This is a ciliate, just a eukaryotic microbe waving its cilia around under our microscope.

And 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. Colors change, details are more apparent. In one case the organism seems to be 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 the life on the other side of our objective, translated through the lens into details, and 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 and 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. 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 a 2000 watt light bulb into your living room and saying, “this is what my home looks like.” Light affects things, and we’re not even 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 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 other ways though 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 almost the opposite of brightfield microscopy. It’s not. The two techniques are actually very similar: light travels from a source through a condenser, goes through the sample, and then it 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. So, in darkfield microscopy, a circular disk is placed inside the condenser, blocking the central part of the 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 see is black.

But the disk doesn’t block all of the light: there is still a hollow cone of light that travels around the disk, unable to reach the objective or our eyes, but that still hits the sample. When it does, those 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.

Well, 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 in this that we are not going to get into, but the result was so important that it would eventually win Zernike the Nobel Prize in Physics. And of course, selfishly, we here 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, and this one can make 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 just one plane, and when that happens, the light is said to be polarized.

We can’t see the difference between polarized and unpolarized light. Now you might see the difference in how the world looks when you’re wearing polarized sunglasses, but these changes are brought about by changes in color or intensity, not the polarization of the light itself. So when does polarized light help microscopy?

Well, a lot of materials stay the same optically-speaking, no matter what direction you shoot light at them from. 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 that light to split, we can see more of these optically anisotropic materials in action. In our case, it often takes the form of shiny internal crystals. So we’ve given you the big overview of what these different techniques can do, let’s go back and look at what that means for that original ciliate we started with.

Here it is 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 visible and vibrant. Now, of course, there are many other microscopy techniques that use light in many different ways, but we think it’s incredible that with just these four, the world of the microcosmos looks almost like different universes, wrapped up into one invisible world around us.

The journey, it seems, is not just about what you see, but also 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 and our brains than with objective reality.

Asking what a microbe actually looks 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. This is the last episode of our first season of Journey to the Microcosmos.

It’s been really wonderful, and don’t worry, we’re just going to take a week off and then we will be back with our second season, featuring more of our microbial buddies and their various antics. Thank you for coming on this journey with us as we explore the unseen world that surrounds us. And a special thank you to all of these people, our patrons on Patreon.

Who make it possible for us to take such a deep and interested look at this wonderful world. Thank you everybody for being a part of that. If you want to see more from our Master of Microbes, James, you can check out Jam and Germs on Instagram.

And if you want to be here and ready for next season, go to YouTube.com/microcosmos