microcosmos
The Microscope Upgrades We've Made Along The Way | Compilation
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Uploaded: | 2022-08-01 |
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Go to https://microcosmos.store before August 8th to get 10% off of almost everything in the store and to pick up a limited edition Dark Mode Hydra T-Shirt!
This channel wouldn’t be what it is if it weren’t for one very key invention: the microscope. Everything we see, we see with the aid of light and lenses, expertly deployed by our master of microscopes, James. And if you’ve been on this journey from the beginning, or if you’ve ever gone back to revisit our earlier videos, you may have noticed that things have changed a bit around here.
Shop the Microcosmos:
https://microcosmos.store/
Follow Journey to the Microcosmos:
Twitter: https://twitter.com/journeytomicro
Facebook: https://www.facebook.com/JourneyToMicro
Support the Microcosmos:
http://www.patreon.com/journeytomicro
More from Jam’s Germs:
Instagram: https://www.instagram.com/jam_and_germs
YouTube: https://www.youtube.com/channel/UCn4UedbiTeN96izf-CxEPbg
Hosted by Hank Green:
Twitter: https://twitter.com/hankgreen
YouTube: https://www.youtube.com/vlogbrothers
Music by Andrew Huang:
https://www.youtube.com/andrewhuang
Journey to the Microcosmos is a Complexly production.
Find out more at https://www.complexly.com
Stock video from:
https://www.videoblocks.com
SOURCES:
https://micro.magnet.fsu.edu/primer/anatomy/magnification.html
https://micro.magnet.fsu.edu/primer/anatomy/sources.html
https://www.geog.ucl.ac.uk/resources/laboratory/light-microscopy/bright-field-microscopy
https://micro.magnet.fsu.edu/primer/techniques/darkfield.html
https://micro.magnet.fsu.edu/primer/java/darkfield/cardioid/index.html
https://www.microscopyu.com/techniques/phase-contrast/specimen-contrast-in-optical-microscopy
https://micro.magnet.fsu.edu/primer/techniques/phasecontrast/phase.html
https://www.microscopyu.com/techniques/polarized-light/introduction-to-polarized-light
https://www.microscopyu.com/techniques/polarized-light/polarized-light-microscopy
https://www.microscopyu.com/tutorials/comparison-of-phase-contrast-and-dic-microscopy
https://www.microscopemaster.com/differential-interference-contrast.html
https://www.microscopyu.com/techniques/fluorescence/introduction-to-fluorescence-microscopy
https://www.microscopyu.com/microscopy-basics/introduction-to-microscope-objectives
https://www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/magnification/
https://www.microscopyu.com/tutorials/nuaperture
https://www.microscopyu.com/microscopy-basics/refractive-index-index-of-refraction
This channel wouldn’t be what it is if it weren’t for one very key invention: the microscope. Everything we see, we see with the aid of light and lenses, expertly deployed by our master of microscopes, James. And if you’ve been on this journey from the beginning, or if you’ve ever gone back to revisit our earlier videos, you may have noticed that things have changed a bit around here.
Shop the Microcosmos:
https://microcosmos.store/
Follow Journey to the Microcosmos:
Twitter: https://twitter.com/journeytomicro
Facebook: https://www.facebook.com/JourneyToMicro
Support the Microcosmos:
http://www.patreon.com/journeytomicro
More from Jam’s Germs:
Instagram: https://www.instagram.com/jam_and_germs
YouTube: https://www.youtube.com/channel/UCn4UedbiTeN96izf-CxEPbg
Hosted by Hank Green:
Twitter: https://twitter.com/hankgreen
YouTube: https://www.youtube.com/vlogbrothers
Music by Andrew Huang:
https://www.youtube.com/andrewhuang
Journey to the Microcosmos is a Complexly production.
Find out more at https://www.complexly.com
Stock video from:
https://www.videoblocks.com
SOURCES:
https://micro.magnet.fsu.edu/primer/anatomy/magnification.html
https://micro.magnet.fsu.edu/primer/anatomy/sources.html
https://www.geog.ucl.ac.uk/resources/laboratory/light-microscopy/bright-field-microscopy
https://micro.magnet.fsu.edu/primer/techniques/darkfield.html
https://micro.magnet.fsu.edu/primer/java/darkfield/cardioid/index.html
https://www.microscopyu.com/techniques/phase-contrast/specimen-contrast-in-optical-microscopy
https://micro.magnet.fsu.edu/primer/techniques/phasecontrast/phase.html
https://www.microscopyu.com/techniques/polarized-light/introduction-to-polarized-light
https://www.microscopyu.com/techniques/polarized-light/polarized-light-microscopy
https://www.microscopyu.com/tutorials/comparison-of-phase-contrast-and-dic-microscopy
https://www.microscopemaster.com/differential-interference-contrast.html
https://www.microscopyu.com/techniques/fluorescence/introduction-to-fluorescence-microscopy
https://www.microscopyu.com/microscopy-basics/introduction-to-microscope-objectives
https://www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/magnification/
https://www.microscopyu.com/tutorials/nuaperture
https://www.microscopyu.com/microscopy-basics/refractive-index-index-of-refraction
Starting today, we are having a 10% off Back-To-School sale over at microcosmos.store.
So whether you are decorating a new dorm room or classroom, or you’re just trying to decide what to wear for your first day of school, microcosmos.store has you covered. We’ve got t-shirts, sweatshirts, pins, and posters. And we’ve got our Microcosmos microscope, slides and coverslips, and even a Microcosmos microfiber cloth to keep everything nice and clean.
And this week only, from now through August 8th, almost everything in the store is 10% off! And that’s not all, we’ve also got this new Dark Mode version of our Hydra t-shirt that's only going to be available during this sale and then never again. So, head over to Microcosmos.store to check out the sale and to pick up your limited edition Dark Mode Hydra T-Shirt This channel would not be what it is if it weren’t for one very key invention: the microscope. Everything we see, we see with the aid of light and lenses, expertly deployed by our master of microscopes, James. And if you’ve been on this journey from the beginning, or if you’ve ever gone back to revisit our earlier videos, you may have noticed that things have changed a bit around here.
Everything we can see is a function of the tools we use. And as we’ve explored new microscopes along with other new tools and techniques, that means we’ve been able to see the microcosmos differently. Colors have changed, contrast has deepened, and sometimes things even sparkle.
So today, we’re tracking our journey… through our journey. We have compiled the videos we’ve made that focus on how our microscopes work and the ways we have improved our methods over time so you can see how much of microscopy is an art—an art that uses light in incredible ways that only physics could predict. So to start, let’s go back to an early video, where we went through the different methods we used at the start of our channel to illuminate our samples. Think of it like an introduction to optics, told through the bodies of tardigrades and algae.
This is a ciliate, just a eukaryotic microbe waving its cilia around under our microscope. This is the same ciliate. And yup, here it is again… …and again.
But as you’re probably noticing, while the rough outline of this organism seems the same from shot to shot, the ciliate itself and the world around it clearly look very different from shot to shot. Colors change, details are more apparent. In one case the organism seems lit from within.
