So you see it uses changes in the phase of light.
Other microscopes use changes in the polarity of the light.
You see when I have unpolarized light, classical light coming from a light bulb,
I have wavelength and I have the light oscillating in different planes.
As you can see here, there are four planes that I indicate
for the oscillation of the light waves.
There are devices that are called polarizers.
It's kind of a filter that will let only light oscillating in a certain plane through.
Then I get polarized light.
And if for example I then would use a second polarizer,
but which would select a wavelength that is like at 90 degree angle from this one,
at the end I would stop completely.
The light would get nothing at the exit.
But here I will use it just to talk about the polarization of light.
Because there is a second device to generate contrast in non-stained specimen,
and that one is called the interference contrast.
Again, the detailed physics are quite complicated of this, so we will not talk about this.
But what is here, you can see you first polarize the light that comes from the light source.
You will polarize it and then you send it to a condenser prism here,
what is called the Wollastononomarski prism.
And what this happens is they are polarized initially like this,
and they will be polarized, it separates it into two polarization planes.
So not actually in two different light paths, but two polarization planes.
So we have the sheared light waves here.
They will go through the sample and they will interact with the sample.
And this leads to the fact that when at the end here,
to a second Nomarski prism or Wollaston prism,
I join my wavelengths again.
They will have shifted a little bit in the polarization plane or in the amplitude,
and it's the interference between these that will generate contrast.
I will then use a second analyzer, polarizer here,
and by turning that one, I will be able to get more or less contrast in my sample.
So it's a bit complicated.
Remember, it uses different polarization planes
that are altered when the light goes through the sample.
And when you then bring the two orientations back together,
this leads to interferences, and these interferences allow you to generate contrast.
This would be an interference microscope.
Actually, this one is a critical fluorescence too.
And you see down here you have the polarizer,
the initial polarizer, you can set the polarization orientation for that one.
A first prism which is here, a second prism which comes here after the objective,
and then you have the analyzer prism up here, which you can move,
and by moving them you can initially, they are oriented in 90 degrees angle,
and by moving them you can choose how much contrast you want to generate.
The images that you get look like this.
These are living human cells from the cheek, which you can easily take yourself.
You just scratch inside your cheek, put it on the glass slide,
put a little bit of water or physiological saline,
and cover slick on it, and you will see this if you have an interference contrast device.
And you can see what is typical of interference contrast is this impression of 3D,
this impression of a relief of a volume.
But you can already guess something is wrong with this.
If I look at it like this, I have the feeling that the nucleus would really
suddenly come out of the cell. We know that this is not the case.
This impression of relief is like an optical illusion because what you have,
if you look at these dots here, there is one dark side and the other side is bright,
so you get the feeling that the light is coming here from the top right.
And this creates this impression of relief.
And what this relief does is particularly accentuated at the transitions from the cell to the rest.
You can see here.
So it is very precise to give you an idea about where the cell stops,
and if we had filopolia here, you would see them much better than with the face contrast.
And you can see this here, the comparison between on the top, you have the face contrast
and the interference contrast of the same sample.
You see here, eritrocytes viewed with face contrast with the strong halo that you can see,
and here you have the interference contrast.
These are cultured cells here, and you can see here the image,
the nucleus actually, the nuclei here, are easily a better visualized with the face contrast,
whereas the edges of the cells are better visualized without the halo of the face contrast when used interference contrast.
These are zignima algae, these are algae visualized either in face contrast or with interference contrast,
generating this impression of a 3D relief.
Interference contrast, so as you can see, is very interesting,
but it is way more expensive to install on a microscope than a face contrast.
So this is why the face contrast is the preferred contrast medium for cell culture.
Now all of this is what we call bright-feed microscopy.
Bright-feed-mocoscopy, why? Because we send light that traverses the sample,
gets into the lens and into the optic system.
There is another mode, very interesting as you will see, which is called dark-feed microscopy.
The previous one is called bright-feed because the background of your image is bright.
Now dark-feed microscopy works the reverse way.
It doesn't use any light, no. It uses light, but you actually generate a light cone,
which is hollow. Inside here it's dark, there is no light.
And this light you send on the sample, and the whole thing is designed in such a way
that if there is no sample, the light, because you generate a second hollow cone here on top.
Well, this light, all the light is sent outside of the front lens.
So it doesn't go into the lens. So this seems a bit strange, if I think, what is the purpose of this?
Well, the purpose is this, the light normally will go not into the objective, so your background will be black.
But if there is a particle on the way, or anything on the way, that deviates the light only slightly,
the light will go into the optic system, and you will see a bright spot in your image.
This is called dark-feed microscopy.
The images that you get with this, here first you see this special condenser called a caduate condenser.
You see it uses a convex mirror that reflects the light on the outside here concave mirror.
This generates this dark cone inside of the cone here that will go through here,
and only light that is deviated by sample on the specimen will be sent into the objective.
The rest will go to the outside.
And these are the types of images that you get with it. These are diatoms.
So this is actually dead diatoms, meaning this is the silica glass box in which the single set of organisms live.
This is a mosquito head visualized in this.
