Most Things Biology

Microscopy

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9–14 minutes

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A microscope is a tool that allows you to see what is REALLY out there living among us. Our puny human senses are too weak to be able to see the compound eye (with 1000s of eyes in one structure) of a flea, the bacteria living on the turns of a guitar string, or the hairs on the skin of a kiwi fruit. A microscope let’s you see things 1,000 times their size. An electron microscope lets you see into someone’s soul.

Using a virtual online microscope, a physical microscope in the lab, or even manipulating an online collection of histological images takes practice. Understanding how the microscope generates what you see is quite important. Also, it really is kinda cool.

Access the Corning Museum of Glass to read about this funky microscope invented by a guy named Leeuwenhoek

Very near to where I live is this place called the Corning Museum of Glass. Corning is the name of the town, but CorningWare is the name of a very durable line of stoneware that was introduced to American households in 1958. I have a good collection of Corningware bowls, plates, cookware, and other vessels for food that are all over 50 years old. I still eat off the Crazy Daisy patterned plates from my childhood. Corning Glass Works also makes the line of Pyrex cookware which is usually clear. So, Corning is not just the name of the town, but the name of the town brand. Impressive.

Check out Corning Museum of Glass’s Optics Exhibition complete with enormous convex lens.

Convex Lenses and Refraction

In a microscope, light rays pass through a series of lenses, including convex lenses, which cause the image to be inverted. A convex lens is a curved lens, as the one above. These types of lenses are thicker in the middle than they are at the edges. This allows them to bend the light rays that pass through them in a phenomenon called refraction. Light rays hit the incoming side of the lens at a stright-on direction, but the curvature of the lens bends the light rays so that when they leave the other side of the lens, they are now going in a different direction. That slight curve at the edge forces all the light rays to converge at a point beyond the lens called the focal point. Have you ever burned ants with a magnifying glass? No? Well, here’s how it works: You put the ant in the focal point.

A microscope, much like a projector, beams light from a small bulb and funnels the light through a convex lens who’s focal point is the exact place on the microscope where your specimen, mounted on a little glass slide, is stationed. As the light rays converge onto the focal point, the light rays headed toward the top, end up at the bottom and vice versa. The light rays at the left end up at the right and vice versa. This is what creates the inverted image that you see when you look into a microscope. Actually, the image is inverted more than once as the light passes through the objective and ocular(s).

Inversion

Inversion is the effect of flipping the image on BOTH its horizontal and its vertical axis. I guess you can say that it is backwards and upside down. The image you see when you look into the microscope is a projection and that projection is inverted. This can be very confusing when using a physical microscope in the lab. With virtual microscopes, its no problem because the images are displayed to you in the correct orientation.

Magnification

It is important to know the total magnification of your microscope so that it is easy for you to compare your images with images in the literature. Compound microscopes have two magnifying lenses: the ocular lens (in the eyepiece) and the objective lens. To find the total magnification, you multiply the magnification of the two lenses. If you are looking at something like a water bear with an objective that magnifies things 40 times and you also have oculars that magnify things 10 times than that water bear is being magnified to 400 times its size. That’s a big water bear.

Most compound microscopes found in a biology labortory for students have 10x oculars and 4 different objectives that can be rotated to fix one of them into place at a time. The objective with the lowest magnification is called the scanning power, indicating that it can really only be used to scan the slide and generally locate your area of interest. The scan power’s magnification is usually 4x. Combined with the 10x oculars, the scanning power can magnify your specimen 40 times. The objective with the next highest magnification is called the low power, has a magnification of 10x, and magnifies your object 100 times. The objective with the next highest magnification is called the high power, has a magnification of 40x, and magnifies your object 400 times. Are ya picking up the pattern I’m laying down there? The last objective is called an oil immersion objective. Although it has a stunning total magnification of 1000x, it requires your specimen to be submerged in oil.

Resolution

If magnification makes your object of interest larger (or smaller), resolution is the sharpness of the object. When I went to school in the 1980s and 1990s, I’d have teachers who would have these handouts that had been made from a copy, that was made from another copy, made from yet another copy. The typed letters on the pages would have blurred so much, it would look as if the letters had bled together. This is an example of low resolution. This can happen when your microscope is not in focus. If you are looking at two separate cells, they could look like one, big, blurry blob of a cell.

Microscopes have these two dials called the coarse and fine adjustment. These can be thought of as focusing dials. Have you ever taken pictures with your phone and you move your phone closer to your object of interest quickly? There are these few seconds where the phone is focusing and you can see your picture go blurry, then focus, then blurry, then focused. Your camera is changing the resolution, bringing your item into focus, and making the image that you see on your screen sharper. These adjustment knobs do that. The coarse adjustment knob makes changes to the resolution very quickly by moving the specimen on your slide closer or farther away from the lenses. The coarse adjustment can only be used on low magnifications. The fine adjustment knob does the same thing, but the increment are so small that our puny little human eyes can’t see it. You can use the fine focus on all magnifications to sharpen your image.

