Microscopy School Lesson 2 – Transmitted Light Microscopy
Although recent advances have offered researchers many sophisticated imaging technologies, transmitted light microscopy techniques are still applicable to a wide variety of research areas. Transmitted light microscopy images are useful to analyse the morphological features of biological samples. Furthermore, transmitted light techniques also they deliver an “extra channel” that can provide context to the fluorescence stainings. Importantly, due to the very low energy use in transmitted light microscopy techniques, they are extremely adequate for live imaging experiments. Therefore, knowledge of transmitted light microscopy techniques; its differences and applications are a valuable tool for researchers.
In this seminar, Dr Claudia Florindo (Life Science Specialist, Andor Technology) will review the anatomy of the microscope and essential concepts in microscopy, such as numerical aperture and resolution. We will explain the bases of the differences between the different transmitted light techniques. Finally, to give a clear understanding between the optical requirement for the techniques and the resulting image, we will show images acquired with the different techniques.
Overall, we aim that students attending this class, can both understand what is transmitted light microscopy, and discover what the best technique for their application is.
Learning Objectives: 1. Recall the anatomy of the microscope. 2. Recognize different transmitted light microscopy techniques. 3. Choose the best-transmitted light microscopy technique for your application.
Questions Answered: - How does the different correction in objectives impact your image? - What is the NA (Numerical aperture)? - How can I have even illumination of my sample?
Hello. Thank you very much for joining the Andor Microscopy School. I will be talking to you today about transmitted light microscopy. So what is transmitted light microscopy? Transmitted light microscopy means that the light passes is transmitted from the source to the lens. And by doing so it also passes through the sample. This method is very useful to distinguish morphological characteristics and optics of the observed material. It also provides an extra channel that offers the context to fluorescence staining. And most importantly, due to the very low energy used in transmitted light microscopy techniques, they are extremely useful for live imaging experiments. So it's a good technique to bear in mind. And when chosen appropriately, the transmitted light microscopy technique to use can be extremely useful for your research applications.
In today's webinar, I will talk about...I've divided it in two parts. And the first part will be on general concepts in microscopy. And I will talk about optical equipment, anatomy of the microscope, what is inside an objective, optical aberrations, and resolution in microscopy. And then I will talk about transmitted light microscopy techniques. I will explain the brightfield, darkfield, phase contrast, and DIC techniques. So, starting from the first part of our webinar, general concepts in microscopy. Before starting to introduce most of the general concepts in microscopy that I would like to talk to you today, I would please stop first to talk to you about the precautions you need to take with the optical equipment.
The optical equipment is prone to damage and is sensitive and once damaged, you can destroy the quality of your images forever. So please be aware, never stain, twist, or drop the objectives. Do not force the controls of the objective of the condenser. And always watch the lens surfaces as they approach the specimen in order not to damage irreversible, an objective. And do not touch the optical surfaces with your own hands. So please be aware, when you're doing a microscopy experiment, there's a sentence that's garbage in equals garbage out. You will not be able to have a nice image if you don't have a very well prepared sample. And to have a very well prepared sample, everything counts, from when you fix your sample, when you stain your sample, and where you mount your sample. So the cover slips of your slides and the thickness of your slides and cover slips will count. So choose glass slides with one millimeter thick.
And the objectives are designed in a way that they are optimized for 0.17 millimeter thickness of glass. So the ideal grade for most of the objectives is 1.5. And you can always check which kind of objective you're using if by any chance it's different. For super resolution, be aware to choose to 1,5H objectives, high quality objectives, which will have a thickness even more stringent for 0.17 to 0.18 millimeter thick. So please remember, everything counts when you're preparing your sample. The thickness of your cover slips and slides will count and will influence the final good quality of your image.
So going through the anatomy of a microscope, what are the names of all these parts on the microscope? So these are the eyepieces. This is the turret or nosepiece. And here are the objectives. This is the microscope stage and the sample holder. The large button, it's the coarse focus, and the small one is the fine focus that will allow you to coarse focus, move the stage quickly. And the fine focus, move the stage more slowly, when you're getting close to focusing your sample. Then you have the condenser, which is a lens that...for transmitted light is underneath of the stage. And we'll focus the light in a way to increase the resolving power. But I will talk a bit more on the condenser. So, the diaphragm of the condenser and the condenser itself.
