Edited in Kakoune + .md + Antigravity
Microscopes
Welcome to the wonderful world of microscopes.
This guide is probably best read while listening to music by Carbon Based Lifeforms -- the author was listening to their albums whilst writing this.
Almost all of the information discussed in this guide will apply to any optical microscope, regardless of type, quality, brand, year, or model -- this information is about the ways of optics and mechanics itself and how those should affect your purchasing decisions.
There is no such thing as a microscope
The term microscope is actually a misnomer.
There is no such singular thing as a "microscope", but rather there are microscope stands, and microscope objectives.
The microscope stand is the physical frame that holds the optics, and accounts for the mechanical part of the microscope. Your ergonomics, structual stability, resistance to stray vibrations, ease of use, and future upgrade options are all determined by the microscope stand.
This will be addressed further in the next parts of guide, but for now, know that your decisionn of microscope stand will affect much of your quality of life when using the tool.
The microscope objective is the primary lens of the system, and works to capture and magnify the image of your sample. The objective controls both the magnification, resolution, and much of the optical quality of the microscope.
Objectives can be removed from your microscope, and new ones fitted in their place, assuming the new objective is compatible with the stand.
Compatibility is determined by:
- Tube length (Does the objective's tube length match that of the microscope? If not, the optics might not form an image at all. Learn more in the section on Optical Systems.)
- Thread size (This is a mechanical attribute. Generally, if the threads dont match, the tube length and optical properties definitely wont.)
- Abberation corrections (Certain optics manufacturers would not fully correct the lens defects in the objective alone -- the tube lens or eyepieces were required to finish the second half of the optical corrections. Learn more in the Objectives section)
Any optical microscope is composed of a combination of these two parts.
Even though the term 'microscope' will be used to describe the combination throughout this guide, the two of these devices should be kept serperate in your mind.
Anatomy of a microscope
As mentioned above, a microscope is not a singular object, but rather a combination of multiple parts.
Let's look at all these parts now, so we can familiarize ourself with the anatomy of a microscope.
We wouldnt want any anatomy mishaps or mistakes in your microscopic rendevous.
Types of microscopes
There are two generally accepted catagories of microscopes: Inverted and upright.
At their core, the two types are differentiated by the orientation of their light paths when used with transmission illumination.
Upright microscopes: Light travels from the consender upwards through the sample and into the objectives located above the stage.
Sharing a design lineage back to the earliest microscopes in existence, upright microscopes from the mid 20th century until present represent the peak of microscope design. These are widely adaptable and commonly used both in lab and research applications.
- Allows the highest resolution objectives.
- Compact, adaptable and affordable.
- Less space to manipulate sample.
- Requires a coverslip on samples, glass slides commonly used.
Inverted microscopes: The condenser shines light downwards through the sample, and into the objectives which are below the slide.
Inverted microscopes are generally used for clinical work due to an ability to work with petri dishes without the use of coverglass, and where ease of handling the sample is more important than being able to see the deepest magnifications. They also seem provide the most modularity and access to external modules for advanced techniques when required.
- Uses long-working distance objectives
- Optics tend to be more expensive.
- Allows free access to the top of the sample.
- No coverslip required, petri dishes commonly used.
Optical quality:
Alright, so you want a microscope, and you want one that gets you good views or pictures. So, let's discuss optical quality. This determines how good an image you get, and is affected by every single piece of glass that the light passes through between your light source, to the sample, and up to your camera or eyes. Optical quality is a bit hard to measure, but you can see the difference. The main affecting attributes are generally catagorized as follows:
- Optical imperfections: these are results of the machining process in the creation of the lenses.
You know how the mirrors of state-of-the-art space telescopes like the James Webb space telescopes cost millions to make, because each mirror has to be precision polished to insane detail, or the entire image will be warped or unusable? Well, optical microscopes are also affected by both the quality of the glass and the the precision machining used to make their lenses. A more refined manufacturing process will yield less distortions or imperfections in the final image.
- Abberation: A good optical system will work to correct the natural abberations created by the warping of light through lenses.
Even the most theoretically pefect lenses will create distortions or abberations in the image. However, any good objective will try to cancel out these distortions atleast partially. Some microscope objectives rely on the upper optical elements of a microscope, e.g. tube lens or eyepices, to aid in this abberation-canceling.
- Coating: A coatings is a thin layer of special materials with (generally) anti-reflective properties that has been applied to certain lens parts.