Working Distance

The position of the objectives relative to your specimen on the slide is called the working distance.  Since the coarse and fine adjustments move the stage up and down, they control your working distance. Each objective lens has a specific working distance which is the distance between the specimen and the objective lens in which your image will appear sharp. The distance between specimen and objective lens decreases at higher magnifications. The objective and the glass slide are but millimeters apart (if that). This is why it is important to only use the fine focus control when viewing with high-power lenses. Using the coarse focus even just a little bit could move the slide right up to the objective and crack them both.

Click here to explore ALL the parts of the microscope

Field of View

All of these aspects of the microscope, the magnification, the resolution, the working distance and the depth of field contribute to creating what you see when you look in the microscope and see the field of view. This is the maximum area you can see when looking through the microscope. Think about looking through a toilet paper roll at the computer screen right now. That’s scanning power. Now, swap it out for a straw. That’s low power. Now swap it out for one of those red cocktail straws that you are not supposed to drink from (it’s for stirring). That’s high power. Every time you increase your magnification, you see less of your object.

Let’s extend this analogy with the water bear. If you were to start out on scanning power with your water bear right smack dab in the middle of your field of view, it will still be in the middle of your field of view when you switch to a higher magnification. But, if your water bear is even just slightly off center, it might be so far off center that when you switch magnifications (from toilet paper roll to straw) it’s completely out of the field of view. The offset gets to be more and more the higher in magnification you go. Bye Bye water bear. Don’t try to search for it, you’ll just make it worse. The only way to fix this when working on either a physical microscope or on a virtual microscope is to go back to a lower magnification and be absolutely certain that your specimen is centered in your field of view. Go back to scan power and start again.

More Light Please

As you move through the objective, magnifying your object of desire, you will need more light intensity. Microscopes usually have something like that dimmer switch in the formal dining room at my parents’ house. My mom always wanted to dim the lights and light the candles for holidays. I could never see exactly what I was eating. Now that I think of it, maybe that was the point.

At low magnifications, cells and tissues can be spread out in your field of view, often with large gaps of space among them. These gaps allow light to shine through unimpeded by the stains of the tissues. At these low magnifications, the dimmer switch needs to be turned down so that you don’t burn your retinas (you can’t actually burn your retinas with a microscope, it just feels like it). As you increase your magnification, the stained cells take up more space in your field of view. At the highest magnification, you be only able to view one cell and possibly not even the entire thing, just some of it, as pictured below. The cell and the nucleus in the picture of the 400x magnified field of view take up the entire field of view with its stain absorbing most of the light. Because of this, more light is needed.

Artifacts

What is an artifact, other than something that Indiana Jones hunts? An artifact is an image that is affected by the process of making the slide. Remember that what you see in the microscope is not what it looks like in your body. When cells are harvested (biopsied) from the body, they are dried and stained in order to fix them to the glass slides we use in the lab. Any water content is evaporated from the tissue in the drying process. Tissues rich in the water-binging protein collagen, such as the dermis of your skin, dry up and crack open just like an old leather belt. This leaves open spaces in the tissue that are artifacts of the drying process. These open spaces aren’t present in the dermis, but when we make a slide of dermal tissue, these open spaces are unavoidable.

Dense irregular connective tissue full of pink-stained collagen. Because collagen is a water-binding protein, evaporation of the water leaves the white spaces.

Stains

It’s important to get to know the types of stains used on histological slides. Knowing the stain can help you identify structures more easily. For example, knowing that the commonly used H&E stain colors collagen a pink color and understanding that Masson’s trichrome stain colors collagen a blue color can help you quickly locate the collagen of the dermis on these two pictures of skin below. The purple and red stained cells of the epidermis are superficial to the dermis. You can even see some skin cells sloughing off as a sheet on the H&E stained slide.

Stratified Squamous Epithelial cells lining the vagina.

The commonly used H&E stain colors elastic fibers red, but they can be hard to see if the tissue has a lot of collagen. Using a stain calls Verhoeff’s will stain the elastic fibers black, making them much easier to see. You can use multiple stains to color a tissue, making what you create a work of art.

Nervous tissue has qualities that allow it to interact with other types of stains such as metal-based silver and gold stains, methylene blue and iodine, and Luxol fast blue stain which is capable of staining myelin.

Gray and White matter of the spinal cord.

View an actual water bear at:
https://allthatsinteresting.com/electron-microscope-images

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