Captions are on! Click CC at bottom right to turn off. “Pinky, what is on the kitchen table?” “Oh, it’s my protists. I’m hoping to find some euglena, maybe some paramecia. I took this sample from—” “Stop, I mean, WHY is it on the KITCHEN table?!” “Oh, I got distracted. I was going to take some drops and put it on a slide while I set up my microscope.” “Yes, well, please get your green water away from where we eat.” “Green water? Don’t be ridiculous, Petunia, there are tons of protists in here. We just have to find them.” “Well, I think you’re out of luck.” “The protists that I’m looking for are unicellular, meaning, they’re made up of one cell. And generally, you need a microscope to see them.” “Huh. Never used one of those.” “You’ve…never used a microscope before?” “No.” “Well you know what that means?” “I’m afraid about what that means.” “We must explore the world of…microscopes!” First of all, let’s focus on a few terms that are important in order to understand microscopes. Magnification is one. That’s really what most people think of when they think of a microscope. And we’re going to talk more about that in a moment. But another term that is important is resolution---basically, are you able to distinguish between two objects? This is important as otherwise two objects could actually look like one object, because you can’t distinguish between them. So, for example, if a microscope had a resolution of 0.2 microns, that means two objects needs to be 0.2 microns or more apart in order to see that they are two different objects and not one. If you want to understand the physics of how resolution works and how it’s calculated, we have a great further reading suggestion in the video details. So let’s talk about some different types of microscopes that all take into account this need for magnification and resolution in order to see specimens. First, light microscopes. If you’re in a classroom, this is likely what you have. And, as its name would suggest, it uses light in order to see an image. Brightfield light microscopes tend to be what people are familiar with- typically producing a darker image on a light background. With a darkfield light microscope, you have a piece that blocks the light source, called a light stop. The idea is that most light has been blocked so the only light you see is light reflected or refracted from structures within the specimen that you’re looking at. So, you tend to see light images on a dark background. There are many other types of light microscopes too. We have a great link for further reading suggestions to learn how each of these work, and some of these are fascinating---some rely on interference, some rely on using a laser…but the thing to keep in mind is, there are certain times when one of these types of light microscopes is more ideal than another. For example, a phase-contrast microscope can give you a very detailed image of a living specimen without requiring any stain, and the detail it provides can be far better than the typical brightfield microscope. So light microscopes use light in some form to see a specimen. But not all microscopes are light microscopes. For example, what if we wanted to see a virus? Viruses are generally very small- typically much smaller than the average bacterium. How can we increase magnification and resolution abilities to see them? That’s where electron microscopes come in. Electron microscopes use electron beams to visualize images. Transmission Electron Microscopes (TEMs) tend to be most ideal for visualizing structures within a specimen while Scanning Electron Microscopes (SEMs) tend to be most ideal for visualizing the 3D surface. Let’s come back to the typical microscope you might see in a classroom, which happens to be the one that I own. A brightfield light microscope. Here is our light source. Light passes through this, the condenser lens to focus the light on the specimen. The level of light can be adjusted by this, the diaphragm. Our specimen---likely to be on a microscope slide---will be placed on the stage here. Light will be reflected, refracted, or absorbed by the specimen. But we rely on these lenses here---the objective lens and the eyepiece lens---to get our total magnification. In fact, this is often referred to as a compound microscope, because it has two sets of lenses. You can see that the objective lenses are attached to this revolving nose piece which allows them to be rotated so you can select a certain one that you want to use. With my particular microscope, I have three objective lenses. A scanning objective lens that magnifies 4 times, the low power objective lens that magnifies 10 times, and the high power objective lens which magnifies 40 times. But don’t forget the eyepiece lens because that also magnifies images, and my particular eyepiece lens magnifies 10 times. So let’s say I use the scanning objective lens. I would multiple that objective lens magnification level by the eyepiece lens magnification level so that my total magnification is 40 times. Now, you’ll also notice there’s a lot of knobs here. Let me introduce you to the knobs. This large knob here is known as the coarse focus and then this smaller knob here is known as the fine focus. Both of these knobs raise or lower the stage, which helps with focusing. The coarse focus knob will move it more dramatically whereas the fine focus knob will move it in smaller increments. These knobs- the stage knobs- don’t move the stage up and down but rather from side to side instead. It helps you explore what’s on the slide itself, but it isn’t adjusting the focus of it. Not all microscopes have those stage knobs; sometimes you have to move the slide manually. This is the microscope arm and the base. Whenever you pick it up, you want to make sure you have one hand underneath supporting the base and the other hand holding the microscope arm. Since it plugs in, you want to make sure there isn’t water around it. So, let’s take a look at what’s in this water sample here. I’m going to do something called a wet mount to prepare my microscope slide. I’m going to drop a drop or two of my water sample onto the slide using a disposable pipette. It’s like a very fancy eyedropper. Kind of. I’m going to put a cover slip on top. There’s some techniques to doing a wet mount to avoid air bubbles that you can check out online. Air bubbles actually can look kind of beautiful under the microscope---which can lead to great disappointment when you find out they’re not some amazing microscopic organism but rather just an air bubble--- something I may or may not know by experience. I’m going to put the slide on the stage. I’m going to use the stage clips here to secure the slide. Light is on, and I already have the scanning objective lens here ready to go, which is good, because I want to find the specimen first. I can adjust the light level if I want---but just know that super bright doesn’t always mean the best image. I can look through the eyepiece and use the coarse focus knob to move the stage up or down slightly to focus. Patience. Euglena! I can also fine-tune focus it with the fine focus knob. I can use the stage knobs to center my image. Now that I found it with the scanning objective lens, I’m now going to move up to the next magnification level. So, remember, total magnification would be 100 times. Since I had already focused it with the scanning objective lens, it’s likely I only need to use the fine focus knob to adjust the focus at this level. I could continue to move up to the high power objective lens, which would give me a total of magnification of 400 times. Now 3 additional tips about using microscopes that I just want to mention. 1) Most microscope slides are glass so they can break if dropped. And if using cover slips, keep in mind they are extremely easy to lose if not careful. 2) Always be aware of where the slide is when you’re moving the stage up and down with the focus knobs. It’s possible to move the stage too close to the objective lens and actually crush the slide. 3) If it’s hard to see your image even when focusing, you may need to clean the lens. But you need to do this by wiping it with lens paper. Not regular tissue. When you’re done with the microscope, make sure the slide is removed, turn off the light, lower the stage to its lowest position, and return the lowest power objective lens over the stage. Unplug it and wrap up the power cord. And if you have a cover, cover the microscope before putting it away. Keep in mind there are some techniques to better visualize some specimens such as using different stains or using immersion oil at high magnification to increase resolution. Definitely something to explore that this video doesn’t go into. Microscopes open up an entire world that you can’t see with the naked eye. Microscopes can also complement what you might be learning in life science. Studying mitosis? Check out the cross section of an onion root tip, where lots of mitosis happens. Studying plant responses? Take a look at stomata from a thin sample underneath a leaf. Studying osmosis? Take a look at how aquatic plant cells respond to different salt concentrations. Endless possibilities to explore endless curiosity. Well that’s it for the Amoeba Sisters, and we remind you to stay curious.