One of the defining features of a high quality objective is it's coatings. These coatings are designed to improve image quality by reducing unwanted reflections and glare, and increasing light transmission through the lenses.
Note that none of these attributes directly affect resolution (the ability to see smaller objects or details). The determining factor of resolution will be discussed in a later chapter.
Optical systems
We have now learned that microscope frames are distinct from the objectives (lenses) they employ, so perhaps you might be wondering about interchanging objectives between microscopes.
This is possible, but limited under the constraints imposed by the different optical systems (known as 'tube lengths') used by different manufacturers and microscope series. There are two main optical systems utilized in microscopy: Infinity tube length and 160mm
What you need to know is that these two types are completely and wholly incompatible with eachother.
Once you buy a microscope built for one tube length, you will be locked to the objectives compatible with that microscope.
Infinity tube length:
Infinity tube system is the state of the art in modern microscopes. Each manufacturer has their own specifications of how their proprietary infinity systems work. As a result, you cannot mix and match microscopes and objectives of different brands.
The name infinity comes from how these objectives focus light. All infinity systems have objectives that focus light infinitly far away. Then this projected image goes through a 'tube lens' that focuses this down into an actual image. Note that the tube lens is specifically calibrated to each brand's optics.
The key here is that there can be a large number of added modules between the back of the objective and the tube lens. Thus, modern infinity microscopes have far superior upgradability and modularity -- and often also a more accessible and up-to-date ecosystem of attachements.
This upgradability will let you access advanced techniques like confocal, fluorescence, DIC, and many others with ease.
160mm tube length:
160mm objectives, which make up most of the 'finite tube length' designs, are so called because the distance between the back of the objective and the image plane is 160mm.
These systems have been used for the atleast last century up until the late 1990s.
Note though that there have not been significant advances in microscope lens optics within the last 80 years or more. As a result, even antique 160mm objectives may have equal or even superior quality optics to modern day objectives -- and are often sought after for the careful craftsmanship that went into their making.
160mm objectives tend to be significantly cheaper than infinity objectives of similar type. These lenses, and even their premium versions are available for very affordable
Unlike infinity tube-length objectives, 160mm objectives are compatible with any other 160mm microscope. This allows you to mix and match objectives of different brands and varying qualities made across a time span of 100 years.
A caveat: many brands of 160mm objective manufacturers would not fully correct all the lens abberations inside the objective itself. Rather, when paired with a matching eyepiece from the same manufacturer and time period, the eyepiece would compensate the remaining abberations. This means that if you use the objective without the matching eyepice, your image may have minor defects or a decrease in performance. When used with a camera system instead of your eyes, a specialized type of lens called a 'Photoeyepiece' would be placed inside the trinocular head, which would correct the remaining abberations for the camera's view.
Abberation: An aspect or distortion introduced into the image by a lens. All lenses bend light, and many times in imperfect ways -- the result is that certain parts of the image from the lens will look a bit 'off'. Complex lens systems will often partially or fully 'correct' for these distortions by using many different lenses in combination. Learn more in the section on Objectives.
Some thoughts about older microscopes
If you are budget constrained, older 160mm microscopes will allow you a great entry path to the field of microscopy.
Their objectives, being 160mm can be freely switched out for objectives of other brands and models, allowing you to mix and match the types you need with ease. You can also more easily source these objectives, even high quality ones, for cheap -- as collectors and refurbishers will be trading them around ebay.
This is definitely the path for those who like the idea of piecing things together from a kit the size of the internet, or are into tinkering and learning the mechanisms of the tool itself.
Reliability
Ergonomics
Once you've been sitting in front of the microscope for an hour, if your microscope has poor ergonomics, you're going to feel it.
Ergonomics is one of those things where it's hard to get a feel from images. Luckily, in modern design, comfortable usage has begun to be a strong area of focus for microscope design. You can take a look at designs from the big four microscope manufacturers for examples of key focus points, and many smaller manufactuers base their designs on models from the big world leaders.
Unfortunately, I don't have too much more to add here than "Make sure it looks comfortable to use".
Well, actually...
One of the most important factors in microscope ergonomics is the eyepieces.
Now, we could discuss the intricaties of back and neck kinematics, and the most comfortable placements...or we could just skip the actual putting your eyes up to the eyepieces part: This is the 21st century, and after hours of sitting there at even a well designed microscope, you're definitely going to want a camera.
Choosing a camera:
Cameras are affected by three primary factors.
