Applied microscopy for theriogenologists
Stallion
Techniques in Reproductive Examination
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<p>Hello, my name is Stuart Myers.<br>I'm a professor at University of California, Davis.<br>For this video, I'll be presenting a guide for microscopy that veterinarians and theriogenologists may find helpful for navigating the vast array of microscopy techniques that are useful for practicing theriogenologists.<br>I will briefly discuss some more advanced imaging methods after that, that you may be exposed to and may need to understand how some of those things work.<br>My objective is to go over the basic operation of a compound microscope so that you'll have the best image capabilities for most light microscopes that you'll be exposed to.<br>I will not be talking about optical physics or about light and vision.<br>Those topics are for another time.<br>So with that, I'd like to jump into the a number of other slides that I have prepared for you.<br>So the goals of this webinar are to review how microscopes work.<br>Particularly, I'm going to focus on compound microscope because most every scope that we use is a compound microscope with variations.<br>We want to understand the use of objectives, light sources, condensers, and basic illumination, as primary components of microscope.<br>We also want to understand how unstained reproductive cells can be imaged using contrast and also staining.<br>And I want to talk about the differences between and the uses of DIC phase contrast and dark field microscopy, because those are used very commonly in reproduction.<br>And to understand how fluorescence, laser confocal, and electron microscopes can be used to image reproductive cells.<br>This slide also demonstrates that there are some really useful resources for microscopy.<br>Some you can go to the main four microscopy manufacturers' websites.<br>There's a lot of information available to you there, Leica, Zeiss, Olympus, and Nikon, and some others for certain.<br>There's also a Molecular Expressions website that I wanted to point out to you that is very useful for all sorts and all levels of understanding of microscopy.<br>The first one is an interactive website from Florida State University, and the second one is a PDF manual called \"Microscope Basics and Beyond.\" I think you'll find it really useful.<br>So the most common types of microscopy is the first thing I want to talk about.<br>Light sources are many.<br>Most microscopes work on halogen or tungsten bulbs in their microscopes.<br>But there are also more recently, a whole variety of LED lights that are available.<br>They're very bright.<br>They don't burn out.<br>They last a long time.<br>But also for fluorescence microscopy, one can use mercury or xenon arc lights.<br>And then lasers obviously are really important for things like confocal microscopy and other types of fluorescence microscopy.<br>Now, most common types of microscopy are bright field and phase contrast, or just contrast enhanced microscopy that primarily the two are phase contrast and DIC, which is differential interference contrast.<br>These are important, and also Hoffman modulation contrast is another type of contrast microscopy that you might find useful that's typically used in cell culture situations that use plastic.<br>DIC is really designed for glass.<br>I'll also be talking briefly about dark field microscopy, which we all know, you probably see it on CASA systems that you've seen.<br>And also, we're going to be talking about dissecting stereo microscopes, a little bit about CASA and its reverse phase.<br>And then I'll end up talking about electron microscopy and fluorescence.<br>And then virtual microscopy is really the newest mode for teaching microscopy, but also for viewing information.<br>So what is a compound microscope? A compound microscope is the microscope with two eyepieces that we're used to looking through for almost everything that we do.<br>It is a high power magnification, high magnification microscope that uses more than one lens.<br>So compound microscope has multiple lenses.<br>As you know, they can be a variety of objective lenses, but also an eyepiece.<br>So we have two levels of optical influence when we look at through a microscope.<br>The compound microscope just simply means a double lens system.<br>As I said, we have nosepiece objectives, but also eyepieces.<br>This provides an enlarged, clear image of a very small specimen.<br>But I'm going to talk a little bit about the way to optimize even the cheapest microscope for getting the best image quality that you can get.<br>The compound microscope in its most simplified version is really a tube with two lenses and with a lens on either side of the tube.<br>So the image is formed by a combination of two lenses.