Then you have this knob over here, which allow you to focus the condenser, and this one and this one are the condenser centering screws, the field diaphragm, and the transmitted light control. And also you can align the lamp. Generally the alignment controls on the lamp are on the back of the microscope. So, I will talk a bit more on this part here of the microscope, which is the condenser. So, if you look on the sideways you have the diaphragm of the condenser and the condenser. So, the diaphragm is like an iris that will open and close allowing more light to go through the condenser to the sample or less light. So what is the condenser therefore? So in order to increase the aperture of the objective and the resolving power of the microscope, the condenser is added on the bottom of the stage. And what the condenser does is that it will increase the angle of the light rays captured that goes to the sample and therefore, it will increase the angle of the lights that will be captured by the objective increasing therefore the resolving power.
So, what is the relationship between the condenser and the numerical aperture of the condenser? So the condenser, the light that goes through the condenser, and the numerical aperture. If you increase the numerical aperture of the condenser, what would you expect to observe on the cone of light that goes through the condenser? So when we increase the numerical aperture of the condenser, more light passes through the condenser to go to the sample. And the numerical aperture of an objective is defined by the equation. The numerical aperture is N, which is the refractive index sin of alpha, which is one half of the wider angle that is captured by the objective. Therefore, the numerical aperture is directly related with the resolution. Higher numerical aperture should correspond to higher resolving power.
So I have this image of something. It was beads that were used to image, and then it was tested, different numerical apertures to image exactly the same sample. What do you think it will happen when you increase the numerical aperture of this objective to visualize the sample? As you increase the numerical aperture of the objective, you increase the...you wider the angle of light that is captured by the same objective and therefore you increase the resolving power. So we pass from a blob, that we don't know what's there, to find out that we have four beats in this blob. Higher values of numerical aperture will allow higher resolution. Smaller structures will therefore be visible with higher quality, and resolution is in fact the ability to distinguish two close dots as separated.
And continuing to talk about resolution, why do you think is oil used in the objectives? So the oil is used to extend the resolving power of the microscope. But why? Why does it happen? It happens because the glass has a refractive index of 1.51. And the oil that is used also has the same refractive index as the glass, and therefore there will not be any diffraction of the light rays, and the wider angle of light will be captured resulting in an increase of the resolving power. So I will explain what I just mentioned with an image. You have the light source here, the slides and the sample, the coverslip. This is an air coverslip and here is an objective. And when the light source is here, this is the wider angle that the air objective will be able to capture, and the others will be refracted or diffracted, and will not be able to be captured by the objective.
On the other hand, if we use oil, the refraction is minor. And there's a wider angle of light that will be captured by the objective, and therefore the cone of light that is captured by the objective is greater, and this will increase the resolving power of the objective. And this is a reason why oil is used in microscopy. So now going through the relationship between resolution, numerical aperture, and focal distance. As I mentioned before, the numerical aperture is related with the refractive index and the sine of alpha, which is half of the angle of the wider angle that can be captured by the objective. And if you increase the numerical aperture, you decrease the focal distance to be able to capture a wider angle of light. Therefore, you need to be aware that some samples will fit to be analyzed with a lower numerical aperture, but won't fit when the numerical aperture is extremely high and the working distance is quite small.
Other practical, very simple advices but sometimes people forget, to increase the resolution and the resolving power of your microscope, please be aware that you should clean the objective. When you're imaging the objective should be spotless clean and your sample preparations should also be clean. Please be aware, as I mentioned before, to have the coverslips and slides with the correct thickness and to use the correct oil with the correct refractive index and to use it correctly. So talking a bit more about objectives. The objectives are essential components on the microscope. And the objective is much more than this front lens. It has a lot of single parts inside. And when you look to objective here, you will have the objective type. Here you have the golden reference where you can go with this number and check on the manufacturer website exactly what is that objective for.
This is the immersion media for this objective. So this objective would allow three different immersion medias with the correction color aligned to the respective immersion media. So by slicing an objective in the middle and looking at their skeleton, we can see that they are actually quite different inside and much more than just a single front lens. They also have the fluorite and apochromat, meniscus lens, then they have the blades of lens, and the amount of the blade lens will depend on the corrections of the objectives as well as the triplet lens. And how will all of these influences in the final result of what an objective can deliver? So the question is why so many lens and what happens to the rays of light when they are passing through the glass? Will it affect the microscope image? How? One of the aberrations that is corrected in the objectives is the field's curvature. What is field curvature? If the objectives are not corrected for field curvature, it is not possible to focus the entire field at the same time, meaning that either we focus the center of the sample or the edges because there are different focal points of rays of light because this objective is not corrected for field curvature. This is an off-axis effect of the lens when it's not corrected.