- Camera resolution. This is the number of pixels the camera has and is generally measured in megapixel or MP. A larger pixel count is important for getting clear images when cropping or digitally zooming in, but is not the whole story.
- Sensor size. This is the size of the physical chip.
- Pixel size. This is related to the ratio of sensor size to camera resolution. Larger pixel size or 'pixel pitch' will allow more light to be absorbed by each individual pixel, reducing noise.
Now let's understand how these work together. A camera is a device that tries to 'sample' or capture an analog(continous) 'signal' using a finite number of 'positions' or pixels, and thus is subject to the Nyquist-Shannon Sampling Theorem.
The Sampling Theorem is commonly used in audio, but it works here too. It says that to accurately capture an image, you must have a resolution beyond that of the sample. Normally this is quite hard to achieve, but as you will learn in the next section, microscopes are able to approach a universal 'maximum resolution'.
When choosing a camera, make sure it statisfies this equation:
- Nyquist-sampling-criterion-fulfilling pixel size (micrometers) = Resolution * Obj magnification / 2.3
Let's do a hands-on exercise for example's sake... You might want to come back to this section after finishing the next chapters.
If we have a 40x 0.65NA objective, we can use the formulas a follows:
R = 1.22*500/(0.65 + 0.65) = ~500 nanometers Ideal pixel size = 500nm * 40 / 2.3 = ~0.86 micrometers
If you've ever looked at camera specs you will know that almost all reasonably priced cameras are far below this ideal. So you will technically always be camera limited…
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Resolution
At it's core, a microscope provides the ability to see the unseen.
Thus it is only natural one would want a microscope capable of seeing the smallest object, or giving the best image, or the highest resolution. To understand how to achieve these goals, we must dive into the heart of how images are formed.
To discuss resolution we must first remedy one of the most common mistakes that newcomers to microscopy make: Conflating magnification with Resolution.
Resolution IS NOT magnification.
Resolution: This measures the ability to distinguish two tiny, close together points as seperate points rather than just a single blob.
Higher resolution means you will be able to see finer and smaller details as crisp images instead of just blur.
Magnification: The change in size between the object and how big it appears. Because of diffraction, a higher magnification makes the image more zoomed in, but does not neccesarily give more resolution. Imagine scaling up a photo taken on your phone and finding that it's really blurry and pixelated when you zoom in.
Diffraction: Light acts like a wave, and therefore when it hits an object around the size of that light-wave, it will bend around the object. You might be like "What? No!", but on a microscopic scale, this result in the edges of the object appering to blur. At high magnifications, this blurring can happen so much that the object's details disappear entirely from sight.
You might have heard that optical microscopes (ones that use visible light to see their specimens) are "Diffraction-limited". This 'limited' refers to their inability to see objects and details smaller than a certain size.
Unlike your eyes or a camera, microscopes are not limited by the discrete size of a sensor or how many rods and cones you have -- but rather by the fundamental properties of light itself.
Imagine we have a tiny structure composed of two objects. At large scales (>500um) these two objects might appear to have sharp edges, but at small scales (<100um) each of these objects begin to appear as a small blurry splotch on the image. At a certain point, as we shrink these objects they become so tiny that the light passing by them is blurred together until they can no longer be distinguished as anything more than a single blurry blob. At this point, they are beyond the maximum resolution limits of the microscope.
So that's great, but this takes one thing for granted: The resolution of a microscope is not an absolute. It is limited and defined by one thing only[1]: The objective.
Let's take the example of a brightfield microscope: Light passes through the sample, is blurred by diffraction, enters the objective, and is cast onto the image-plane and seen as a picture of the sample.
But the light that goes straight through is just one part of the story. What about all the light that is blurred, or rather bent away from the objective by diffraction? This light is bent away at a certain set of angles known as the "Diffraction Orders" -- however, if we could capture this light we could get more information about the sample.
To capture some of these diffracted rays, let's make the aperture of our objective wider. Now we can collect a wider angle of light, also known as having a wider Numerical Aperture, we have more data about the sample, and this begins to counteract the effects of diffraction.
Resolution is defined as: 1.22 λ / (NA(obj) + NA(cond)) Where 1.22 is a geometrical constant due to a circular aperture, λ is the wavelength of light used (500nm is mint-green), NA(obj) is the numerical aperture of the objective, and NA(cond) is the condenser's numerical aperture.