On this channel, we're constantly flipping between different ways of capturing images of organisms. So which one of them is what they actually look like? Well...none of them. Anything you see through a microscope is an image —which in our case, means that everything we show you on this channel, every frame, is not the microbial world itself. It’s an interpretation of life on the other side of our objective, translated through the lens into details, shapes, and colors—all affected by the way we light up the life we want to see.
Light is amazing, it’s also very weird. It travels in waves, and as it interacts with particles and materials, it scatters, shifts. Even if we can’t actually see those light waves in motion, so much of what we observe in the world around us is rooted in the physical properties that define those waves—like how we can observe certain frequencies of light as colors. But waves have far more to them than just their frequency, and microscopy has combined the resourcefulness of many different sciences to use light to give us different ways to peer into the microbial world.
So let’s start simple with the good old fashioned white light. Early microscopists used oil lamps and sunlight to see through their microscopes, and while the technology has changed, the simplicity of this has endured into the modern technique of brightfield microscopy. It all starts with a source of light, though modern microscopes have their lamps built in, set up underneath the stage that holds our sample. Light travels from the source through a condenser, which works to focus the light onto the sample above it.
This focused light travels through the sample towards the objective lens, which takes in an image and magnifies it into these bright backgrounds with organisms, sometimes rendered transparent by the intensity of the light. You might say that this is as close as we get to seeing what the microcosmos actually looks like, but that would be like taking 2000 watt light bulb into your living room and saying, “this is what my home looks like.” Light affects things, and we are not shining light on these organisms, we’re shining light through them. Now, brightfield might seem relatively simple, but that simplicity has been incredibly powerful in allowing scientists old and new to wade through microscopic waters.
Still, there are limitations to consider with any scientific technique, and one of the major challenges for brightfield microscopy, particularly when we’re looking at microbes, is contrast. Pigmented organisms are easy to visualize against the bright background, but in cases where the organism has been rendered transparent, it can be harder to distinguish their bodies from the rest of the world they inhabit. Scientists can navigate these challenges using stains that make certain structures more visible, but for our purposes, we like to avoid stains because they can affect the microbes themselves. There are ways to contend with this challenge, one of which is built on one of those simple-yet-strange properties of our world: you don’t always need to shine light directly onto an object to see it.
This technique is called darkfield microscopy, which sounds like it must be the opposite of brightfield microscopy. It’s not. The two techniques are actually very similar: light travels from a source through a condenser, interacts with the sample, and then travels into the objective lens, producing the image we see.
But what we want in darkfield microscopy is for the beam of light to hit the sample, but not our eye. In darkfield microscopy, a circular disk is placed inside the condenser, blocking the central part of the light light from shining through the sample and into our eye, or, our camera. This means that when there is no sample on the slide, all you’ll see is black. But the disk doesn’t block all light: there is still a hollow cone of light that travels around the disk, unable to reach the objective or our eyes, but that light still hits the sample. When it does, their microscopic, transparent bodies scatter those hidden rays into our view. And as they do, an image of their bodies forms against a dark background, providing us with this almost cinematic footage.
Another method to get better contrast than brightfield microscopy is called phase contrast microscopy, and it’s built on working with a property of light that we can’t actually directly experience. Microbes , or really anything, that is easily visually observed with brightfield microscopy are called amplitude objects because as light passes through them, the amplitude of the light wave changes, which we see as changes in light intensity. But there is another class of specimens: these are called phase objects.
As light passes through these objects, the waves slow down and shift slightly in phase compared to the unaffected light around it. And if you’re wondering what that means in terms of what we can see, that’s the issue: our eyes don’t process these differences in phase. And so in the final image, these objects, or in our case, organisms, are very difficult to see.
In the 1930s, a physicist named Frits Zernike developed a method to shift the direct light just slightly enough so that these changes in phase could actually be translated into changes in amplitude, producing an image of these formerly hard-to-see phase objects by essentially treating them as amplitude objects. There is a lot of physics to this that we are glossing over, but the result was so important that it would eventually win Zernike the Nobel Prize in Physics. And of course, selfishly, we appreciate his work because it lets us see more of our more hidden microbial friends. And for the last type of microscopy we’ll go over today, we’re going to be getting into another property of light that we can’t directly see, but that makes the microcosmos glow. Most of the light we see has an electrical field that vibrates in all sorts of planes relative to the direction the light is traveling in.
But that vibration can be restricted to one plane, and when that happens, the light is said to be polarized. We[8] can’t see the difference between polarized and unpolarized light. You might notice the difference in how the world looks when you’re wearing polarized sunglasses, but these are changes brought about by changes in color or intensity, not the polarization of the light itself. So when does polarized light help us in microscopy? Well, a lot of materials stay the same optically-speaking, no matter what direction you shoot light at them with.
But there are certain materials where specific properties, like how fast light travels through them, can vary depending on which way the light is striking them. These materials. called optically anisotropic, can also take in a ray of light and divide it into two separate beams. By aiming polarized light at our sample and then reconstructing an image based on how the various parts of the organism interacts with that restricted light, particularly by how it might cause the light to split, we can see more of these optically anisotropic materials in action. In our case, it often takes the form of shiny crystals. So we’ve given you the big overview of what these different techniques can do, let’s go back and review what that means for the original ciliate we started with. This is the ciliate under brightfield microscopy. The background is bright, the image produced by changes in light amplitude that allow us to see the overall shape. But some of the detail is hard to make out.
Under darkfield though, the contrast increases and some of these details become more obvious, displaying compartments and cilia in greater detail that are also apparent under phase contrast. And then under polarized light, the crystals that blended in with the organism previously are now quite visible and vibrant. There are, of course, many other microscopy techniques, but we think it’s incredible that with just these four, the world of the microcosmos looks almost like different universes, wrapped up in one invisible world around us.
The journey, it seems, is not just about what you see, but how you see it. And ultimately, none of these views are what the microcosmos actually looks like, either that, or all of them are. Our brains play tricks on us to make us believe that the world looks one way, but the world looks different at night than in the day, and both of those things have more to do with the physiology of our eyes than their objective reality.
Asking what microbes look like is, to some extent, forcing our own experience onto something that is beyond it. Which is not something I ever would have thought of if it weren’t for this little YouTube channel. Now we have been really fortunate over the years to have the support of so many people, and to be able to direct that support back to the microscopes we use. In 2020, we introduced James’ new microscope, which he was able to buy thanks to crowdfunding. This was our first major microscope upgrade on the series, and it’s incredible to see the jump in quality and how it compares to the different techniques we showcased in the previous video.
This golden algae is swimming around in water and white light, illuminated from below using a brightfield microscope. And here we have that mallomonas sample, filmed a day later using a different technique thanks to a brand new, freshly installed microscope. When James, our master of microscopes, saw them for the first time, he got a little emotional because, to quote him directly, “the thing I know so well, was looking so so much different than usual.” Those differences in how the mallomonas look are due to a type of microscopy called “Differential Interference Contrast.” Or…DIC.