Other aberrations corrected in the objectives is spherical aberration. The spherical aberration happens in monochromatic light and it is the uneven focus of monochromatic light due to the curvature of lens. And you can see that these rays of light, they are not focused all exactly at the same place. And this will cause the image to appear as a bit hazy or slightly out of focus. Sometimes this can be also corrected by adjusting the coverslip's thickness or adjusting the refractive index of your sample. Other correction in the objectives is the correction for chromatic aberration. What happens is that the different wavelengths of light if the objective is not corrected will focus in different planes.
And therefore, on a non-corrected lens, on the Z-axis, a dot will appear that should colocalize, a multicolor bead that should colocalize, all the colors in the same plane will appear as if they are in different Z planes. On the other hand, if the objective is corrected, all the rays of light in the different wavelengths will focus on the same point, and therefore the multicolor beads, all the colors will appear in the same Z plane. So, if your objective is corrected, please check if you're using the correct oil to correct for refractive index mismatch, which also could cause some chromatic aberration. So, when joining all of the knowledge together, what happens in different objectives and what is corrected in terms of color? For an achromatic objective, the field curvature is not corrected and it's corrected for spherical aberration for one color and chromatic aberration for two colors.
A plan achro objective, the field curvature is corrected, spherical aberration, one color and chromatic aberration, two colors. A fluorite, the field curvature is not corrected. Spherical aberration, two to three colors, and chromatic aberration, two to three colors. A pan fluorite and the plan apochromat, the field curvature is corrected. The spherical aberration are corrected to three to four colors in both of them. But the difference is the chromatic aberrations. In a plan achro you have four to five color correction on chromatic aberration, whereas in a pan fluorite you have two to four colors correction. The price of the objectives will increase with the amount of lens inside and the amount of corrections. So you need to consider which objective you really need to your particular experiment or your applications.
So, choosing wisely. What do I mean by that? So, a few years ago I remember when I was working in a facility, one of the PIs came to me and told me, "Claudia, I want a 40X objectives." At the time, I went back to him and say...and told him, "What do you need to do?" Because if you go to the website, you see that there are 42, 40X objectives in 2012. Now, I actually did the same search in the same website and they are 99, 40X objectives. And the problem remains how to choose. Even more surprisingly, if you go for 63X objectives, you have more than 260 objectives that you can choose from. So, how do you choose? With such a large variety of objectives they are obviously different for different applications. It's extremely complex to select an objective.
So please take in consideration the following parameters when you're going to choose an objective. Check the magnification. That was our first part, that's then what numerical aperture do you need? What working distance is your sample very large? Do you need to work very far away from your sample or do you want to work very close to your sample? What corrections do you need for flat field, for color corrections? What transmission, wavelengths transmissions do you need, and the transmitted light techniques that you need? Do you need brightfield? Do you need darkfield, phase contrast, DIC? And for fluorescence, what do you need? What transmissions do you need? Is it a water immersion objective, oil immersion, glycerol immersion? What thickness of coverslips are you going to use? It is corrected? Can it be corrected? So you need to consider all of these parameters when you're choosing an objective.
And now, I'm going to talk about transmitted light techniques itself. So as I mentioned in the beginning of the talk, transmitted light techniques stands for a group of techniques in which the light passes through the source, through the sample, and is captured by the objective. And it's useful. It provides an extra channel for fluorescence microscopy, but it's also...it's a standalone technique that can be used for live imaging or for analyzing morphology of samples when properly selected.
In transmitted light microscopy, this is our Hela cells stained...non stained. It's a brightfield image. But it's actually the cells most of the time are very transparent and they don't have enough structures to deliver the necessary detail that our eyes can visualize. So in order to image some specimens or samples such as bacteria, live cells, or thin tissue slices, researchers sometimes use other transmitted light techniques or stain their samples. And today, I'm going to talk to you about brightfield, darkfield, phase contrast, and DIC, which is differential interference contrast. So one of the things that you need to do...you should do when you're going to acquire any imaging transmitted light microscopy, before starting to acquire image, check that your microscope has the proper Kohler illumination aligned. This method gives a very even illumination of your sample and therefore your resulting image would be much better. And also, one of the things that Kohler illuminations does is that you will not see the filament of the light bulb in the image.
So comparing the illumination plans when you have Kohler aligned, if you have the light source here, you have the focus of light here, it's focused in the sample, it is focused again in the field stop, and it will focus in your eyes, in the retina or when captured. On the other hand, the lamp filament, it's focused here, it is focused on the front focal plane of the condenser, on the back focal plane of the objective, and before you capture the image. And therefore, the focus plane of the lamp filament is different from the focal plane of your sample. So you can take a nice image of your sample without the interference of the image of your filament. So how to align Kohler illumination in a microscope. The first thing you do is using the focus knob, you bring the sample into focus, and then you close down the field diaphragm, and you close the NA of the condenser.