This is one of the most important things to understand in microscopy: with more light collected, we gather more information, and get a higher resolution. Higher numerical aperture = Increased resolution.
Numerical Aperture: If we have a lens focused to a point, this lens can only see light rays coming in from a certain maximum angle. Numerical aperture, or NA, is a measure of the how wide this angle-of-accepetance is for the lens or objective, and of the refractive index the lens in working with. A wider angle means a higher numerical aperture, however this comes at the cost of a shorter working distance. The numerical aperture is analogous to the F/stop number in photography.
Working Distance: The distance between the objective lens and the sample. Due to geometry, if we want a wider collection angle for the objective, while keeping the diameter of the objective the same, our objective will have a shorter 'focal-length'. Thus working distance (WD) and NA are linked. A simple example of this is a 4x objective, which may sit several milimeters above the sample, meanwhile a 100x objective must be touching the sample, and (generally) suspended in refractive-index-increasing oil. The WD is analogus to the focal-length of lenses in photography.
By increasing the numerical aperture, the objective is able to resolve smaller objects.
Numerical apeture is defined as: NA = n sin θ Where n is the index of refraction of the medium in which the lens is working (1.00 for air, 1.33 for pure water, and typically 1.52 for immersion oil), and θ is the half-angle of the maximum cone of light that can enter or exit the lens
Thus, besides increasing the acceptance-angle, there is actually another way to raise the numerical aperture: By increasing the refractive index of the medium between the sample and the lens, we can lower the angle required to get a certain NA.
In practice this is done by adding a drop of oil on top of the slide or coverglass, and letting the objective become submerged in this oil.
Hopefully it is apparent now that microscopes themselves do not have 'a resolution', but rather that is determined by the currently equiped objective.
Different objectives are equipped with different numerical apertures, and thus each objective is capable of achieving a different maximum resolution.
Now we can understand why optical microscopes themselves are diffraction-limited: To achieve a higher resolution we must increase the objective-aperture's angle of acceptance, and this can be done by either having a wider lens, or a shorter focal length and working distance, or a higher refractive index of the medium. With only one objective[2] the angle of acceptance can never go beyond 90degrees, and there is a limit to how high one can reasonably push the refractive index
Here, at around 250nm there is an absolute limit, where widefield[3] microscopes using visible light, no matter how many optimizations we add, can not see smaller. This is called the Abbe Limit.
Notes: 1: There are a number of techniques involving specialized illumination practices and fluorescence that can achieve higher resolution beyond the diffraction-limit. 2: There is a highly experimental set of techniques known as I5M and 4Pi-confocal, which both use a custom-built dual-objective microscope. By using two opposite-facing objectives these techniques are capable of achieving <100nm axial resolution: well below the diffraction limit -- but only in the axial direction. 3: For many super-resolution techniques, instead of seeing the whole field of view at once (known as widefield), only data from a single point of the sample is recieved. This point is then scanned across the sample until an image is formed, such as in confocal-laser-scanning microscopy.
A note on Depth-of-field
Increasing resolution is actually not always what we want.
This may sound suprising but imagine the following:
If we increase the objectie's NA, we increase the ability to resolve objects as seperate in the lateral (or X and Y directions of the image plane) dimensions. But our sample is not flat -- there exists the third dimension, Z, or in microscope terms: the axial dimension.
By increasing resolution, we also increase the ability of the microscope to resolve different planes along the axial direction as seperate. Such a behavior, in an amped up form called "Optical sectioning" is crucial and much sought after in research fields especially when dealing with thick samples.
However, in effect, this increase in axial resolution reduces the depth-of-field of the microscope.
When we are using a microscope to look at a range of almost-transparent objects in thick samples, we oftentimes need a thick depth-of-field to enable us to quickly scan the sample.
Depth-of-field: The range of distances around a lens's focal point where objects appear sharp and in focus. Imagine if instead of seeing a whole room at once, you could only see a thin slice of the room in focus at once. In camera photography, especially portrait photography or macro photography, this effect is oftentimes sought after as it gives a soft 'bokeh' background and sharply isolates the subject. On a microscopic scale, this effect is drastically increased. Indeed this increase in resolution is immensly beautiful, but imagine trying to walk through a room when the entire thing is so blurry that you can barely see all but a few inches of depth at a time.
Decreases in Resolution
Above we have discussed resolution in the theoretical, but now let's take a look at the practical.