<br>So in this case, the human eye looks through the ocular eyepiece lens that extends the light waves through this microscope tube, and the light then converges to and from the objective lens that is sitting there.<br>So we have a magnification and resolution to distinguish two objects or more than two objects, and the image is reversed, as I'm sure you know this from optical physics in your previous lives.This is one that isn't so simple.<br>This is a complex fluorescence microscope, and I put this up here just to show you that there's a lot of thought that goes into more sophisticated microscopes that generate bright field, but also fluorescence images.<br>And we're not going to be talking too much about that, I just wanted to show a gee whiz slide about how great these are.<br>Now, the optical parts of the microscope are twofold.<br>There is the image formation part of the scope, which is above the stage, and then the illumination system, which is below the stage, and this is in an upright microscope.<br>In an inverted microscope, these things are reversed, and so we see the light source on the top of an inverted scope, but they work essentially the same.<br>Above the stage, we have oculars and objectives, so remember our two lenses, for the optical tube.<br>And then below the stage, we have a condenser and a light source.<br>And the condenser is really important.<br>It is a lens that focuses the light beam.<br>Both parts are equally important for good image quality.<br>And you want to use the microscope with the illumination controls facing you.<br>That's sort of the proper way to use a microscope.<br>So if we talk about the determinants of image quality, quality of the objective lens is probably the foremost important thing that provides high image quality.<br>Secondly, the cleanliness of all lenses.<br>So lenses need to be clean and dust-free.<br>And then the appropriate use of the condenser contributes to the really highest quality image that you can get.<br>Now most of us, and I know that I was one of them in my past, is I had no idea really what to do with the condenser, which is this unit underneath the stage.<br>There are several different types of condensers, which I'm going to talk about.<br>And those condensers then, I didn't know what to do with them, but you just sort of turn them around until your image quality gets the best.<br>The oculars contribute little to the image quality, but they improve user comfort.<br>So those are the eyepiece lenses.<br>The light source is really important.<br>Most microscopes have an in-base, variable light source.<br>Some of them can be even wavelength variable.<br>And then there's a field diaphragm in the bottom below the condenser that can be turned for centering the condenser, and also it's often an iris.<br>So it's a closing diaphragm, basically, that you can adjust.<br>The transformer, which controls brightness, is something that you're used to using.<br>You're just going to turn this up to where you get the maximum light through the specimen that you can tolerate, that your eyes can tolerate.<br>Now, the way an illumination system works at the condenser is that it gathers light to go through the sample, through the stage, to the objective.<br>So the more light you use to form an image, the better is the resolution.<br>It is a lens, and it needs to be in focus for maximum effectiveness.<br>And that's what you're doing when you're messing around with the condenser controls underneath the stage.<br>So here's an example to show you that the condenser, which is this lens here, in an ideal situation, it sends light beams.<br>It basically focuses the light that comes out of the light source, focuses them up through the slide where the specimen is, and then you want to get the smallest possible convergence, the least convergence of light beams into the objective.<br>The objective then gathers the light.<br>Now, when the condenser is out of focus, it sends the light beams to scatter before they even get to the eyepiece.<br>So we don't get as many light beams or photons through the specimen into the objective.<br>Excuse me.<br>So that's all really important.<br>And the result of that would be a darker image made by fewer photons and consequently less resolution.<br>So the way that condensers work, there are a couple of different types of condensers.<br>There are sliding condensers.<br>This is an example of a sliding condenser, and it has an auxiliary lens, which is usually zero power.<br>It's just usually a piece of glass in there.<br>And then we have a sliding small objective that you can use to focus that.<br>So for low light level, we're going to use the condenser without this microscope additional lens here, condenser lens.