Earlier this year, James started a crowdfunding campaign to purchase a new microscope, one that would allow him to use differential interference contrast to produce these images you are seeing now. Now technically, “differential interference contrast” is not a type of microscope. Rather, it’s a method that enhances contrast. And as you can see, the final product is an image that seems almost 3-dimensional.
Just watch this stentor coeruleus as it swims across the slide. Its cilia are so prominent that it almost feels like you can touch their vibrating fuzziness. And the striations down its body are so sharp that when the stentor contracts, it’s like you can feel it pushing inward and then back out again.
I don’t know about you, but it’s almost like you could reach out and touch it—like you’re in a movie theater with 3d glasses on, reaching out to something that seems to be reaching back out to you. Differential interference contrast microscopy was invented by Georges Nomarski in 1952, building off the principles underlying phase contrast microscopy, which is a different though similar thing. But both of these methods work to translate invisible shifts in light phase that can happen when studying certain samples into visible changes in light amplitude, which we see as changes in light intensity.
And as a result, both techniques enhance contrast and let us see parts of the microcosmos that might be invisible with regular brightfield microscopy. But phase contrast microscopy and DIC shift and work with light in different ways to accomplish this goal. How do they do it?
Well, if you’ve ever taken an optics course, you will remember how terrible that was. But, very basically, phase contrast and DIC are two ways to take advantage of a strange reality. Light actually travels further through some materials than others.
Kinda. The optical path length is a function of both the distance between two points and the refractive index of the material the light is travelling through. Basically, denser samples have longer optical path lengths. Phase contrast microscopy takes advantage of that, making areas with longer optical path lengths look darker. DIC on the other hand, doesn’t make areas that are denser darker, it uses some very cool optics to create sharp contrast in areas of rapid change in the optical path length. So, the faster the gradient from more to less dense or vice versa, the more contrast you see. But it’s also important to note that while these images are the result of light traveling through a sample, they are not an actual topographical map of an organism. DIC microscopy is a technique, but it does require certain physical additions to a microscope.
For one thing, we need to split light apart. So, first we have a prism that splits polarized light into two orthogonal rays of light that will then pass through the sample and interact with it in different ways. And then there’s a second prism that recombines those two rays after they’ve traveled through the sample, forming an image based on the differences those two beams of light experienced for our eyes and cameras to see.
In addition to these prisms, this technique relies on having higher quality objectives than what we’ve been using in the past. The objectives are the little lenses at the bottom of the microscope that actually do most of the magnification. Not many people would realize this, but the objectives we use for Journey to the Microcosmos are some of the cheapest and most common types of objectives, they’re known as achromatic objectives.
But while these objectives produce so many of our favorite images, James needed a microscope update to be able to do differential interference contrast. So with help, perhaps including some from you, James bought a new Zeiss microscope. It took eight weeks for it to arrive, which is a long time to wait.
But luckily the manufacturers sent him a 150 page manual to occupy his time until it arrived. When the microscope finally showed up, James still had to wait for an engineer to install the nearly 50 pound machine. But he didn’t waste any time. James went out to gather his precious Mallomonas and prepared a slide that night, keeping it stored in a humidity chamber so that he could look at it as soon as the microscope was ready. The next day, the Zeiss engineer took 3.5 hours to put all the pieces together and set everything up.
But from there, well, it was all on James to observe what he could and experiment with his new microscope. Some of his experimenting has been geared towards making sure that he can get the best image possible. These objectives have a much smaller working distance compared to our previous ones, and so you need to prepare the slides a lot thinner to get a sharper image. So even for our master of microscopes, there was somewhat of a learning curve to make sure he was getting videos that were as sharp and vivid as he wanted. But some of the experimenting is based out of that most exciting thing of all: curiosity. He’s removed some of the prisms to see how it affects the footage, and added a magnifying glass over the light source to scatter the light in different ways. That’s the joy of the microcosmos.
It is so infinite. There’s it’s own objective existence, full of so many more organisms than we’ll ever be able to identify. But there’s also the infinite nature of how we experience it, of how our own view of this world is shaped by the many different tools we use to observe it. And maybe that’s the case all the time...that what we see is as much about how we view the world as it is about the world itself. The same thing with a different lens, a different technique, a different base of knowledge, can look completely different.
As we start our third season of Journey to the Microcosmos, we hope you will continue to join us through both our new and old lenses, as we uncover more of this hidden, unending world. In 2021, James went all out on improving his toolkit, investing in higher quality objectives, a polarizer, and the equipment he needed to do fluorescence microscopy. If you’re not sure why those things had all of us on the Journey to the Microcosmos team so excited, well, that’s what our next video is for.
You will get to see just how much of a difference an objective can make, and why electrons jumping up and down can reveal a whole new side to the microcosmos. We’re nearing the end of our fourth season of Journey to the Microcosmos, and over that time, we’ve been able to see microbes through all kinds of lenses—literally—thanks to James, our master of microscopes. For the rest of us on the Microcosmos team, it’s been really cool to not only see the footage that James has recorded, but also see the journey that he has been on with the microscopes themselves. Now, we’ve been able to make this journey because of all of you and the support you have shown to this channel by watching and commenting and sharing with others, or even and especially by supporting us on Patreon. We’ve all been on this journey together, so let’s look back on where it’s taken us, and also get a peek at where we’re going in the future. In the first season, James started out with two microscopes.
One was a microscope that he assembled himself from different parts for under $200. The other microscope was a Motic BA310 that he received from a microscope company shortly after we started this channel. And these microscopes give us so much beautiful footage, thanks in part to the different techniques that James used with them, whether that was bright-field or phase contrast or darkfield, or some other technique to manipulate the light shining on the microcosmos. But these microscopes also had their limits, so we were really excited to see what would happen when James bought a new microscope before the start of our third season: the Zeiss Axioscope 5. This new microscope came with higher quality objectives (which are the lenses on the bottom of the microscope), as well as prisms that made it possible for us to look at microbes with differential interference contrast microscopy, a technique that sharpened their features and made them seem more three-dimensional on our screen. We have videos exploring these different techniques and microscopes, so if you want to learn more about them, we recommend checking those out because light is much weirder than it seems. But we wanted to do a quick summary for reasons that are probably very clear from the title of this video: we have upgraded our microscope!
The Zeiss Axioscope 5 was a very exciting change, but it was also an expensive one. So when James bought it, he was only able to get two objectives: a 20x objective and a 63x objective. Adding that to the 10x magnification the eyepiece gives, let us magnify our samples by 200x and 630x respectively, which is not bad.