In real world situations, microscopes generally struggle to reach the theroetical maximum. Let's see why:
Optical quality: As discussed in a earlier section, the various abberations and lens imperfections present in all materials will -to varying degrees- slowly corrupt and interfere with the final image. This can be remedied with higher quality objectives (discussed in the next section) and reducing the number of glass elements between your objective and the eyepieces/camera. Assume each optical element that light must pass through will degrade ~1% of quality.
Insufficent sampling: This is an issue of the vision of the viewer. But since our eyes are generally optimal for microscopy work, this issue applies just to cameras. If the camera used does not satisfy the Nyquist-Shannon Sampling Theorem, the mismatch in camera resolution and signal resolution will cause information to be lost. See the Ergonomics section for more information.
Out of focus light: Perhaps the single most destructive cause of decreased resolution is out-of-focus light. As we've learned, all transparent objects scattee and diffract light, and this scattering is sometimes useful to form an image. However, there are images being formed by the entire depth of the sample.
We can only clearly see the slice of the sample we are focused on, but the scattered light from the rest of the sample is still layered onto our image as blurred double-image remnants. On fluorescence images of thick samples the destructive effects of out-of-focus light becomes even clearer. This is why 'optical sectioning' is so sought-after in research fields. Thus, following the principles of optical quality above, the sample itself can also be considered an optical element, and so thinner samples will give better image performance.
Contrast: A microscope with high resolution becomes useless if you cannot distinguish a transparent sample from the background. While not directly an impact on resolution, the pursuit of higher contrast often leads people to choices that decrease resolution. The field of visibility enhancing effects is wide and ranges from simple brightfield staining to darkfield, DIC, and fluorescent-tagging.
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affectors definition
Objectives
At the heart of the microscope is the objective. This is the actual lens of the microscope.
The objective controls both the magnification, resolution, and much of the optical quality of the microscope.
Remember from the section on Resolution, that only one type of optical element in your microscope has an affect on the maximum resolution of your microscope: The objective. And, more specifically, only the objective's Numerical Aperture or NA can increase your resolution.
There are probably dozens of different types of objectives.
However, for ordinary brightfield microscopy work, these are the primary classifications of objectives, with each type having a different improvement on the quality of image it can provide.
Simple Archomat
This is the most basic type of microscope objective, and will get you -- depending on the manufacturer and quality of machining on the lenses -- a poor, to decent image.
These objectives are the cheapest type of objective, and commonly found on entry level scopes.
Ordinary simple achromat objectives have approximately 50-60% of the field of view in focus. Towards the edges of the field, however, the becomes unfocused. This is due to an affect called Sphereical Abberation.
The following two types of objectives are also acrhomats, as this refers to being affected by chromatic abberation, but unlike those, the simple achromat is often left unmarked.
Semi-plan
These, or better, will be outfitted on any scope meant for serious work, and will provide good images.
Semi-plans begin to have their spherical abberations corrected, and these objectives have about 70-80% of the field in focus.
This type may be marked as Semi-plan.
Full planar
Planar lenses have significantly improved performance with over 95% of the field of view being perfectly in focus at once. Also known as the field-of-view being flat -- hence the name: Planar means flat.
If bought from a reputable manufacturer, these objectives will provide excellent, sharp images due to the precision manufacturing that must be put in.
Many companies will offer two types of plan objectives, a 'normal' cheaper version (e.g. Dplan) for everyday lab work, and a 'premium' version (e.g. Splan) with the absolute highest care put into their manufacturing.
Any top-tier research microscope should come with plan objectives, or have them easily purchasable.
Typically marked as Planar/Plano, Plan, or PL.
Sphereical Abberation: This is an effect created because most magnifying lenses are in fact semi-spherical in shape. This sphereical nature allows them to collect and bend light rays in such a way that they magnify the image. The issue is, though, that towards the edges of lens, objects are farther away from the lens then in the center -- and so only part of the image can be in focus at once.
The previous three types of objectives improved the flatness-of-field of the image. But there is still another type of abberation to be corrected: Chromatic Aberration.
Chromatic Abberation: This is a common effect, where objects in the sample will appear to have green and purple blurs around or inside them. This occurs because the colors, e.g. red green and blue, are different wavelengths of light. When white light passes through a lens, the different wavelengths of light will be bent or magnified by different amounts. This is the basis of a prism. Sometimes, the colors created by this effect are mistaken for colors appearing in the object -- but do not be fooled: the colors formed by chromatic abberation are fake.
These next types of objectives seek to nullify or 'correct' this chromatic abberation.