This dark-feed microscopy has had a special use, especially in neuroscience.
Because in neuroscience, we often put into our sample radiative molecules.
This can be, for example, a radiative acid case here, a radiative RNA that is complementary to the pre-provisopressin messenger RNA,
which is present in cells producing vasopressin.
So this radiative probe will be able to bind because it's complementary to the messenger RNA to the messenger RNA in these cells.
So the question then is, how do I detect the radiative cells?
Sometimes we use other radiative molecules, like radiative femiting to detect cells that have been dividing.
Okay, today we do this mostly with bromodiac security.
But as a possibility, you can use other radiative tracer, metabolic tracers that get into the cells.
You want to see which cells took up this tracer, which was used to identify neurotransmitter cells.
If you put GABA, radiative GABA on the cells, only cells that have GABA re-uptake mechanism, mostly GABA-eugenic neurons themselves, will take up the GABA.
And so, how do we see where is radiativity?
Well, simply after we do the sections, we put a liquid film onto the slide, and we let that one dry.
We expose it, and after exposure, the radiative has created these black dots, which are actually silver particles that are in the film.
And you can see this is this slide observed in bright field, the same one observed in dark field.
And you can see the dark field allows me to much better see here these dots.
You will see this particular look at this dot here.
I barely see it in bright field, but in dark field, this dot is very visible, as you can see here.
There.
So, this technique, this dark field microscopy allows us to see where this radiative signal is situated in the tissue.
And additionally, it generates tremendously beautiful images like here,
this mid-microstarias rotata cells dividing, so very spectacular.
And many of the images that are each here submitted to the Nikon, small world microphotographic competition,
many of these images are generated with dark field microscopy.
Like here, these are daphnia, so small crystallins that can be visualized, and this one is living,
so you can see them very well here with dark field microscopy.
Then we have the polarizing microscope.
So, I showed this slide already to show you what polarization means.
And in the polarizing microscope, I illuminate my sample only with polarized light.
Light oscillating in a specific orientation, and then I can use a second polarizer to look at,
if maybe in my sample there is something that has changed the polarization of the light that I sent to.
Initially, all the light is oscillating in one specific plane.
And what can happen is, in my sample, there is something that interacts with the light,
and that we switch the polarization plane, may twist it.
You can see here we have this one would be the analyzer, which is the red light,
and the other one is the polarizer.
Blue indicates the initial polarizing orientation.
Now, if this is the original one, if I turn the analyzer now,
you see, if they are in the same direction, I get 100% of the light that goes through.
If I turn it a little bit, the analyzer, the orientation of the analyzer,
I get less and less light going through the analyzer until when the orientation of the two is perpendicular,
90 degree angle, no more light gets through, unless in my sample something
made the polarization turn a little bit.
So, if I have a polarization microscope, I have a polarizer down here,
which I can move a little bit, but I have the analyzer, which is the same type of filter,
a polarizing filter, which is up here.
You have a knob here that allows you to turn, it's called the analyzer,
because this one I can now turn and see if I can extinguish some of the light,
the polarized light that goes through the sample.
Often you will also have a rotating stage here, so that you can turn the sample also.
And this generates images like these.
This is vitamin C.
The polarizing microscope is used a lot in crystallography.
Now, why do I get these different colors?
Because when the polarized light goes through, initially it's a white polarized light of many wavelengths,
well, the wavelengths in the different parts of the crystals are put into different orientation,
and so, for example, with the analyzer here, only the orange wavelength state in the original polarization plane.
The others have been shifted, or he is only the blue one.
This would be another type of crystal. This is rock.
Rock can be polished into thin slices, and that's what people do in geology,
or for that matter, other materials can be sliced.
And usually we biologists have a thinking that we are the main users of microscopes.
If you ask the people who sell microscopes, they will tell you that material science and geologists,
well, they are much bigger users of photonic microscopy, and particularly polarizing microscopy,
because you see here it allows us to analyze the crystal structure of the rock if you make a thin slice,
where light can still go through the rock molecules.
In biology, it can be interesting because here you have the same image viewed in interference contrast,
and as you guess, this is a mitotic spindle here, with the crumble zones here.
In the polarizing microscope, if I turn these mitoses properly
so that the orientation here of the spindle microtubules corresponds to the plane of polarization
of the light, and I am sending to it, here in the mitotic spindle acts a little bit like a polarizing filter.
Here it lets the light through, but here we have microtubules that go at 75 at 90 degree angle,
and you can see it's dark because the light here does not get through,
and these microtubules, they stop all the light, and so this allows us to analyze,
to see, for example, very organized structures in photonic microscopy.
One particular use in neuroscience in neuropathology is a stain called Congoret,
which is a stain that stains amyloid deposits, one of the Halmer collisions among others above Alzheimer's disease.
On the top you have the bright field view, and you see this pink staining of the Congoret of these protein deposits,
but the real diagnostic criterion appears in polarized microscopy,
because then you get what is called the b-refringent, and you see you get images like these from this one,
which is called a multiscross, and we get these multiscross b-refringents in polarized light,
then this is a diagnostic criterion to say this is indeed an amyloid deposit.