<br>And then for the condenser iris, you can see that here the iris is closing and then closing even more.<br>And this causes a real focus ability for the light beams to then go strictly through the condenser, and it actually cuts off errant light beams.<br>So then if you slide over the lens over this, then you have much more higher power of light beams going through your specimen.<br>And here's an example of how the condenser illumination forms cones of light, depending on whether the iris condenser is open or closed.<br>So here's an example of where there might be a problem.<br>The condenser on the left is out of focus, or the condenser iris is closed.<br>So this means that the objects on the slide are not getting the proper light-In focus.<br>So the condenser is out of focus.<br>So what we often do is then focus the condenser, which usually it has a knob, and we can turn the condenser up and down, closer or further away from the stage until we get sharpness of our sample on the stage.<br>So that's basically all you need to know about how to work with a condenser.<br>It's really important.<br>This also is part of the Kohler light illumination that we'll talk about a little bit.<br>But Kohler illumination is a type of illumination that is very, very important for making sure your light beams are optimized.<br>So magnification is simply calculated by the ocular power times the objective power.<br>So if you had a 10X objective and a 10X ocular, we're looking at a total of 100X, which is actually low power.<br>In high power, we would have a 100X objective and a 10X ocular, which would give 1000X power for higher power imaging.<br>Resolution is the ability to see small details.<br>It's the ability to visualize two things separated by a tiny, small distance.<br>High-resolution objectives are the most expensive part of the microscope image.<br>In the left side, you can see that you can see sharpness of two images, and the resolution is the highest power you can use to resolve two separate objects.<br>If you can't resolve them, we get fuzziness at the edges of those things.<br>And remember, again, the condenser focus is really important to make this work, along with high-quality objectives.<br>The higher the better.<br>Now, the contrast is the ability to differentiate two different specimens from the background.<br>The color in the specimen is the contrast when you're using staining.<br>So staining adds contrast.<br>Some specimens have very little contrast.<br>As you know, sperm, endometrial cytologies, red blood cells, and white blood cells, they generally have little inherent contrast.<br>So we can add contrast in the microscope by either staining or by adjusting the condenser properly.<br>And you folks are probably more familiar with endometrial biopsy and cytology that you might look at using a number of different stains.<br>And this is the kind of color contrast that you get when you add color stains to specimens that have been either fixed or unfixed in certain cases.<br>This is an endometrial biopsy on the left, and we see vaginal or uterine cytologies on these images A and B here.<br>And you can see the neutrophils, and you can see a lot of other cells and debris that are in there quite nicely when you enhance with color.<br>Objectives are the most important part of the microscope, and resolution depends on the quality of the objectives.<br>It is generally also, as it were, the most expensive part and the most easily damaged part.<br>So when you're dealing with objectives that are just sort of run-of-the-mill, average objectives, your light quality and your object resolution may be good enough for you to see.<br>But if you're looking for very small, finite differences, you're going to need higher quality objectives and higher powered objectives.<br>These are just showing a number of different objectives from 2X, 10X, 20X, 40X, and 100X, and the 100X is an oil lens.<br>You can get a high, dry high power lens, which is 60X, which is almost as good as 100X, but does not require oil.<br>These objectives are always color-coded, and they have information printed on the side or engraved into the side of the objective that has meaning for the types of use that you'll want to use.<br>So let's talk a little bit about types of contrast in light microscopy.<br>When we're looking at specimens that don't have a lot of inherent contrast, we want to use contrast-enhancing techniques.<br>The two most common ones are phase or DIC, or differential interference contrast microscopy.<br>DIC is also known as Nomarski optics.<br>So if we look at the top row of images here, these are phase contrast images.<br>So phase, they get this appearance of a halo effect around the specimen because of the way the objectives and the eyepieces work using phase rings.<br>And then on the bottom three images, these are DIC images.