Plan Fluorites
These objectives are able to partially correct their chromatic abberation, providing better and clearer images.
Fluorites are made from glass containing higher levels of fluoride, and are commonly used in Fluorescence applications where having different colors focused to the same spot is important. Hence the name.
Commonly found marked Fluor, FL
Plan Apochromat
These sought-after objectives represent the highest tier of objectives.
Apochromats attempt to fully correct both chromatic and sphereical abberations, causing almost the entire field to be in focus at once, while simultaniously focusing visible colors to the same point in the image.
Also, these objectives will often have increased numerical aperture over their plan counterparts, giving impressive increases in resolution abilities.
However, be mindful of the incredibly thin depth-of-field, and close working distances that come from a high NA. For example, the Olympus SplanApo40x has a miniscule working distance of just 130 microns -- less than a common coverslip!
Due to this, Apochromats of high resolution (beyond 20x) should be avoided for common microscopy work.
These will be noticable from their 'bulk' in size and shape, and are strongly marked APO.
Note: You do not need an apochromat. As beautiful as they seem, they are both expensive and fiddly. Many premium-version plan objectives from reputable manufacturers will partially correct chromatic abberations, making them unnoticable up until 40x or above magnification.
Here are links to valuable resources with more information on objectives, their types, and how to choose them:
Upgrading 160mm Microscope Objectives by Diet Tom's Diatoms https://drive.google.com/drive/u/1/folders/1gEznVlvIQweLmvfmXg0eIkOjX8xaeuuk
Microscope Objective Specifications by Nikon MicroscopyU https://www.microscopyu.com/microscopy-basics/microscope-objective-specifications
Illumination
If objectives are the eyes of the microscope, illumination is the light needed for those eyes to work. All optical microscopes need light to come from somewhere and interact with the sample. The direction the light comes from, and the way the interaction happens, is what will be discussed here.
In the first section, we introduced the idea of inverted vs upright microscopes. Both of these designs can be further classified by their ability for transmission/reflection.
Transmission means that the microscope's light is passed through the semi-transparent sample and then enters the objectives. The vast majority of optical microscopes currently in service are transmission microscopes, as the set up is simple: light source > condenser > sample > objective.
Compare this with reflected or 'epi-illumination' as it is often called, where light is beamed at the sample and then reflects off the opaque parts of the sample and into the objectives. While most stereoscopes use reflected illumination in the form of adjustable lamps, epi-illumination puts the light source behind the objective using a collection or mirrors or optics.
Brightfield
Brightfield is the default mode of most optical microscopes. This is the "black specimen against white background" that you will commonly see in most photos.
Brightfield is caused by transmitting light through the sample, and so you are essentially looking into the light source, giving that white background (called the 'field'). Imagine holding up a leaf in front of the sun, you can see the veins in the leaf as the sunlight passes straight through the leaf and into your eyes.
Notes on condenser aperature
There are many ways of utilizing the diffraction of light to increase contrast and thus effective resolutionq, including many more techniques that we will not go into here such as Hoffman Modulation. But keep in mind, that all microscopes are diffraction-limited instruments, and therefore all of these methods will inevitably decrease the actual resolution of the microscope.
Darkfield
If brightfield is black against white, darkfield is the inverse. The light source is blocked from entering the objective, and only light that is scattered by the sample is visible.
Since we are blocking most of the input light, we need to use a stronger light source to get a good image. This can have the effect of heating, making it dry out easier.
While dedicated darkfield condensers are preferred for many lab applications, you can a nearly perfect approximation up to around 0.6NA (typical of a 40x objective) by using a handmade or 3D printed circular 'stop' in the center of your condenser. These darkfield light-stops can be made or found relatively inexpensively, and block the center of the light path, preventing direct light from entering the objective, and only allowing light that is scattered by the sample to enter the objective.
Think of darkfield like holding up a leaf in the evening sun, but this time, looking 90 degrees away from the sun. The leaf glows in the sunlight against the far away background, and you can see the structures in the leaf as they scatter the sunlight 90 degrees toward you. Because of this, darkfield often gives spectacular sights, as the sample glows brilliantly against the black, or 'dark', field.
There is an issue with darkfield, though. The black background tends to make cameras overexpose, and so the contrast of the glowing sample becomes too much contrast. This can form blur and light bleed around the details in a sample like a photo taken between broad daylight and complete shadow on a sunny day, and often makes it difficult to see fine details.
