Hello, everybody welcome to week six of our Coursera course in Experimental Methods in Systems Biology. This we're going to be talking about life cell imaging and fluorescence microscopy. So just an outline of the talks for the lecture portion of, of this week. First we're going to have two components of fluorescence microscopy, first a little bit about the implementation and in particular the instrumentation that also makes fluorescence microscopy compatible with live cell imaging over a time course. And then we are going to talk about the types of different fluorescent probes that you, can use to actually monitor things in life cells in real time. You can actually report on many different things, that are going on in cells in that way. And then, we are going to spend some time going over how to be quantitative about all of this. I just want to make a few notes up front about what we are or are not going to cover. a, a, pretty big revolution in, fluorescence microscopy that's been going on to so called super revolution microscopy which lets you Image past the so-called diffraction limit of normal fluorescence microscopy. So, you know, because light has a wavelength and visible light, the wave lengths between say around 400 to 700 nanometers, you can't really see things much smaller than that. But people have developed techniques a variety of techniques that actually allow you to get past this diffraction limit and see things that are smaller. But is not used every often is a life cell imaging, so we are not going to cover it here but it's it's actually a nice thing to do a little bit more research on if you're interested. Lots of people do single-molecule imaging so instead of, you know, in, in this course we're going to be talking about populations of many, many molecules that we're looking at the fluorescence of. You know, there's lots of things that you can also do by looking at single the fluorescence of single molecules but we're not going to have time to go into it in detail on this course. Another technique that people do in live cells, is something called fluorescence recovery after photobleaching called FRAP. a, and this is a technique where you shine very bright light onto a, onto your cell, your sample. Essentially bleaching out, the fluorophore so it's no longer fluorescent. And then watching how that fluorescence recovers back into the spot that you bleached out. And this is typically by diffusion although that could be active transport as well. so, you know, it, it's a very useful technique but we're not going to go into it, in this course. So, first just a little about, about the instrumentation that you need to do fluorescence microscopy in live cells. So, of course, the first thing we need to understand, or what are the general components of the fluorescence microscope itself. And this is illustrated here on the left it's pretty basic, but, diagram, but it, you know, really hits the the main parts of, of the microscope. The first thing, of course is a, is a light source. You need to be able to excite the fluorescence that are in your sample. So there are several types of light sources one could have. Lasers of course, most people know what lasers are. They have very defined wavelengths, usually just like you know, one nanometer, maybe even less. There,s been recently developed a so-called white laser which actually contains a whole variety of wavelengths, but you can select very finely which wavelength you want, that's kind of neat thing that's becoming more and more available. There is a broad coverage lamps, so xenon and mercury lamps that just emit a whole range of wavelengths of light. And there is LEDs which can have defined wavelengths, but not as defined as a laser usually it has a broader spectrum like on the 20s or 30s of nanometer. Of wavelengths, and like white lasers you can also have white LEDs that will emit a whole wide range of wavelengths of light that you can then select. So how do you select what wavelengths of light you want, coming from your excitation source? That is accomplished with a so called excitation filter. We talked a little bit about filters and the lectures. But essentially it's, it's just a, a tool that only allows certain wavelengths of light through. And they come in two varieties a bandpass filter, which lets only a defined range. With a lower and upper cutoff of wavelengths of light or a longpass filter which will let all wavelengths of light through past, above a certain wavelength cutoff. So once you pass your excitation light through this excitation filter, now you're only selecting for a certain range of wavelengths which need to be routed to your specimen. And the way that that's done is, with a so-called dichroic mirror. So when the excitation light this dichroic mirror is at a 45 degree angle to the excitation light. And typically the way that it works is that it will reflect the excitation in the direction of the objective and the specimen on the stage. But then it will let through the emission light that's coming back from your specimen. It is kind of a interesting thing, but it's a, you know, a very common component in all of these, types of fluorescence microscopy experiments. So once that light gets reflected, the excitation light gets reflected of the dichroic mirror. It goes through the objective to your specimen, whatever fluorophores are in your specimen will get excited will emit. the, their fluorescence emission. And, then, it goes back through the dichroic mirror, but as I explained the dichroic mirror lets through this emission light. And then it goes through a so-called emission filter, which is similar to the excitation filter, it can be a bandpass or a longpass. But it's just designed to now, select on a different set of wave lengths. So then, the light that's going through to the detector is very well defined as, you know, however you according to however you set up those filters. Okay, and a just a note about these how these filters are arranged in the microscope typically, you