If it appears, an hexagon, and with your sample inside, everything is fine, is sharp, so you have proper Kohler illumination. Many times when you close down the field diaphragm, it looks dark, and you can barely see the sample. So what would you do? You need first to center the field diaphragm with these screws here. So I'll center the field diaphragm. And then using this focus knob of the condenser, you will go with the condenser up and down until you focus very sharply these edges. Once the edges are focused, you open slightly the condenser and the field diaphragm and everything should be fine. If you're using DIC, you also want to adjust the Wollaston prism on top of the objective to increase your contrast.
So the contrast in a bright field image is determined by the differences in light in absorption, refractive index, or color. And it's acquired as light passes through the sample altering its direction. So in very thin samples, they will be very transparent. And one of the ways to increase the visibility of structures when imaging with brightfield is use light absorbing dyes such as eosin and hematoxylin. And please be aware that you need to do proper brightfield image. Besides doing good Kohler illumination, you need to match the numerical aperture of the condenser to the numerical aperture of your objective. Because if you put too much light from the condenser to the objective, the objective will not be able to capture everything. I envision it as if looking at the sun, we have a lot of light, but if you look directly, we have so much light that besides being blind, we cannot see any details. So you need to match the numerical aperture of your condenser to the numerical aperture of your objective as it's what the amount of light that your numerical aperture of your objective can capture is matching the numerical aperture of my eyes to the amount of light that the sun is sending to me, to all of us.
So, this is a 40X objective with the numerical aperture of the condenser open to its maximum. And this was the image that I was able to acquire. But if I decrease the numerical aperture of the condenser, you can see this was exactly the same image taken one after the other. There's actually something there. Maybe here if you look very strongly now that you have some structures, you can see that there are something here. Can I increase this? Yes, I can. If I put the numerical aperture of the condenser to match the numerical aperture of my 40X objective, I can see much better the same sample. So, this is what I mean that you need to match the...when doing brightfield image, you need to match the numerical aperture of your condenser to the numerical aperture of your objective. And when you change objective, you need to adjust that again in a way to have the best image possible. It will always be a very transparent sample and then brightfield. But in this case, I can see whereas in this case, I couldn't.
And this is the same image that you can see a wider field of view of a brightfield element properly adjusted with the Kohler illumination and the numerical aperture of the condenser matching the numerical aperture of the objective using to acquire the image. Advantages of brightfield microscopy renders in its simplicity and the easy adjustments required to do a very nice brightfield image. It's also a very cheap technique in terms of the optic required to do brightfield. The disadvantage is that many samples are actually nearly transparent to brightfield. We can avoid that by staining the samples with light absorbing dyes such as hematoxylin or eosin. Or if we do not want to stain the samples, we might need to recur to other transmitted light techniques that I will further explain.
And now, talking about darkfield image. So these are two images of the eye of a mouse in a darkfield with color cameras and pollen in darkfield, darkfield images, darkfield techniques. These very beautiful colored images is what you're observing now. And with a black and white camera, you can see the same sample as before being acquired in brightfield or in darkfield. So actually, the cells are the ones that shine, whereas the background is black. So the principle of darkfield is similar to the principle of an eclipse. When you look the moonlight, the stars in the sky will appear visible. The dark background will enhance the visibility of fainter objects. So using darkfield, transparent samples can be easily seen without any staining. And this is a graphic showing what happens to the light rays in brightfield illumination. We have the background and diffracted light, the diffracted wave, and then the specimen wave. And this is why it's really hard to see in background...in brightfield in same samples because the specimen wave is quite close to the background wave.
On the other hand, in order to generate contrast to see an object, on darkfield illumination, the specimen wave and the diffracted wave are quite close one to the other. That's what's captured by the objective. Therefore, the background wave is selectively illuminated. That's why you see it on a black background. And how does it happens? What happens is that before the condenser there's a field stop that will make the light rays that enter the condenser to be in an angle and when they go into the sample, here is the objective, here is the sample plane, only the lights that is diffracted will be captured by the objective and the other light as it entered in an angle it is excluded from the objective. Therefore, you see only what passes through the sample and diffracted some light, and all the rest will have...will be shown in a black background.