Oblique
Ahhh, oblique. This is a fun one. Almost every amatuer microscopist will accidentally discover oblique in some form or another. The key is knowing how it is made, as then you can utilize it.
Similar to how brightfield with a low numerical aperture light fed via the condenser can increase contrast via diffraction effects, oblique is another way to to utilize those diffraction effects, this time creating a 3D-like effect in the image. Despite working on wildly different principles, this 3D like effect leads oblique-based methods to often be called a poor man's DIC.
Oblique just means the light comes in at an angle, so there are many varients of oblique. All of these can be achieved using patches or 'stops' placed in the condenser that block some of the microscope's illumination.
Linear Oblique: This is what most people will refer to as 'oblique'. It is created by blocking one side of the light path, causing the light to enter the sample from an angle, creating a 3D-like effect in the image as features are cast in bright light on one side, and darkness on the other.
While these may look like shadows cast from one side, they are not so simple.. Rather, light from the condenser is only allowed to enter the sample from one side, and when this light passes through the sample it is diffracted into multiple rays or "diffraction orders". Because the light is coming in at an angle, some of these rays will miss the objective entirely in certain areas, while in other areas, the rays will be diffracted into angles that allow them to enter the objective. This creates interference patterns at the camera plane, forming the 3D effect.
Note that since this is diffraction based, the resulting image will be lower resolution than ordinary brightfield, despite having higher contrast. Compare this with DIC, where the 3D effect is generated using pure interference patterns via polarization mechanics, and thus does not lose resolution.
Diffraction order: A diffraction refers to the different angles that light is diffracted at when it passes through a material capable of splitting it. Every color has a different angle that it is diffracted by, causing the different colors to be split apart by e.g. the diffraction grating of a spectrometer. But also, every color will be split to multiple instances of that angle, creating a series of dots spaced by that color's angle apart. A common example is the continous rainbow effect seen on the surface of CDs, caused by the microscopic grooves on the surface of the CD splitting the light into different angles.
Circular Oblique: Whereas linear oblique only lets light in from one side, circular oblique lets light in from all angles, balancing contrast with a greyish background and creating a beautiful soft 3D effect in the image as no one side is too strongly lit. In fact the easiest of way of creating circular oblique is to use a darkfield stop with an insufficent numerical aperture for the current objective.
I highly recommend experimenting with oblique techniques. Perhaps the easiest way of beginning experimentation is by placing your finger in the light path of the condenser to block some of the light, and then slowly moving it around to see how the image changes. Note that the closer your finger is to the focal plane of the condenser, the sharper the shadow cast by your finger will be, and thus the more pronounced the effect will be, especially at high numerical apertures.
Phase Contrast
The previous techniques have all relied upon the diffraction of light to create contrast. With phase contrast, we utilize a much more elegant method.
Since light is a wave, it has 'peaks' and 'valleys', or more technically, it has a 'phase'. If I were to take two waves of light, and perfectly align them so that the peaks of one wave lined up with the peaks of the other, they would amplify each other, creating a brighter light. And the inverse would cancel the light out, creating darkness. This is what is called an interference pattern. The core of phase, is harvesting this interference pattern.
Phase contrast works by placing a special ring or 'phase plate' below the condenser, and a matching 'phase plate' one in a specialized objective. When light passes through different materials of varying thickness, like the phase plate or the sample itself, the 'phase' of the light will be shifted as it slows down within that material.
Since we know that light that is refracted by the sample will both be shifted in phase, and coming in at an angle relative to the background light, the phase rings work to shift the incoming light, creating an interference pattern. This shows up as dark or light halos tracing the edges of objects in the sample.
Due to this massive contrast-increasing effect, phase can let you see the ordinarily invisible, and is used to be sought-after in clinical settings for viewing unstained cells. However, the halos it creates can be very distracting, and so it is not always the best choice for viewing samples.
Note that while we are not using diffraction to generate the contrast, the use of phase rings neccesitates special objectives with annuli around the edges that reduce their numerical apertures. Therefore, compared to normal brightfield, a phase contrast objective of the same magnification will be lower in resolution.
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Differential Interference Contrast
This is regarded by many as the holy grail of microscope contrast methods. Differential Interference Contrast, or DIC, works on unstained specimens of many kinds and creates unmatched visual clarity, because the contrast it provides is not at the expense of resolution.
It does this by splitting the light into two beams, and then recombining them at the camera plane, creating interference patterns that result in a 3D-like effect in the image.