have these so called filter cubes, which contains all three of these, of these things. The emission filter, the dichroic mirror and the excitation, filter in these defined angles. All right, so you can pop in and pop out these filter cubes, and sometimes they're they're contained in separate regions of a microscope, and this can be useful if you want some flexibility or if you want some additional speed. But you know, there's a whole wide range of arrangements that these filters can have. Okay? so, of course, a very important part of the microscope is the objective. as, as we all probably already know, this is the lens that allows the magnification of the image of the specimen. So depending on the so-called X value of the objective, you get that fold magnification of your image. Typical objectives are 4X, 10X, 20X, 40X 60X, 100X and these objectives can come in different varieties of they way they need to interface with your sample. So, the easiest is an air objective where you don't need anything. Many of the more high quality objectives require oil emursions. So, you have to put oil on the objective, in order to to make it work properly, and then the oil is the layer between the objective and your specimen. but, you know, there, there's, there's a couple of different options there. And of course, there's the stage this is where the specimen sits in a simple microscope it's just a fixed stage that can't be moved around. It can also be movable by hand on a slightly more advance microscope. So you can move around what part of the sample you're looking at, or in even more advanced microscopes it can be motorized, so you can control where the stage with a computer and that enable you to do for example multi position imaging, so where your looking at multiple parts of your sample over the same time course. And one of the most pieces of course, the ocular, and that routes the light from the florescence admission into a detector which, the simple simplest of which is your eye. You just look at what's what's coming out. But typically, this is some sort of advanced camera some of which are you know, some of the more advanced models are cooled CCD cameras, but there's more advanced scientific-grade CMOS cameras coming out that seem to be quite good. And so this schematic here that I found is actually for an upright microscope which means that the objective is above your specimen. But really for live cell imaging typically you're, you're using an inverted microscope, which means that the objective is below the specimen. So with all that being said about the basic components of a fluorescence microscope you know, there, there's several additions that need to be made to that, in order to make it compatible with live-cell imaging. And of course, the most important part about that, it's just the ability to maintain the cells in a healthy state. Meaning that you have environmental control on the microscope. That means that you're keeping the the media or the solution that the cells are living in at the correct pH, with the correct balance of ions or osmotic pressures, at the proper temperature. Most mammalian cells like to be at 37 degrees Celsius, so you need the ability to heat up and then keep that temperature. And most cell cultures need a particular set of atmospheric conditions, for example, most cell growth medias for mammalian cell cultures rely on a carbonate buffer to keep the pH. Which means that you need typically a 5% carbon dioxide mixture in the atmosphere. also, humidity if you don't want your culture media to evaporate, you need to have a humid atmosphere in there. If the media starts to evaporate, you change your pH, you're going to change osmora, osmo osmolarity and you're going to make the cells unhealthy. Some experiments are done under conditions of hypoxia or normoxia, and so you would need the ability to alter that as well. So this is what a typical environmental chamber might look like where here on left. Some of the setups just take a typical microscope, like this, this one here and they just enclose it with this kind of plexiglass enclosure that's, usually has to be custom fit around your microscope. Has a couple of doors that you could you know, to which you could take in and put out your sample. But then input into this chamber are, you know, heated streams of air or humidified air that have been mixed with the proper amount of carbon dioxide or other sorts of gases that you might need. And other, more simple setup is this right here where it's kind of zoomed in on the objective. For this is an inverted microscope, so you see the objective on the bottom ,and the light kind of coming through that way. And then the cells on the stage are just enclosed in a very small environmental chamber here, where usually you know, the proper humidified C-O two mixture is kind of fed into something like, like this here. So you have a nice atmosphere in there, and then there's some sort of heating heating element around the outside that keeps the proper temperature. And of course another important aspect of live cell imaging is making sure that the microscope doesn't move. And this is really non-trivial, because most buildings have slight vibrations or slight movement in them, which can really become visible when you're doing you know, looking at things at high magnification and really wanting to keep that same vertical position, so a, a z position keep the focal plane, but also the same X Y position. And so usually you'll see micro, microscopes set up on these kind of specialized tables that have that have special dampening elements in them that isolate them from, from from vibrations or they have, you know, sitting on special pads. Special types of rubber that absorb vibrations, or keep them from moving too much when these normal sorts of things happen. Okay, so that's really all I wanted to introduce in terms of the components of the microscope. The next series of slides will talk about the types of imaging that are, that one can do in live cells.
