have a bandwidth of it. In this case, it's 500 slash 50, meaning that it goes 50 across. So 25 on one side, 25 on the other. And it's really only letting through 475 to 525 nanometer light. Of course it's not you know, it's not perfect and different filters have different characteristics in terms of this percent transmittance plot as a function of wave length but in general. Arrangement of these filters in different patterns allows you to look at the emission profiles of, of various fluorophores you might have in your cell as a function of excitation of different wavelengths of lasers. Okay, and flow spectrometry, typically these behind these filters are photomultiplier tubes commonly referred to as PMTs. Kay? These allow essentially a conversion of photons of light into an electronic signal which can then be processed and converted into a format which a computer can store. And by putting different filters in front these different photomultiplier tubes you can then get information about the intensity of light at different wavelengths for each different cell that's going by your laser and in, and in quite a rapid format. And in fluorescein optometry, these are typically referred to as FL1, FL2, FL3, FL4, FL5, et cetera, et cetera if you have more channels. And you know, you'll see this, this kind of nomenclature a lot and typically it's okay. And usually they have pretty standard meanings. They're like FL1 typically refers to a, kind of a green fluorescence channel here. Although if, if one really wants to be precise about the channel that you're looking at, you won't just put FL1. You'll actually put what are the spectral characteristics of the filter that's, that's passing light through to this detector channel. So just a little bit about what is a fluorophore. So you know, in order to do flow cytometry typically you need one or more fuorophores that bind to something in your cells that's indicative of the thing you want to measure. And for that you need fluorophores there's a wide range of fluorophores available now. A very common one that's been used for many years is called FITC or fluorecein. And it's a green fluorophore. So it's excited by blue light typically by a 488 nanometer laser. Which is a very common laser line to have on flow cytometers or microscopes. So, if you shine 488 nanometer light on cells which contain this fluorophore FITC. You can see here, on the bottom the excitation spectra of FITC follows this curve here. So if we look at 488 nanometer wavelength light, come up here, we're really exciting FITC very, very well. Okay, then we can collect light over here according to the emission profile. So the emission maximum is around 430 nanometers but it can really be collected at a whole range of wavelengths in order to figure out you know, if when we excite at 488 nanometers. We get a certain intensity of FITC emission by collecting light, according to this emission spectrum over here. And we can apply this sort of logic to really a whole wide range of fluorophores that span essentially this whole ultraviolet, infrared wavelength range. So, when we're collecting fluorescence we have two different options, typically, on the flow cytometer. as, as I mentioned before, you have some electronics that, that are between the photomultiplier tube and the computer which is storing the information. And historically these were kind fixed components and you could, you could collect them either on a linear amplifier or with a logarithmic amplifier. And once you set that you really couldn't, it was very hard to change later on. With modern instruments it's not so much of a problem. This can be changed later on with, with, with more relative ease. But it's still important to understand whether the fluorescence emission you want to collect should be with a linear amplifier or with a logarithmic amplifier. And all that means is that. What you're going to do is whatever that fluorescence emission intensity is coming into the photomultiplier tube, you're actually going to pass it in an electric signal to the computer that's at a linearly proportional to that emission, or you're going to amplify it logarithmically. so, just some general considerations. The forward and side scatter channels are typically collected on a linear scale. Just kind of a standard. Whereas florescence emission is typically displayed on a log scale. And the reason for this is that usually with fluorescence emission you're, you're concerned with kind of large scale changes. Is is the population positive or negative for a particular fluorophore? And you can kind of see the difference here that if you were to collect a fluorescent emission, this particular experiment here is trying to identify the levels of a protein called CD8 with a with a, with a FITC fluorophore tag. And if you collect it on a linear scale and you look at the histogram of events. So, this is essentially the number of cells. And then the intensity of FITC that was in. That number of cells across a whole population. And you can see here that, you know, there's a whole lot of cells here which have very low FITC levels. And then it just, it, it's very hard to tell what's going on here at higher levels of FITC emission. But if you put that on a log scale you can very clearly see here that there's two populations of, of cells in this experiment. You have these, so called, FITC negative cells, which are, and displaying a very low FITC fluorescence. And then you have these FITC positive cells, which are displaying a very high level of fluorescence. About, you know, two orders, two or more orders of magnitude higher fluorescence emission than FITC. So this is, you know, really why, often we collect fluorescence emission on the log scale in flow cytometry. But there are ex, some exceptions for that. One very big one is when you're looking at DNA content quantitatively. Like, for example cell cycle analysis is one very common application of flow cytometry in order to determine what fraction of cells in a population are in different phases of the cell cycle. In this case your DNA content over the cell cycle was really only varying twofold. As opposed to over here, you know, you have a signal that's varying say a 100 fold or more. when, when your signal is only varying two-fold it's very difficult to see that difference on a log scale. So the standard for, if you're looking at DNA content quantitatively, this should be examined on a linear scale as opposed to a log scale.