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Reflected-illumination
If brightfield is transmission, this is the inverse. Light is shone from above the sample, and the objective collects the light that is reflected off the sample.
There are two general forms of reflected-illumination:
Incident/external illumination: In incident illumination, you have a light source like a lamp or
Epi-illumination: This method is a more advanced version of incident illumination, and requires a module to modify the light path of the microscope.
Ordinary microscopes have their light sources and condenser opposite the objective. The 'epi' in epi-illumination means 'same', and this is because here, the objective becomes the condenser. This works by having a beamsplitter
You can find modules that provide reflected illumination of white-light using 50/50 half-silvered mirrors, but in the modern day, the most common use for the epi-illumination light path, is for fluorescence microscopy.
Fluorescence
Let us understand the basics first. Normally when light hits a molecule, it might be bounced off, giving the molecule it's color. Othertimes, and often for specific colors, the light is absorbed by the molecule. This energy must go somewhere, so it is converted into 'vibrating the molecule faster', or as we know it at larger scales, heat.
Certain types of molecules have unique structues that make them capable of absorbing light of one wavelength becoming temporarily 'excited' as they store this energy, before re-emitting this light with somewhat lower energy and therefore a longer wavelength. This is called fluorescence.
The mechanics of this involves a deep understanding of electrical states and molecular structure, but the important part is: All fluorescent molecules have specific 'wavelengths' or color ranges that they can absorb (excitation bands), and then cooresponding bands that they will reemit that light at (emission bands). You may find this refered to in a syntax like 470ex/680em (the peaks of chlorophyll fluorescence, which glows a deep red when exposed to blue excitation light).
Since most of the sample will be dark, while the fluorescent compounds will be glowing, this means you can use specific wavelengths of light to see only the fluorescent molecules, and nothing else. This is done by replacing the typical 50/50 beamsplitter in the epi-illumination module with a dichroic mirror, which is a special type of filter that reflects certain wavelengths of light while letting others pass through.
It so happens that in nature there is an incredibly large number of these such compounds, known as 'autofluorescent compounds'. Sometimes, this can be used to derive useful identification information about the compound, but this should be done with caution. However in individual cells, this property is rare enough that we can use specially engineered dyes and proteins, called fluorophores, to tag specific structures of interest. Hence, much modern biology relies on fluorescence microscopy.
Fluorescence but fancy
It was mentioned that fluorescence allows you to target specific structures and nothing else. When using the dichoric mirrors and color filters of fluorescent microscopes, you can quite literally filter out everything except the light emitted by the fluorophores you are interested in.
This concept can be expanded upon to great effect in high level research microscopy, making it one of the most diverse illumination and contrast methods in it's many forms.
Confocal, and Structured Illumination microscopy are two such techniques, and allows you to bypass one of the primary limitations of optical microscopy: blurry light. By only triggering fluorescene in certain parts of the sample, you can effectively counter the distortion caused by thick samples, and begin to see with much higher clarity.
Remember our discussion of microscope resolution, where the absolute resolution is limited by physically not being able to resolve two points that are closer than half the wavelength of light? With fluorescence-utilizing super-resolution microscopy, we can get around this limitation using complex arrays of lasers. But this is beyond the scope of this guide...
Fits your purpose
The key to purchasing a microscope is to know your intended purpose, and do a bit of research.
So that said, what is the ideal microscope for a given purpose?
If you plan to be working with well slides, petri dishes, or deep containers of the sort, you will benefit greatly from the extended working distance of an inverted microscope.
On the other hand, if all you are doing is looking at cilliates in pond water, any upright microscope will do.
Once you have a brightfield microscope, you can experiment with different contrast methods to see what you like, as most microscopes will allow simple modification like this.
For clinical work, or looking at small objects like bacteria, and identifying hard-to-see flagella, you may want a system capable of phase microscopy.
If you are working with roots or soil directly, having reflected capabilities, incident or epi, is crucial. Otherwise you will be looking at a dark silhouette, giving you little information.
Fluorescence can be very useful for obtaining extra information about a subject, identifying key structures, and narrowing down some compounds like microplastic (which fluoresces vibrant neon colors at 490nm excitation), but it is by no means a silver bullet.
Conclusion
The field of microscopes is a complex one. But now that you know the basics, you can begin to make informed decisions about what you want in a microscope.
Thank you for coming on this journey with us.
Good luck with your microscopic adventures!
