Okay. So now that we understand a bit about the components of a fluorescence microscope and how you can outfit a fluorescence microscope to do live cell imaging. That is to keep the cells happy on the microscope. We're going to talk about the types of imaging that one can do. I like this this picture here because it kind of concisely says in a pictorial format, what are the different common types of imaging that one does, and how does it look in the context of a cell? So we're going to go over all of these. We'll go over TIRF microscopy, which allow you to look at a very thin layer at the bottom of a cell. We'll do different types of confocal microscopy. Laser scanning or spinning disk confocal microscopy we'll, we'll touch on. And we'll also talk a bit about wide field fluorescence microscopy which is the easiest, and probably the most common used technique. You know, in thinking about all these different techniques that are available it, it's really nice to have some sort of logic as to what techniques are, are appropriate, or applicable, depending on your particular question or system of interest. And this publication here has, has provided a very nice flowchart diagram of this kind of a logic. So first of all, you know, if you're interested in fluorescence most of the time you are, but if you're not then you can usually rely on transmitted light microscopy here abbreviated TLM. We'll, I'll just briefly mention about that in a slide later. But most cases we are interested in fluorescence to look at things going on in cells. And, if you're interested in phenomena that are going on at the cell membrane, in a very small slice close to that membrane, typically around a 100 nanometers from the basal surface of the cell, then you can use a technique called TIRF microscopy to look at that. If you're not interested in only those events, you need something different. If you have a very thick sample, say like, something like that, you have to use something called multi photon microscopy, which allows you to, to go deeper into tissues than things that I'm going to talk about here. so, we, we won't touch on this. But you, you should just be aware that there is a technique called multiphoton microscopy that allows you to look at samples that much thicker than a typical mammalial cell culture. So most of the techniques that I'm going to be talking about not only here but throughout this course just deal with cell cultures and most of these are thinner than about 20 microns. So and if they are, if you have kind of cell monolayers then a lot of times. Wide field fluorescence microscopy. WFM is an appropriate technique in that it allows you to see a lot and it's relatively simple to implement. We're actually going to be doing our lab based video on wide field microscopy applied to livescore imaging. If it's not thinner, if you if you need to do something different, maybe if you need to, a little bit more, axial resolution or, you know, you don't want to image the whole cell, you want to image something in the slice of cells, then you need to ask yourself, do you need to look at it very, very quickly or not? And if you don't you can usually use a technique called confocal laser scanning microscopy. It's just one way of obtaining optical sections instead of looking at a whole you know, a whole stack of light coming out of your cell you can really just look at one slice in that cell using this technique. Using any confocal technique. If you need to look at things very quickly then you need to ask yourself, do you need to look at them quickly and take many, many slices or is it, is it sufficient to just maybe look at one line out of the slice. So. Depending on the answer to that question, you can, again, go back to confocal laser scanning microscopy. Or if you need to look at things very quickly, and look at many, many slices within a sample, a so-called Z-stack, because you're going up in the Z direction and taking many images. Then you can use a tech, technique called spinning disk confocal microscopy. Which in many cases is, is considered as better suited for live cell imaging as, as compared to confocal laser scanning. All, although both are really compatible with it. So just very briefly, I mentioned transmitted light microscopy as something if you don't really care about fluorescence. But it can also be useful and be used in conjunction with fluorescence microscopy. and, and give you additional information you can't see such as fine details of cell morphology or. You know, really allow you to track cell movement or small processes that are, that are developing on the cells etcetera. There's a couple different types that are commonly used. Brightfield is the easiest one. But the, the resulting images can sometimes be very difficult to see the cells on. This is actually a very, this is a very good one here. It's not usually so easy to see the cells in pure Brightfield. Two of the more advanced methods are called DIC or Phase Contrast. Phase contrast is. The simpler of the two just includes putting some rings, some so-called phase rings into your light path, which really helps you to see a lot more differences than you would see in brightfield and, and identify cells better. But it does, because you're putting these phase contrast rings into the light path, you actually lose a lot of light. So this, this can be quite detrimental when you're dealing with fluorescence microscopy, especially if you're, if you're worried about things like signal to noise or kind of week fluorophores it's really something that, that is often just a killer. So. You know, even though it is compatible you know, you, these types of issues need to be thought through when you are trying to combine transmitted light with fluorescence microscopy. So the first technique I am going to be talking about in detail is called TIRF or Total Internal Reflection Microscopy. And as I mentioned before, this is a technique that you can use when you're interested in looking at only about the bottom 100 nanometers above the cover slip. So, the, the very bottom of the cell that's attached to the surface. So naturally, this is good for monitoring events on the plasma membrane, such as cell adhesion processes or. Sometimes signaling events that happen on the, on the platform membrane. And the way that it works is just by changing the angle of the laser of an excitation laser. So when you change the angle of tha excitation laser as, as illustrated here. If you're at a so-called critical angle, the laser ends up generating what's called an evan, evanescent wave. Which is, what just means that you're illuminating only a very small portion of your sample. Close to where that, that light is being essentially reflected around in this area. so, it's localized excitation, which means that you're really only exciting the floor force that are within this evanef, evanescent wave region. So that's essentially what allows you to then look at events that are very close to that cell surface. So this is a very useful and pretty widely used technique. Okay. The next technique I'll be explaining a little bit about is laser scanning confocal microscopy and this has been around since the 50's. Here's a picture of the original patent for laser scanning confocal microscopy. It's quite complicated looking here but the principle really isn't isn't that difficult. It's essentially an apparatus that uses lots of angles and mirrors and optics. That allows illumination of only one spot at a time. So, if you have an excitation source that's reflected off back. Kind of a curved back mirror here. It's going to go through kind of a, a, a point of expingement here, and then expand back out again, go through your objective, hit a certain spot on your sample. And then the emission light is