But a standard microscope usually comes with 5-6 objectives for a reason: the microcosmos is full of things both small and large to observe and there isn’t a one-size-fits-all objective that you can use to observe everything. So one of James’ priorities was expanding his set of objectives so that he could have the flexibility to see the microcosmos at more scales. And thanks to his recent upgrade, he’s now gone from a set of 2 to a set of six that range from 5x to 100x and that gives us a magnification range from 50x to 1000x. Since I have gotten my own prototype of the microcosmos microscope that we are manufacturing thanks to backers of our kickstarter, I’ve been amazed at how important it is to have this variety of options, and how much time I spend on the lower magnifications and well as going deeper .
So without further ado, let’s introduce these different objectives by looking at a ciliate called Tetrahymena that’s taken up residence in a rotifer exoskeleton. With the 5x objective, we can see little dots swimming in what used to be empty space, but it’s hard to make out the details. When we move to the 10x objective, we can start to make out shapes of the Tetrahymena and more of the emptiness around them, but it’s difficult to see the exoskeleton. Then as we start to go up in magnification, the details of the exoskeleton become clearer. It's cool to see more and more of those details as we zoom in. And especially when we get to the 100x objective, which gives us 1000x magnification, you can make out so much of the insides of the Tetrahymena, that they look like bags of very resilient bubbles. Here’s another ciliate called a Pseudoprorodon that James looked at through all of these objectives.
At first it just looks like a little green thing floating around and knocking into stuff. But as we go up in magnification, we can start to make out the subtle undulations of its body, which shifts the different shades of green. And as we look even closer, the green itself takes on a new life, revealing the perimeters of the individual algae inside the pseudoprorodon. Revealing those details inside the tetrahymena and pseudoprorodon comes at a cost. We can’t see as much of the world around them, whether that’s the emptiness around the rotifer exoskeleton or the objects that the pseudoprorodon keeps bumping into. Whether or not you need those details depends on what you’re trying to learn or what we are trying to show, and that’s why having the flexibility to look through a wider range of objectives is such an exciting upgrade for us.
Objectives are our gateways to the microcosmos, and we’re excited to have all of these new scales to look at. We’ve also made some other upgrades so we can revisit techniques we used on our older microscopes. Like a polarizer, because who doesn’t love this shiny, iridescent stuff?
But we’re not just sticking with what we’ve known. Thanks to this upgrade, we’ve got a whole new way to look at cells: fluorescence microscopy. And we are so excited for this one.
The techniques we’ve highlighted so far are all clever manipulations of white light, a mixture of many visible wavelengths of light. But in fluorescence microscopy, instead of illuminating our sample with that mixture, we choose a specific wavelength of light. That light is called the excitation light because, well, it excites certain structures based on their chemistry. And those excitable structures then emit some light of their own.
The emission light will then travel back through the objective, get past barriers that filter out the excitation light, and then show up in our eyes as vivid color. With our upgrade, we have four different wavelengths of light that we can use for fluorescence microscopy, and the parts of the sample that we see fluoresce will depend on which light we use. In this footage, we’re looking at the Pseudoprorodon that we saw earlier, only this time it’s being hit with 385 nanometer light. The red we see is the fluorescence from natural structures, this is called autofluorescence. The rest of the organism is dark because it did not respond to that 385 nanometer light, making the color all the more vibrant. But the power of fluorescence microscopy is not just finding fluorescent structures that exist in nature. Chemists and biologists have developed all sorts of ingenious techniques to highlight different parts of cells using fluorescent dyes and proteins. James is working on building up his arsenal of fluorescence microscopy tools and techniques so that soon, we’ll be able to dive even deeper into the microcosmos to seek out things that have been hidden from us before, uncovering the unseen world inside the unseen world that surrounds us The upgrades we’ve talked about in the last few videos have involved fancy equipment and exciting techniques.
But light is so weird and powerful that sometimes you don’t need anything particularly fancy to make it do something useful. Sometimes you just need the right objective and a little bit of oil. If you’ve been following along with us lately, you probably know that we’re pretty excited about our latest microscope upgrades.
And can you blame us? It's like having a new toy that’s actually a spaceship. It’s pretty hard to shut up about something that is shiny and new, and also takes you into a whole new world.
And that’s kind of what it feels like to see our familiar friends at this magnification. Even though we’ve seen tardigrades many times before, in so many different ways, we have not seen them like this. And bringing this level of magnification gives us a new layer of the microcosmos to explore. It’s like we’ve descended into a new level of a cave, getting further from our world—though fortunately for the easily scared among us, the cave is metaphorical and the risks are negligible. All we need for the journey are some objective lenses and a tiny drop of... oil? Yeah, oil.
But to explain what that oil does, we’re going to have to get a little technical first. Because yes, we love our microbes, but we’re only able to see them thanks to light, and light is complicated, but we need to give it its due. So let’s start with the objective lens. The main job of the objective lens is to take light leaving the sample and focus it into an image for the us to see.
And there are two main numbers that describe the objective lens. The first one’s pretty obvious: it’s the magnification. But the magnification you see written on the objective lens is not the same as the magnification we write up on the corner of the videos.
That’s because the objective lens isn’t the only source of magnification in our microscope. We’re also looking through an eyepiece that has its own magnification. So the final magnification that we see is the product of the objective lens’ and eyepiece’s magnifications.
Now we don’t usually do math on this channel, but this is one of the rare cases where the math and optics is straightforward. In our microscope, the eyepiece has a magnification of 10x. So if we’re looking through a 63x objective, the final magnification is 630x. And that is the number you’ll see in the corner. But you should never trust an optics lesson that seems simple. How could it be when the image we’re seeing is the product of so many things at once, some tangible like a lens, others much less so. And what is less tangible than light, which is, as we’ve seen before on this channel--a very strange thing.
We cannot touch it, but we can manipulate it, using what we know of the way it travels and bends and reflects to see our world differently. But light… has its limits. The thing about making things larger is that at some point, it’s not enough to just zoom in. You need to be able to capture the detail that’s there. And this leads to the second important number you’ll find on an objective lens: the numerical aperture.
Unlike magnification, “numerical aperture” is probably not super self-explanatory. What the number describes is the ability of the objective to take in light. As light travels from the sample and to the objective, it radiates outwards like a cone. The higher your numerical aperture, the wider the cone of light that's going into your objective.
And that allows for more rays to enter the objective from all sorts of different angles, helping to illuminate more details and give greater resolution to your final image. But of course, that’s still not all that goes into capturing the perfect microscopic image. Let’s take a look at this glaucoma spinning around at 1000x magnification. It’s visible, and you can see some of the details. It looks…fine.
But let’s take a look at it again here. Same glaucoma. Same objective. Same level of magnification.
What was “fine” before now just looks dull in comparison to the image we were seeing before with the detailed striations and vivid pockets of green on the glaucoma’s body. To get from one of these images to the other, James—our master of microscopes—didn’t change any of the technical settings on his microscope or pull some kind of video editing wizardry. He just simply added a drop of oil to the coverslip encasing the glaucoma, and dipped the objective into the oil. Now this is because the cone of light going from the specimen into the objective isn’t just going straight from the sample to the lens.