<br>So DIC uses prisms and polarizers, so it uses polarized light.<br>It does not have a halo effect, but it gives you a really high cell surface detail.<br>And here you can see this really nice cell surface detail on these DIC images that you can't really see on the phase contrast images.<br>The phase contrast images also gives you a lot of information.<br>They're both really good ways to look at unstained specimens.<br>And this is an example that you can see if you go to that website that I pointed out earlier.<br>You can actually play around with simulations of how these phase rings work.<br>Now, just as a preview to the next slide, the way that phase contrast works is you need a phase ring in the objective, and you need a phase ringUnderneath the condenser.<br>And so what you do is you align these two phase rings up to where they're overlying one another, and this gives you the perfect phase image.<br>This slide shows a little bit more information on phase and DIC specimens.<br>So on the left side of the slide, we see the phase contrast light pathway.<br>And what we see is a phase ring in the objective.<br>Here's the specimen, and then the condenser also has a ring or an annular ring in the condenser unit.<br>This is what gives us the halo unit, and this is the part where I'm not going to talk about physics or vision because there's just different ways these images are generated by cutting out the center part of the light beam with the phase ring diaphragm.<br>DIC microscopy, as I said, uses prisms and polarizers, and it gives you a high surface detail.<br>So these two images are DIC images on the right side of this picture.<br>And then this diagram here shows the light path using optical prisms and polarizers.<br>So for DIC microscopes, there are three things that are in the light path.<br>There's a polarizer and two prisms, and those have to be aligned properly in order to see that.<br>If you're working with a DIC microscope, you have to be able to adjust three different things, and these are set up differently in different brands of microscopes.<br>Here's what we're used to looking at with sperm morphology.<br>Phase contrast is fine for doing high power 1,000X magnification for sperm morphology.<br>But you can see that the Nomarski or DIC image also gives you a lot more information about the surface.<br>You can sometimes see things like vacuoles and irregularities in the plasma membrane.<br>Sometimes you can see the acrosome edges, but mostly you cannot see that with light microscopy.<br>This also is using oil immersion, but also there are new lenses now that use water immersion.<br>They're a lot easier to clean and keep track of.<br>But water immersion lenses use a different refractive index than oil, and they are very expensive.<br>Here's the classical eosin-nigrosin or therriogenology stain showing sperm morphology, and this is an exclusion stain, so it stains the background, and the intact cells exclude the dye, and damaged cells take up some of the dye.<br>When we look at the contrast between DIC and phase contrast using embryos, you can see that the phase contrast of embryos shows a lot of detail about the edges of the cells and the light refraction of the cell cytoplasm.<br>But DIC at 40X shows a lot more surface contrast, and it shows a lot of the granularity of the plasma membrane itself.<br>And in this particular specimen, you can see very, very nicely, this is a human embryo showing the pronuclear stage of the embryo.<br>So we have the male and the female pronucleus that you can see very, very nicely, and you might not be able to see that with phase contrast.<br>Another contrast method is called Hoffman modulation contrast or HMC.<br>Hoffman is used in a lot of ICSI and IVF programs because it is basically a low-cost version of DIC.<br>It costs a fraction of what DIC costs, maybe about 20 to 25% of what DIC costs for a microscope.<br>And you can usually buy an additional Hoffman condenser that fits on most microscopes to convert microscope to a Hoffman optics.<br>So the nice thing about Hoffman optics is that you can use it directly with plastic dishes.<br>So that's why you see a lot of labs that have cell cultures.<br>Here's an example of fibroblasts growing in culture, and you can see really nice contrast and detail of the cell nucleus and the cell surface as well.<br>DIC happens to have really weird effects on plastic, and so you generally need to have culture in glass for using DIC.<br>So it's kind of a trade-off.<br>This is a human embryo, a human zygote, again, showing two pronuclei, but this is also with Hoffman.<br>So it's not quite the detail that you might see with DIC, but it's quite good, and it's often used for embryos in ICSI.<br>And in fact, it's difficult to do ICSI without at least some kind of Hoffman or DIC optics.