reflected back at a certain angle, and then by using. The series of mirrors here. You can then reflect that light, such that it passes through only one certain pinhole point, so you can then reject light that's coming from all places that are not that point you wanted, and only let in light from that point that you wanted. And that, that, this is a so-called pinhole. And then the detectors behind this pen hole. So by changing the position of this pen hole, by changing sometimes the size of the pen hole, by changing very rapidly the the angles of these mirrors, you can do so called rastoring. So, you can just go. Kind of across your sample in both the X and Y direction. And then build up a series of points that give rise to a a so-called confocal section which is only the emission light that's coming from a certain Z plane of your sample. And it's filtering out the out of focus out of plane light. So you know, it's really a nice technology to, to get better images of what's going on only in certain sections of cells. but, it leads to large losses of emission light, of course. Because, you know, you're only. Letting in light that's, that's from really one small point. And you're getting rid, rid of a lot of the other light that's not coming there. It really requires quite, can require a quite strong excitation. And you'll also need to move this across. Every point in the image that you want to that you want to look at. Things like photo bleaching where youre fluorophores you know go to a state where they cannot be excited anymore so kind of an irreversible loss. Or phototoxicity where. Which is related to things like free radical production that are, that's just damaging cellular components leading to, kind of, a stress responses of cells. And can lead to things like cell death. now, these things are much more of a concern with laser scanning approach. So another type of confocal microscopy is a so-called spinning disk. And this is essentially using the same approach as the laser scanning confocal, except instead of one pinhole, you have series of pinholes on, on a ring, that are arranged kind of in this pattern. And then this disc just spins around very, very fast. So as the excitation light passes into these pinholes. And as this disc spins around, you're, you're kind of. Doing the same thing that the confocal, that the laser scanning confocal does, but in parallel and very rapidly, much more rapidly than the than the la, laser scanning machine. And one thing that makes a spinning disk even better for live cell imaging is the so-called microlens array, which is a component of the Yokogawa spinning disk unit. Where the excitation light is actually you, there normally can be lots of losses of the excitation light, unless you use this micro lens array to kind of to focus this excitation light and really send more of it through the objective to your sample. so, just to summarize, the spinning disc confocal allows you to acquire images a bit quicker and in a, in a way that's more light efficient than the laser scanning approach, which makes it more amenable to live cell imaging in many instances. Although this does come at the cost of a reduced ability to do confocal sectioning. So you have a reduced resolution in the Z direction but often times, this isn't too much of an issue. Those are a little bit about the technologies on you know, how do you actually acquire some of these images. But what, what are some practical things about it? Well the ideal acquisition system for live cells, really. You, you should think about several things. So, first of all sensitivity is very important. You need to be able to acquire images of sufficient quality from oftentimes weakly fluorescent specimens. And, you really need to worry about things like photobleaching and phototoxicity, which I explained before, so. You want to be able to collect as, as much light as possible with the minimum amount of excitation power. And this is something that often just needs to be tuned on a case-by-case basis. To figure out how much your fluor fours and how much your cells can handle, with this regard. It needs to be fast often times. Depending on the speed of, of the thing that you, that you care about. Can really dictate what sort of approach you take if you're interested in imaging calcium transient and, and cardiomyocytes for example, you need something extremely fast. If you're interested in, in looking at proliferation responses of cells in culture, you don't need something that fast. But, regardless, you need to suit the speed of your system to the speed of the biological process you w, you want to image. Sufficient resolution of course. Sometimes you just care about. Kind of an integrated measure of the, over the whole cell, so you might not need very fine resolution, but sometimes you really want to capture some cellular processes or localizations, such as things happening on the plasma membrane. So this is another thing that needs to be taken into account. And dynamic range. So often times you want to do multicolor imaging. So, you know, perhaps looking at several things within the same cell. Or you want to look at things across multiple cells in the population. And, and often times the signals from these, from different cells or from different markers in the same cell can have widely varying intensities. So this is something that's, that's an important factor. How, what is the difference in intensities that I'll really be able to resolve with my setup? Another very important thing, if you're doing live-cell imaging over a time course, which is a typical application. You need to be able to keep the focal plane. And usually you'll want to look at multiple positions in your sample over that time course. So there are two pieces of hardware that really help that. That should be on any of these microscopes. One is called a an auto focus system which uses typically an infrared laser to measure the distance between this, the the sample and the objective. And it keeps that distance constant. As perturbation might change it over time. It could something as simple as the air conditioning turning on in the, in the room with the microscope, which causes very subtle temperature variations which causes focus drift. Or it could be building vibrations as much or as good as those vibration dampening tables are sometimes you just can't avoid it. So you really need some active piece of hardware that accounts for that over long time courses. And the motorized stage is important because usually within any time course there's some dead time between the the distance between your time points and the amount of time it takes to actually acquire an image. So that means you can actually look. At more cells during the same experiment and get more data out of it if you are able to move the microscope stage around to different positions and acquire those images. And that's facilitated by this so called motorized stage which is controlled by software in the acquisition. And and of course really important and it goes to a lot of those first points that I was talking about. Sensitivity, that's Speed, resolution, dynamic range. A lot of it's about the camera and photomultipliers or photomultipliers that, that that your system has on it. So there's actually a huge body of research on those. We're not going to cover it here but if you're really ,. You know, if you're really serious about trying to do some live cell imaging or thinking about your particular system, you need to take a hard look at what kind of cameras or, or detectors are available and what's going to do the best job for what you want. Okay, so the next series of lectures, I'll just be talking about, what are the types of probes that are available to let you visualize things in living cells?