It’s passing through something—it’s passing through air. Light travels at different speeds through different materials and when it changes from material to material, it will bend. This is called refraction and materials like oil slow light down more. And that matters to our microscope because it gives us the wider cones of light we need to get a higher numerical aperture. Whether we’ve got air between the sample and the lens or oil when we’re viewing our samples depends on the objective we’re looking through. Our lower magnification objectives have lower numerical apertures, and they’re meant to be used with air.
Our higher magnification objectives have higher numerical apertures, and they’re meant to be used with oil. Using air with oil objectives gives us poor images, and using oil with our dry objective would damage the objective. So…don’t do that. The numerical aperture and its relationship to resolution is important because it puts a limit on just how much magnification we can actually do with a given objective.
There’s a general estimate if you are looking into microscopy yourself, that you’ll get a good magnification when you’re working in the range that’s 500 to 1000 times the numerical aperture of the objective. So our 100x objective and 1.3 numerical aperture can reach a maximum of 1300x magnification depending on your eyepiece. With our 10x eyepiece, we get 1000x magnification, and it works great.
But if we tried to get more from this set-up by increasing the magnification of our eyepiece to 20x, we could not actually get 2000x magnification. Because of the constraints of our numerical aperture, we would just be losing half of our image without actually gaining more detail in what’s left to see. But that’s what happens any time we want to take an image: we have to make a decision between what we can and cannot see. There are always choices that have to be made, and details that have to be lost.
We simply cannot see all of the world, in all its entirety, all at once. But we can see more of it by tracking the choices we make as we dive deeper, choices in techniques and materials that affect what we can see and how we see it, and enrich the story further, even when those choices impose constraints. At some point in history, we wanted to see more, and lenses with their magnificent manipulation of light have helped us do that. We’ve used them to see light from distant stars, and to peer into the most mundane surroundings on earth.
And whatever image has come back to us has brought the universe closer to view, even if it’s just in fractions. Thank you for coming on this journey with us as we explore the unseen world that surrounds us. And remember, if you’re watching this before August 8th, you have until then to check out our back to school sale at microcosmos.store. Almost everything in the store is 10% off right now and we have a limited edition Dark Mode Hydra t-shirt that’s only available during the sale.
So check out microcosmos.store before August 8th if you want to get your hands on one. All the people's whose names are coming up on the screen right now, they are our patrons on Patreon. They are the reason that we are able to continue exploring this beautiful unseen world that surrounds us.
There is so much left to explore and I am so excited to continue exploring it with you and all of our patrons. And if you would like to become one of these people you can go to Patreon.com/JounryToMicro. If you want to see more from our Master of Microscopes, James Weiss, and why wouldn't you?
You can check out Jam and Germs on Instagram or on TikTok. And if you want to see more from us, there is always a subscribe button, somewhere nearby.
So whether you are decorating a new dorm room or classroom, or you’re just trying to decide what to wear for your first day of school, microcosmos.store has you covered. We’ve got t-shirts, sweatshirts, pins, and posters. And we’ve got our Microcosmos microscope, slides and coverslips, and even a Microcosmos microfiber cloth to keep everything nice and clean.
And this week only, from now through August 8th, almost everything in the store is 10% off! And that’s not all, we’ve also got this new Dark Mode version of our Hydra t-shirt that's only going to be available during this sale and then never again. So, head over to Microcosmos.store to check out the sale and to pick up your limited edition Dark Mode Hydra T-Shirt This channel would not be what it is if it weren’t for one very key invention: the microscope. Everything we see, we see with the aid of light and lenses, expertly deployed by our master of microscopes, James. And if you’ve been on this journey from the beginning, or if you’ve ever gone back to revisit our earlier videos, you may have noticed that things have changed a bit around here.
Everything we can see is a function of the tools we use. And as we’ve explored new microscopes along with other new tools and techniques, that means we’ve been able to see the microcosmos differently. Colors have changed, contrast has deepened, and sometimes things even sparkle.
So today, we’re tracking our journey… through our journey. We have compiled the videos we’ve made that focus on how our microscopes work and the ways we have improved our methods over time so you can see how much of microscopy is an art—an art that uses light in incredible ways that only physics could predict. So to start, let’s go back to an early video, where we went through the different methods we used at the start of our channel to illuminate our samples. Think of it like an introduction to optics, told through the bodies of tardigrades and algae.
This is a ciliate, just a eukaryotic microbe waving its cilia around under our microscope. This is the same ciliate. And yup, here it is again… …and again.
But as you’re probably noticing, while the rough outline of this organism seems the same from shot to shot, the ciliate itself and the world around it clearly look very different from shot to shot. Colors change, details are more apparent. In one case the organism seems lit from within.
On this channel, we're constantly flipping between different ways of capturing images of organisms. So which one of them is what they actually look like? Well...none of them. Anything you see through a microscope is an image —which in our case, means that everything we show you on this channel, every frame, is not the microbial world itself. It’s an interpretation of life on the other side of our objective, translated through the lens into details, shapes, and colors—all affected by the way we light up the life we want to see.
Light is amazing, it’s also very weird. It travels in waves, and as it interacts with particles and materials, it scatters, shifts. Even if we can’t actually see those light waves in motion, so much of what we observe in the world around us is rooted in the physical properties that define those waves—like how we can observe certain frequencies of light as colors. But waves have far more to them than just their frequency, and microscopy has combined the resourcefulness of many different sciences to use light to give us different ways to peer into the microbial world.
So let’s start simple with the good old fashioned white light. Early microscopists used oil lamps and sunlight to see through their microscopes, and while the technology has changed, the simplicity of this has endured into the modern technique of brightfield microscopy. It all starts with a source of light, though modern microscopes have their lamps built in, set up underneath the stage that holds our sample. Light travels from the source through a condenser, which works to focus the light onto the sample above it.
This focused light travels through the sample towards the objective lens, which takes in an image and magnifies it into these bright backgrounds with organisms, sometimes rendered transparent by the intensity of the light. You might say that this is as close as we get to seeing what the microcosmos actually looks like, but that would be like taking 2000 watt light bulb into your living room and saying, “this is what my home looks like.” Light affects things, and we are not shining light on these organisms, we’re shining light through them. Now, brightfield might seem relatively simple, but that simplicity has been incredibly powerful in allowing scientists old and new to wade through microscopic waters.
Still, there are limitations to consider with any scientific technique, and one of the major challenges for brightfield microscopy, particularly when we’re looking at microbes, is contrast. Pigmented organisms are easy to visualize against the bright background, but in cases where the organism has been rendered transparent, it can be harder to distinguish their bodies from the rest of the world they inhabit. Scientists can navigate these challenges using stains that make certain structures more visible, but for our purposes, we like to avoid stains because they can affect the microbes themselves. There are ways to contend with this challenge, one of which is built on one of those simple-yet-strange properties of our world: you don’t always need to shine light directly onto an object to see it.
This technique is called darkfield microscopy, which sounds like it must be the opposite of brightfield microscopy. It’s not. The two techniques are actually very similar: light travels from a source through a condenser, interacts with the sample, and then travels into the objective lens, producing the image we see.
