When the magnifier itself was the cutting edge of technology, the microscope has been always the leading technology. After that, microscope contributed a lot to medical and biology studies such as finding and observing virus itself. Moreover, an atom can be observed with a certain microscope and the application is getting more and more. Microscope developed with the basis of new theory is also launched in the market.

Here, we try to use easy words and explain microscope, especially optical microscope including technical words and leading technic. In 「Terms for Optical Microscope」we explain the features and the meanings for the users who just started to use optical microscope.

History of Optical Microscope

Optical lens and Optics that existed from BC

The microscope is also regarded as a kind of magnifying glass, the lens has a long history from BC. Lens was made by polishing crystal, and it has been discovered from the ruins of Iran. The lens was used to collect solar heat, the applications of this era, it seems the doctor had used to burn the wound closed.
 It was used as a magnifying glass after the 11th century. Aruhazen scientist Arabic (Egypt) is in the “optical” his book, there was a presentation describing the structure of the eye lens that looks at optics principles and compensate for the vision in it. The book had been translated into Latin, to be read in a number of monks, manufacture of the lens by the monks became popular throughout Europe. In the 13th century, was used as a magnifying glass (leading stone) placed directly on top of the book.
 Also found describing the lens to evolve, in Venice, Italy, and from becoming like glass colorless and transparent, unlike colored glass until it is made active, that there were glasses already in the 13th century are.

Invention of the microscope in the Netherlands at the end of the 16th century

 Janssen parents, the Netherlands around 1590, has the invention of the microscope by combination of the two convex. They looked through a telescope in reverse, and was discovered by chance. They used the Kepler telescope, which is now commonly used as an astronomical telescope and it shows reversed image. 
 Robert Hooke, as the famous Hooke’s law, has published a sketch of microorganismsby using the microscope in the 17th century. Magnification of the microscope at that time is said to have been most about 150x. On the other hand, Leeuwenhoek, Dutch hook introduced to the British Royal Society, has achieved a magnification of 200x or more in a single lens microscope with polished. It was kind of like a lens with a prepared slide (glass plate for observation for fixing the sample) this microscope preparations, of Leeuwenhoek. Around this time, materials for the glass lens has not yet been well studied, and two pieces fit lenses appear to be distorted just one sheet, the image is no longer visible.

Establishment of the modern microscope

 It was from the 19th century, The current microscope that combines multiple lenses started evolving. Carl Zeiss, Germany, realized magnification of 6-700x with Shot, glass blower, and Abbe, physicists, and made a significant contribution to medicine and biology. Performance is improved almost to the limit of the optical theory, the end of the 19th century. 
 Then, the device and lighting, such as polarized illumination, the sample processing techniques such as fluorescence has been developed, there was an extension of the optics. In that it is possible to know the three-dimensional structure (see more clearly) to raise the resolution, confocal microscope began to be studied in the late 1950s.

Beyond the optics

 On the other hand, more than using light (image performance that is clearly visible even when expanded) the resolution of the optical microscope is limited, theoretically. Hideyo Noguchi could not be considered the cause of yellow fever is a virus, because of the lack of the spatial resolution of the optical microscope. Thus, electron microscopy (transmission type), appeared by replacing light into electrons. The theoretical spatial resolution of electron microscopy is 1000 times higher than optical microscopy. It can observe the 1nm structure. Developed in Germany in the 1930s, a transmission electron microscope, in 1938 before the war, had already been released from Siemens. 
 In the same electron microscope, the principles of the scanning electron microscope are different. Rather than enlarge the image in the lens, as thin needle tip (probe) scan the surface of the sample, it can be said that it is the prototype of a scanning probe microscope. Its development prototype was made from the 1960s.
 The first scanning probe microscope (SPM) was scanning tunneling microscopy(STM), that was developed by IBM in 1983 and able to measure the image of atoms. 
 After that, SPM also utilizes the atomic force and scan the atomic information by using X-ray diffraction or reflected electrons. Also, the development of a computer helps the device control and image processing.
 In optics, scanning probe type has been developed. Recently, microscopy that utilize the near-field light has been being developed.