So when to use darkfield illumination. Darkfield illumination is very useful for low magnifications up to 40X objective because the numerical aperture of the condenser needs to be higher than the numerical aperture of the objective in order to have darkfield illumination. And if you wish to see everything in a liquid sample, darkfield is the perfect solution for it...for you. The disadvantage sometimes is that you see really everything. Even tiny dust particles are highly obvious. And therefore, if your sample...if you want to image a sample that is not that clean, the image might appear confusing because every little thing in your sample will be visible. They are easy to obtain images in the correct focal plane at a very low magnification and it's good for very small or very low contrast specimens.
So phase contrast microscopy as well as darkfield and DICs were Nobel Prizes in microscopy. And I must say microscopy is a fantastic area because it's constantly evolving. And the way scientists discover new ways to approach and to see the unseen is for me outstanding. Going back to phase contrast microscopy, phase contrast microscopy allows to visualize samples that would otherwise be invisible by using interference instead of absorption. And you can see much clearly when compared to brightfield, extremely transparent samples. And here, again, the same image that before it looked like we were seeing it very well. In brightfield and exactly the same, you can see it's the same cells here is this telophase here and that one over there, it's exactly the same picture taken...not the same picture, the same sample taken with brightfield or with phase contrast. And you can see that with phase contrast, you see it much clearly.
The phase contrast will have a phase annulus underneath the diaphragm in order to create the required interference, and then a phase plate on the top of the objective. So phase annulus, the condenser, sample objective, and the phase plate. So this graph will now compare what happens to the specimen wave under phase contrast illumination. So this is the specimen wave in brightfield illumination, and as I mentioned before, is quite close to the background wave. And that's why it's hard to see structures in brightfield illumination. But with phase contrast illumination, the interference causes a further separation from the specimen wave from the background wave. And this will allow the samples to be more visible.
So advantages of phase contrast microscopy is that it's possible to visualize structures that would be otherwise invisible in brightfield. And this would include certain organelles that could not be previously seen. And therefore, the images will look better due to the details that this technique is able to capture. Moving on to another transmitted light technique, I will now talk about DIC, differential interference contrast. In there, in DIC, invisible features of the sample are made visible. It has a very complex optics that allows to obtain information on the optical density of the sample. The image will be similar to phase contrast microscopy, but it doesn't have the phase halo that is characteristics of the phase contrast images.
And this phase halo, it's a bright halo that appears quite often around the sample and can be very destructive, destructive. And this technique was...theory was published in 1955. And here, you can compare a bright field image here in a DIC image. I truly love DIC. If you can see quite clearly and if you look with the details, you can see that these are the chromosomes, these are the centrosomes. And here, you could even see the spindle. When I did this image, actually, I costained it with DN DAPI and alpha tubulin and gamma tubulin. And I could quite clearly confirm that what I'm saying is actually true. This is a mitotic cell, chromosomes, microtubules, and centrosomes.
So the DIC, how does it work? It works by separating the light into orthogonally polarized rays. And they go through the sample at the same plane and they are combined before observation. And the interference of those two parts when they are combined will be sensitive to the optical path that the light had went through and will cause kind of a 3D structure that allows you to visualize very well the details inside of a seen...previously transparent sample and unstained. So in terms of optics, you have the light, there's a polarizing filter, and there's a Wollaston prism that separates the light rays. They go through the condenser and through the sample separated, will be captured by the objective separated. And then there's a Wollaston prism that joins them again, and the polarizing filter, and then the image will be captured by our eyes or by a camera.
And this is the difference on adjusting the Wollaston prism or not. I don't know if you still remember in the beginning of the talk when I was talking about aligning Kohler illumination. Then I mentioned that after aligning Kohler illumination in DIC to get further contrast on the image, you might want to adjust the Wollaston prism that's on the top of the objective. And here is exactly the same sample that I have acquired with Wollaston prism non adjusted and the Wollaston prism adjusted. So when you compare DIC with phase contrast, it actually produced higher resolution images. It shows very good contrast. DIC can be used in thick samples and it does not have the distracting halo of phase contrast microscopy. So DIC is actually an extremely good technique for transmitted light microscopy but the optics is complex and it's actually the most expensive of them all.
So in summary, from our lesson today, I would like you to remember to use the equipment carefully and respectfully, to choose the correct objectives, oils, and coverslips for your particular application, to choose the correct transmitted light contrast that you're going to use, darkfield, brightfield, phase contrast, DIC. If you're not seeing it well, check your Kohler illumination and properly align your Kohler illumination. And if your image is still not good, try to adjust the numerical aperture of the condenser, the light filters, clean the lens and clean your sample.