But what we want in darkfield microscopy is for the beam of light to hit the sample, but not our eye. In darkfield microscopy, a circular disk is placed inside the condenser, blocking the central part of the light light from shining through the sample and into our eye, or, our camera. This means that when there is no sample on the slide, all you’ll see is black. But the disk doesn’t block all light: there is still a hollow cone of light that travels around the disk, unable to reach the objective or our eyes, but that light still hits the sample. When it does, their microscopic, transparent bodies scatter those hidden rays into our view. And as they do, an image of their bodies forms against a dark background, providing us with this almost cinematic footage.
Another method to get better contrast than brightfield microscopy is called phase contrast microscopy, and it’s built on working with a property of light that we can’t actually directly experience. Microbes , or really anything, that is easily visually observed with brightfield microscopy are called amplitude objects because as light passes through them, the amplitude of the light wave changes, which we see as changes in light intensity. But there is another class of specimens: these are called phase objects.
As light passes through these objects, the waves slow down and shift slightly in phase compared to the unaffected light around it. And if you’re wondering what that means in terms of what we can see, that’s the issue: our eyes don’t process these differences in phase. And so in the final image, these objects, or in our case, organisms, are very difficult to see.
In the 1930s, a physicist named Frits Zernike developed a method to shift the direct light just slightly enough so that these changes in phase could actually be translated into changes in amplitude, producing an image of these formerly hard-to-see phase objects by essentially treating them as amplitude objects. There is a lot of physics to this that we are glossing over, but the result was so important that it would eventually win Zernike the Nobel Prize in Physics. And of course, selfishly, we appreciate his work because it lets us see more of our more hidden microbial friends. And for the last type of microscopy we’ll go over today, we’re going to be getting into another property of light that we can’t directly see, but that makes the microcosmos glow. Most of the light we see has an electrical field that vibrates in all sorts of planes relative to the direction the light is traveling in.
But that vibration can be restricted to one plane, and when that happens, the light is said to be polarized. We[8] can’t see the difference between polarized and unpolarized light. You might notice the difference in how the world looks when you’re wearing polarized sunglasses, but these are changes brought about by changes in color or intensity, not the polarization of the light itself. So when does polarized light help us in microscopy? Well, a lot of materials stay the same optically-speaking, no matter what direction you shoot light at them with.
But there are certain materials where specific properties, like how fast light travels through them, can vary depending on which way the light is striking them. These materials. called optically anisotropic, can also take in a ray of light and divide it into two separate beams. By aiming polarized light at our sample and then reconstructing an image based on how the various parts of the organism interacts with that restricted light, particularly by how it might cause the light to split, we can see more of these optically anisotropic materials in action. In our case, it often takes the form of shiny crystals. So we’ve given you the big overview of what these different techniques can do, let’s go back and review what that means for the original ciliate we started with. This is the ciliate under brightfield microscopy. The background is bright, the image produced by changes in light amplitude that allow us to see the overall shape. But some of the detail is hard to make out.
Under darkfield though, the contrast increases and some of these details become more obvious, displaying compartments and cilia in greater detail that are also apparent under phase contrast. And then under polarized light, the crystals that blended in with the organism previously are now quite visible and vibrant. There are, of course, many other microscopy techniques, but we think it’s incredible that with just these four, the world of the microcosmos looks almost like different universes, wrapped up in one invisible world around us.
The journey, it seems, is not just about what you see, but how you see it. And ultimately, none of these views are what the microcosmos actually looks like, either that, or all of them are. Our brains play tricks on us to make us believe that the world looks one way, but the world looks different at night than in the day, and both of those things have more to do with the physiology of our eyes than their objective reality.
Asking what microbes look like is, to some extent, forcing our own experience onto something that is beyond it. Which is not something I ever would have thought of if it weren’t for this little YouTube channel. Now we have been really fortunate over the years to have the support of so many people, and to be able to direct that support back to the microscopes we use. In 2020, we introduced James’ new microscope, which he was able to buy thanks to crowdfunding. This was our first major microscope upgrade on the series, and it’s incredible to see the jump in quality and how it compares to the different techniques we showcased in the previous video.
This golden algae is swimming around in water and white light, illuminated from below using a brightfield microscope. And here we have that mallomonas sample, filmed a day later using a different technique thanks to a brand new, freshly installed microscope. When James, our master of microscopes, saw them for the first time, he got a little emotional because, to quote him directly, “the thing I know so well, was looking so so much different than usual.” Those differences in how the mallomonas look are due to a type of microscopy called “Differential Interference Contrast.” Or…DIC.
Earlier this year, James started a crowdfunding campaign to purchase a new microscope, one that would allow him to use differential interference contrast to produce these images you are seeing now. Now technically, “differential interference contrast” is not a type of microscope. Rather, it’s a method that enhances contrast. And as you can see, the final product is an image that seems almost 3-dimensional.
Just watch this stentor coeruleus as it swims across the slide. Its cilia are so prominent that it almost feels like you can touch their vibrating fuzziness. And the striations down its body are so sharp that when the stentor contracts, it’s like you can feel it pushing inward and then back out again.
I don’t know about you, but it’s almost like you could reach out and touch it—like you’re in a movie theater with 3d glasses on, reaching out to something that seems to be reaching back out to you. Differential interference contrast microscopy was invented by Georges Nomarski in 1952, building off the principles underlying phase contrast microscopy, which is a different though similar thing. But both of these methods work to translate invisible shifts in light phase that can happen when studying certain samples into visible changes in light amplitude, which we see as changes in light intensity.
And as a result, both techniques enhance contrast and let us see parts of the microcosmos that might be invisible with regular brightfield microscopy. But phase contrast microscopy and DIC shift and work with light in different ways to accomplish this goal. How do they do it?
Well, if you’ve ever taken an optics course, you will remember how terrible that was. But, very basically, phase contrast and DIC are two ways to take advantage of a strange reality. Light actually travels further through some materials than others.
Kinda. The optical path length is a function of both the distance between two points and the refractive index of the material the light is travelling through. Basically, denser samples have longer optical path lengths. Phase contrast microscopy takes advantage of that, making areas with longer optical path lengths look darker. DIC on the other hand, doesn’t make areas that are denser darker, it uses some very cool optics to create sharp contrast in areas of rapid change in the optical path length. So, the faster the gradient from more to less dense or vice versa, the more contrast you see. But it’s also important to note that while these images are the result of light traveling through a sample, they are not an actual topographical map of an organism. DIC microscopy is a technique, but it does require certain physical additions to a microscope.
For one thing, we need to split light apart. So, first we have a prism that splits polarized light into two orthogonal rays of light that will then pass through the sample and interact with it in different ways. And then there’s a second prism that recombines those two rays after they’ve traveled through the sample, forming an image based on the differences those two beams of light experienced for our eyes and cameras to see.