<br>Stereomicroscopes, most of you are familiar with stereoscopes.<br>They're used for dissecting, like embryo searching, oocyte searching.<br>You can use them for looking for parasites and things like that.<br>So it's larger image specimen that you're looking for with a dissecting scope.<br>These are, as I said, they're designed for larger objects.<br>They generally use an LED or incandescent, but there are fluorescentDissecting microscopes that can be gotten.<br>Here's two Leica microscopes that we have.<br>One has the fiber optic illumination from the top, and this one has the transmitted light base, which is much more useful for embryos and oocytes.<br>These types of microscopes are also fitted for surgical microscopy, and you'll see this very commonly for microsurgery techniques.<br>As I said, these scopes usually require mirrors to enhance the contrast.<br>They can be bright field or fluorescence, and the magnification is lower than a typical compound microscope, but they are a type of compound microscope, and they do a very nice job with 3D view of specimens or samples for looking at more details.<br>Dark field microscopy is something that you're probably familiar with.<br>It uses an aperture iris in the lens.<br>It actually separates the light within the objective.<br>So I don't want to go into great detail about it.<br>But these would be for specimens that are often too delicate for bright field or have a refractive index that's too close to the surrounding environment.<br>So it provides a dark background, and the specimen becomes illuminated.<br>So it requires blocking out of the central light path, which ordinarily passes through and around the specimen.<br>So it allows only oblique rays from every azimuth or from every angle to strike the specimen mounted on the slide.<br>So these are some examples of three different, some sort of a plant material or pollen or something.<br>Radiolarian in bright field.<br>And so in bright field looks like this, in dark field looks like this, and then we have dark field fluorescence that shows that.<br>More commonly, we are used to using dark field microscopy for computer-assisted sperm analysis or CASA.<br>On the left side of this slide, we see a group of embryos growing with a negative phase contrast image.<br>These happen to be primate embryos at approximately the two-cell and four-cell stage using a dark field microscope.<br>And then with CASA, we see that CASA really allows you to actually use the computer to then track the movement of these cells, because the cells are lit and they're lit brightly, and so they can be tracked easier by a computer system.<br>So now I just want to talk a little bit about fluorescence microscopy.<br>Using a fluorophore, which is an organic molecule with the ability to absorb light at a particular wavelength and then emit it at a different or higher wavelength.<br>Very commonly now used, particularly for looking at sperm acrosomes in all domestic livestock males.<br>And it has a really growing use in referral institutions and tertiary hospitals that have the ability to do imaging with multiple fluorophores.<br>These microscopes require an excitation source, which would usually be an LED or a mercury lamp, or even a laser.<br>And then they require a fluorophore to enhance different cell compartments, or those cell compartments may be autofluorescing.<br>It requires wavelength filters and a detector, including photomultiplier tubes is one type of a detector.<br>So what you can see here that these are intact equine sperm, where the bright yellow is isolating the outer acrosomal membrane, and the red is a DNA stain of propidium iodine that is staining the DNA-containing material.<br>Here we can see an image with two intact acrosomes and two that have already acrosome reacted.<br>So you don't see any bright green or yellow fluorescence on those because the acrosome has been lost.<br>And here's just another example of acrosomes, equine acrosomes with PI or propidium iodide, and a FITC-PNA.<br>So this is a fluorescinated lectin peanut agglutinin.<br>And what we see with fluorescence, we require excitation of the fluorophore at one wavelength, and then the excitation at another wavelength that can then be picked up by a detector device of some sort.<br>So just going over these acrosomes, they are very difficult, if not impossible, to visualize without fluorescence.<br>This diagram you've probably seen along the way, that is showing the acrosome laying over the sperm nucleus and surrounded by the plasma membrane.<br>At the time of acrosomal exocytosis or the acrosome reaction, the outer acrosomal membrane and the plasma membrane fuse, and that causes a release of the acrosomal contents.