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
“Our brain plays tricks on us to make us believe that the world looks one way, But the world looks different at night than in the day”
Love that.
Also, never heard Hank speak so slowly. Didn’t think it was possible!
This is such a cool video!
I would also add that light doesnt just affect how we see them, it also affects the microbes themselves. Under white light for instance some organisims become very active and you can see they chloroplasts working. The heat they recieve from the lamp also makes them more active (or in some cases, less active). So its not just how we see them that is a constructed image simmilar to reality but not reality itself, the microbes we see are also not behaving or reacting the same way they would in their natural environment. Its all really simmilar and close to reality, yet it's never reality itself that we see.
Thank you so much for sharing this it was so amazing. I study biochemistry and absolutely adore this kind of content so thank you for letting me find it!!
Very beautiful illustration of microscopy methods! This should be shown in school & university to generate interest and of course for the educational part.
This was truly beautiful, as a mechanical engineer i can now say i'm even more curious about biology than i ever was.
I love that channel
I like fluorescent microscopy.