In addition to these prisms, this technique relies on having higher quality objectives than what we’ve been using in the past. The objectives are the little lenses at the bottom of the microscope that actually do most of the magnification. Not many people would realize this, but the objectives we use for Journey to the Microcosmos are some of the cheapest and most common types of objectives, they’re known as achromatic objectives.
But while these objectives produce so many of our favorite images, James needed a microscope update to be able to do differential interference contrast. So with help, perhaps including some from you, James bought a new Zeiss microscope. It took eight weeks for it to arrive, which is a long time to wait.
But luckily the manufacturers sent him a 150 page manual to occupy his time until it arrived. When the microscope finally showed up, James still had to wait for an engineer to install the nearly 50 pound machine. But he didn’t waste any time. James went out to gather his precious Mallomonas and prepared a slide that night, keeping it stored in a humidity chamber so that he could look at it as soon as the microscope was ready. The next day, the Zeiss engineer took 3.5 hours to put all the pieces together and set everything up.
But from there, well, it was all on James to observe what he could and experiment with his new microscope. Some of his experimenting has been geared towards making sure that he can get the best image possible. These objectives have a much smaller working distance compared to our previous ones, and so you need to prepare the slides a lot thinner to get a sharper image. So even for our master of microscopes, there was somewhat of a learning curve to make sure he was getting videos that were as sharp and vivid as he wanted. But some of the experimenting is based out of that most exciting thing of all: curiosity. He’s removed some of the prisms to see how it affects the footage, and added a magnifying glass over the light source to scatter the light in different ways. That’s the joy of the microcosmos.
It is so infinite. There’s it’s own objective existence, full of so many more organisms than we’ll ever be able to identify. But there’s also the infinite nature of how we experience it, of how our own view of this world is shaped by the many different tools we use to observe it. And maybe that’s the case all the time...that what we see is as much about how we view the world as it is about the world itself. The same thing with a different lens, a different technique, a different base of knowledge, can look completely different.
As we start our third season of Journey to the Microcosmos, we hope you will continue to join us through both our new and old lenses, as we uncover more of this hidden, unending world. In 2021, James went all out on improving his toolkit, investing in higher quality objectives, a polarizer, and the equipment he needed to do fluorescence microscopy. If you’re not sure why those things had all of us on the Journey to the Microcosmos team so excited, well, that’s what our next video is for.
You will get to see just how much of a difference an objective can make, and why electrons jumping up and down can reveal a whole new side to the microcosmos. We’re nearing the end of our fourth season of Journey to the Microcosmos, and over that time, we’ve been able to see microbes through all kinds of lenses—literally—thanks to James, our master of microscopes. For the rest of us on the Microcosmos team, it’s been really cool to not only see the footage that James has recorded, but also see the journey that he has been on with the microscopes themselves. Now, we’ve been able to make this journey because of all of you and the support you have shown to this channel by watching and commenting and sharing with others, or even and especially by supporting us on Patreon. We’ve all been on this journey together, so let’s look back on where it’s taken us, and also get a peek at where we’re going in the future. In the first season, James started out with two microscopes.
One was a microscope that he assembled himself from different parts for under $200. The other microscope was a Motic BA310 that he received from a microscope company shortly after we started this channel. And these microscopes give us so much beautiful footage, thanks in part to the different techniques that James used with them, whether that was bright-field or phase contrast or darkfield, or some other technique to manipulate the light shining on the microcosmos. But these microscopes also had their limits, so we were really excited to see what would happen when James bought a new microscope before the start of our third season: the Zeiss Axioscope 5. This new microscope came with higher quality objectives (which are the lenses on the bottom of the microscope), as well as prisms that made it possible for us to look at microbes with differential interference contrast microscopy, a technique that sharpened their features and made them seem more three-dimensional on our screen. We have videos exploring these different techniques and microscopes, so if you want to learn more about them, we recommend checking those out because light is much weirder than it seems. But we wanted to do a quick summary for reasons that are probably very clear from the title of this video: we have upgraded our microscope!
The Zeiss Axioscope 5 was a very exciting change, but it was also an expensive one. So when James bought it, he was only able to get two objectives: a 20x objective and a 63x objective. Adding that to the 10x magnification the eyepiece gives, let us magnify our samples by 200x and 630x respectively, which is not bad.
But a standard microscope usually comes with 5-6 objectives for a reason: the microcosmos is full of things both small and large to observe and there isn’t a one-size-fits-all objective that you can use to observe everything. So one of James’ priorities was expanding his set of objectives so that he could have the flexibility to see the microcosmos at more scales. And thanks to his recent upgrade, he’s now gone from a set of 2 to a set of six that range from 5x to 100x and that gives us a magnification range from 50x to 1000x. Since I have gotten my own prototype of the microcosmos microscope that we are manufacturing thanks to backers of our kickstarter, I’ve been amazed at how important it is to have this variety of options, and how much time I spend on the lower magnifications and well as going deeper .
So without further ado, let’s introduce these different objectives by looking at a ciliate called Tetrahymena that’s taken up residence in a rotifer exoskeleton. With the 5x objective, we can see little dots swimming in what used to be empty space, but it’s hard to make out the details. When we move to the 10x objective, we can start to make out shapes of the Tetrahymena and more of the emptiness around them, but it’s difficult to see the exoskeleton. Then as we start to go up in magnification, the details of the exoskeleton become clearer. It's cool to see more and more of those details as we zoom in. And especially when we get to the 100x objective, which gives us 1000x magnification, you can make out so much of the insides of the Tetrahymena, that they look like bags of very resilient bubbles. Here’s another ciliate called a Pseudoprorodon that James looked at through all of these objectives.
At first it just looks like a little green thing floating around and knocking into stuff. But as we go up in magnification, we can start to make out the subtle undulations of its body, which shifts the different shades of green. And as we look even closer, the green itself takes on a new life, revealing the perimeters of the individual algae inside the pseudoprorodon. Revealing those details inside the tetrahymena and pseudoprorodon comes at a cost. We can’t see as much of the world around them, whether that’s the emptiness around the rotifer exoskeleton or the objects that the pseudoprorodon keeps bumping into. Whether or not you need those details depends on what you’re trying to learn or what we are trying to show, and that’s why having the flexibility to look through a wider range of objectives is such an exciting upgrade for us.
Objectives are our gateways to the microcosmos, and we’re excited to have all of these new scales to look at. We’ve also made some other upgrades so we can revisit techniques we used on our older microscopes. Like a polarizer, because who doesn’t love this shiny, iridescent stuff?
But we’re not just sticking with what we’ve known. Thanks to this upgrade, we’ve got a whole new way to look at cells: fluorescence microscopy. And we are so excited for this one.
The techniques we’ve highlighted so far are all clever manipulations of white light, a mixture of many visible wavelengths of light. But in fluorescence microscopy, instead of illuminating our sample with that mixture, we choose a specific wavelength of light. That light is called the excitation light because, well, it excites certain structures based on their chemistry. And those excitable structures then emit some light of their own.