<br>That leaves a naked inner acrosomal membrane, which some stains can see, and also some stains can see the outer acrosomal membrane.<br>So there's a number of stains.<br>It's sort of beyond the scope of this conversation to talk about that.<br>But here you see DIC of sperm, and this is the typical eosin-nigrosin stain.<br>And in the DIC, even though you can see the surface of the sperm, you're looking at the plasma membrane.<br>You really can't detectYou cannot detect acrosomes very well with a light microscope.<br>With eosin-nigrosin staining or some sort of triple stain or double stain, you can see what looks like might be the acrosome, but you can't get a really good view of the entire edge of the acrosome to be able to visualize it unless you use fluorescence.<br>Now, this is an alexinated PSA, which is a P-lectin that binds specifically to galactosyl residues in the plasma membrane when the plasma membrane is intact.<br>Now, confocal microscopy, I'm just going to say a couple words about this.<br>It's an optical imaging technique that has very high optical resolution and a very high contrast, and it uses lasers to focus very specific wavelength light beams on your specimen.<br>So you can see they're complicated, and it requires a computer to process, but you can get these really beautiful images with a confocal microscope.<br>And you'll see a lot of confocal microscopic images, but you may or may not use one in your work.<br>Here's some examples of confocal microscopy.<br>This is an equine blastocyst on the left that shows where you can actually see mitotic figures specifically, because this is stained for anti-tubulin and Hoechst stain, which stains nuclei.<br>So the red is MitoTracker red, so it's staining mitochondria.<br>So you can see where there are conglomerations or accumulations of red mitochondria.<br>And it's beautiful.<br>This is actually a 3D image, and it can rotate in 3D, so it's a beautiful image taken with multiple three-dimensional slices.<br>Another use of confocal microscopy is just using a differential contrast method to stain the inner cell mass and the trophoblast differently.<br>And again, this is an equine blastocyst showing the trophoblast in red with a confocal microscope.<br>If we move on to electron microscopy, electron microscopy is not being used as much as it used to be.<br>It uses very harsh toxic fixatives, including lead and other toxic chemicals.<br>The electrons are the highest resolution particles, so this is even a step higher resolution than laser.<br>But this is an image of what's called scanning electron microscopy, and you can see a bunch of sperm bound to the outside zona pellucida of a mouse egg.<br>And then here we have the other type of electron microscopy, which is transmission electron microscopy.<br>And transmission EM requires sectioning with a very sharp knife, a very tiny knife that sections all these specimens.<br>So these are sperm cells in parasagittal or sagittal sections.<br>And then the lower ones are showing scanning electron micrographs of sperm.<br>So it mostly shows the surface and the 3D nature of the specimen.<br>And I'll finish with this last slide, which I think are some spectacular electron images of a mouse embryo.<br>You can see the cell mites.<br>You can see just incredible detail on the surface with a scanning electron microscope.<br>And then these are some equine sperm with scanning electron microscope.<br>You can actually see the mitochondrial barber pole wrapping around the midpiece of the sperm cell.<br>And here we see a distal droplet on a sperm cell, and it's just exquisite detail.<br>You can actually see this moth-eaten appearance of a cryo-damaged equine sperm that actually shows some cracking here along the edge of the acrosome.<br>So to conclude the talk, I'm just going to do a brief summary of what we talked about.<br>We talked about that most medical microscopy uses compound microscopes, and these provide some of the highest resolution of magnified cells and tissues.<br>We talked about trying to achieve an understanding of the basic components such as objectives, light sources, condensers, and basic illumination that's necessary to provide really good microscopic images that are useful.<br>And I also described the differences between and the uses of DIC, phase contrast, and darkfield microscopy.<br>And lastly, I talked about fluorescence, laser confocal, and electron microscopy and how that can be used to image reproductive cells.<br>So I want to thank you for your attention, and I want to thank the ICER International Committee for putting this series together.<br>I hope the presentation will be useful to you, and I wish you wonderful adventures in microscopy.<br>I will leave you with a beautiful confocal image of a mouse colon for your viewing pleasure.<br>Thank you very much.</p> |