The emission light will then travel back through the objective, get past barriers that filter out the excitation light, and then show up in our eyes as vivid color. With our upgrade, we have four different wavelengths of light that we can use for fluorescence microscopy, and the parts of the sample that we see fluoresce will depend on which light we use. In this footage, we’re looking at the Pseudoprorodon that we saw earlier, only this time it’s being hit with 385 nanometer light. The red we see is the fluorescence from natural structures, this is called autofluorescence. The rest of the organism is dark because it did not respond to that 385 nanometer light, making the color all the more vibrant. But the power of fluorescence microscopy is not just finding fluorescent structures that exist in nature. Chemists and biologists have developed all sorts of ingenious techniques to highlight different parts of cells using fluorescent dyes and proteins. James is working on building up his arsenal of fluorescence microscopy tools and techniques so that soon, we’ll be able to dive even deeper into the microcosmos to seek out things that have been hidden from us before, uncovering the unseen world inside the unseen world that surrounds us The upgrades we’ve talked about in the last few videos have involved fancy equipment and exciting techniques.
But light is so weird and powerful that sometimes you don’t need anything particularly fancy to make it do something useful. Sometimes you just need the right objective and a little bit of oil. If you’ve been following along with us lately, you probably know that we’re pretty excited about our latest microscope upgrades.
And can you blame us? It's like having a new toy that’s actually a spaceship. It’s pretty hard to shut up about something that is shiny and new, and also takes you into a whole new world.
And that’s kind of what it feels like to see our familiar friends at this magnification. Even though we’ve seen tardigrades many times before, in so many different ways, we have not seen them like this. And bringing this level of magnification gives us a new layer of the microcosmos to explore. It’s like we’ve descended into a new level of a cave, getting further from our world—though fortunately for the easily scared among us, the cave is metaphorical and the risks are negligible. All we need for the journey are some objective lenses and a tiny drop of... oil? Yeah, oil.
But to explain what that oil does, we’re going to have to get a little technical first. Because yes, we love our microbes, but we’re only able to see them thanks to light, and light is complicated, but we need to give it its due. So let’s start with the objective lens. The main job of the objective lens is to take light leaving the sample and focus it into an image for the us to see.
And there are two main numbers that describe the objective lens. The first one’s pretty obvious: it’s the magnification. But the magnification you see written on the objective lens is not the same as the magnification we write up on the corner of the videos.
That’s because the objective lens isn’t the only source of magnification in our microscope. We’re also looking through an eyepiece that has its own magnification. So the final magnification that we see is the product of the objective lens’ and eyepiece’s magnifications.
Now we don’t usually do math on this channel, but this is one of the rare cases where the math and optics is straightforward. In our microscope, the eyepiece has a magnification of 10x. So if we’re looking through a 63x objective, the final magnification is 630x. And that is the number you’ll see in the corner. But you should never trust an optics lesson that seems simple. How could it be when the image we’re seeing is the product of so many things at once, some tangible like a lens, others much less so. And what is less tangible than light, which is, as we’ve seen before on this channel--a very strange thing.
We cannot touch it, but we can manipulate it, using what we know of the way it travels and bends and reflects to see our world differently. But light… has its limits. The thing about making things larger is that at some point, it’s not enough to just zoom in. You need to be able to capture the detail that’s there. And this leads to the second important number you’ll find on an objective lens: the numerical aperture.
Unlike magnification, “numerical aperture” is probably not super self-explanatory. What the number describes is the ability of the objective to take in light. As light travels from the sample and to the objective, it radiates outwards like a cone. The higher your numerical aperture, the wider the cone of light that's going into your objective.
And that allows for more rays to enter the objective from all sorts of different angles, helping to illuminate more details and give greater resolution to your final image. But of course, that’s still not all that goes into capturing the perfect microscopic image. Let’s take a look at this glaucoma spinning around at 1000x magnification. It’s visible, and you can see some of the details. It looks…fine.
But let’s take a look at it again here. Same glaucoma. Same objective. Same level of magnification.
What was “fine” before now just looks dull in comparison to the image we were seeing before with the detailed striations and vivid pockets of green on the glaucoma’s body. To get from one of these images to the other, James—our master of microscopes—didn’t change any of the technical settings on his microscope or pull some kind of video editing wizardry. He just simply added a drop of oil to the coverslip encasing the glaucoma, and dipped the objective into the oil. Now this is because the cone of light going from the specimen into the objective isn’t just going straight from the sample to the lens.
It’s passing through something—it’s passing through air. Light travels at different speeds through different materials and when it changes from material to material, it will bend. This is called refraction and materials like oil slow light down more. And that matters to our microscope because it gives us the wider cones of light we need to get a higher numerical aperture. Whether we’ve got air between the sample and the lens or oil when we’re viewing our samples depends on the objective we’re looking through. Our lower magnification objectives have lower numerical apertures, and they’re meant to be used with air.
Our higher magnification objectives have higher numerical apertures, and they’re meant to be used with oil. Using air with oil objectives gives us poor images, and using oil with our dry objective would damage the objective. So…don’t do that. The numerical aperture and its relationship to resolution is important because it puts a limit on just how much magnification we can actually do with a given objective.
There’s a general estimate if you are looking into microscopy yourself, that you’ll get a good magnification when you’re working in the range that’s 500 to 1000 times the numerical aperture of the objective. So our 100x objective and 1.3 numerical aperture can reach a maximum of 1300x magnification depending on your eyepiece. With our 10x eyepiece, we get 1000x magnification, and it works great.
But if we tried to get more from this set-up by increasing the magnification of our eyepiece to 20x, we could not actually get 2000x magnification. Because of the constraints of our numerical aperture, we would just be losing half of our image without actually gaining more detail in what’s left to see. But that’s what happens any time we want to take an image: we have to make a decision between what we can and cannot see. There are always choices that have to be made, and details that have to be lost.
We simply cannot see all of the world, in all its entirety, all at once. But we can see more of it by tracking the choices we make as we dive deeper, choices in techniques and materials that affect what we can see and how we see it, and enrich the story further, even when those choices impose constraints. At some point in history, we wanted to see more, and lenses with their magnificent manipulation of light have helped us do that. We’ve used them to see light from distant stars, and to peer into the most mundane surroundings on earth.
And whatever image has come back to us has brought the universe closer to view, even if it’s just in fractions. Thank you for coming on this journey with us as we explore the unseen world that surrounds us. And remember, if you’re watching this before August 8th, you have until then to check out our back to school sale at microcosmos.store. Almost everything in the store is 10% off right now and we have a limited edition Dark Mode Hydra t-shirt that’s only available during the sale.
So check out microcosmos.store before August 8th if you want to get your hands on one. All the people's whose names are coming up on the screen right now, they are our patrons on Patreon. They are the reason that we are able to continue exploring this beautiful unseen world that surrounds us.
There is so much left to explore and I am so excited to continue exploring it with you and all of our patrons. And if you would like to become one of these people you can go to Patreon.com/JounryToMicro. If you want to see more from our Master of Microscopes, James Weiss, and why wouldn't you?
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