Components of Optical Microscope

Objective lens

 This is a lens located on the side of the sample and the sample image cannot be observed by magnifying the image without this lens. Leeuwenhoek’s microscope had used the only objective lens. Although the early objective lens was made with the only one lens, the latest objective lens is the combination lens eliminating the aberration that is the cause of the distortion. Also there is a lens called oil immersion lens designed to eliminate the influence of the difference in the refractive index between air and lens by filling the oil having the same refractive index as the glass, between the lens and the sample. 
Cause of aberrations is on the shape of the lens itself and also on what the light is refracted at the lens.
Chromatic aberration: Please remember a prism. Newton discovered that the sunlight (white light) splits into the colors of the rainbow if it passes through the prism. When white light passes through the glass, the light has the different refraction angles depending on the wavelengths (color) so that the light having the different color goes through different route and as the result, the colors blur after the light goes through the lens. 
Spherical aberration: Surface of the lens was spherical until recently. If the spherical lens is used, the parallel light enters into the outside of the lens and the parallel light enters into the center of the lens are not focused at the same one point. If the lens is designed to focus the parallel lights on the same one point, the curved surface of the lens is no longer spherical. A non-spherical lens (aspherical lens) has been able to be designed recently and it has been applications/ed to the DVD pickups, CD and glasses. 
Objective lens is designed to make the aberration smaller by the combination of several lenses.
In the electron microscope, the electromagnet to focus the electrons becomes the objective lens. The lens is not used for a scanning probe microscope.

Eyepiece

This is the lens closer to the eye. Since the binocular is used for an optical microscope it is so called binocular or eyepiece. The magnification of the microscope is determined by multiplying the magnification of the objective lens by the magnification of the eyepiece.
Recently, the optical microscope with a CCD camera is becoming popular. With regard to a laser scanning microscope, the image is observed through a detector such as a CCD camera. In this case, the lens in front of the detector acts as the eyepiece. 
The diaphragm is placed in front of the eyepiece for a confocal microscopy to eliminate the ambient light that is the cause of the blurred image. The only one point can be seen in this configuration so the scanning function is required to observe the image.

Illumination

The illumination plays an important role for the optical microscope. 
The most common illumination is the bright-field illumination. This is the illumination to fully enter the illumination light into the diameter of objective lens to reduce the extra diffuse reflection light. The image contrast is created by the light absorption rate of the sample for the transmitted illumination and the reflectance of the light for the reflected illumination.
The dark-field illumination is the illumination to introduce the illumination light into just barely around the objective lens and enter the only refracted or diffuse reflected light by the sample into the objective lens.
In the fluorescence microscope, the sample is dyed with a special dye to generate fluorescence. The fluorescence is emitted by the illumination of certain wavelength of light (excitation light). The wavelength of the fluorescence light is different from that of the excitation light so the only fluorescence light can be observed in the dark field by separating the fluorescence light from the excitation light with the filter.
There are other illumination methods such as a polarized and phase contrast illumination.

Sample

If there is no sample, nothing is observed. But the sample is not always simply placed in front of the objective lens. If the oil immersion lens described above is used, the sample should be created as a preparation to demonstrate the effect of the emulsion oil. Furthermore, if the fluorescent observation is practiced, the sample must be dyed with a fluorescent dye as far as the fluorescent material is not already contained. To increase the contrast, the sample is also dyed with the color. 
In the transmitted electron microscope, the sample should be precisely sliced to make the very thin film to uniformly pass the electrons through it. There is a dedicated machine to make the thin film sample. In the scanning electron microscope, the non-conductive sample must be coated with a metal to avoid the charge up on the sample surface by the irradiation of the electron beam.

Microscopes on the Cutting Edge

Confocal microscope

When we see an ideal point by microscope, it can not be seen proper point. We see rings, which causes blur, around the point. The microscope which cut off the rings by putting stop is a confocal microscope. One more feature is spot illumination via an objective lens. Because both illumination and image are focused together, the microscope is described as confocal microscope. But since in this confocal microscope we can see the image only at the one point, scanning is needed for imaging. When focal spot is misaligned in height direction, focal spot of the illumination is also misaligned. Then the image gets dark and invisible. In the confocal microscope, since only the focal spot can be seen brightly, samples can be captured sterically.
In applications for biology, by observing fluorescing dyed samples via confocal microscope, it becomes possible for us to recognaize blurring image as clear and stereoscopic image. This technique is similar to nonlinear optical microscope that can see only fluorescence induced by laser light.

Raman microscope

Raman effect is that light, which wavelength is different from the illuminating light, comes from materials by the illumination of the samples depending on the its condition. Without fluorescing dyeing, the light differenct from excitation comes from. But the light is very weak, the Raman effect could not be realized until strong laser light would appear.
Since Raman scattering light induced by certain incident light is very unique for the materials, it is possible to identify the materials by checking the spectrum of the scattering light. Although the structure of the Raman microscope is similar to that of fluorescence confocal microscope, the Raman microscope has a spectrometer.

Nonlinear optical microscope

 Nonlinear optical microscope can see the different light from illumination. Although illumination light is reflected and refracted in the materials, small part of the light is changed its wavelength depending on the condition of the materials. It it also known that the condition of the materials can be changed by strong illumination.

Second harmonic generation microscope

The second harmonic generation (SHG)is a frequency doubled light to an incident light generated in the materials by the incident light. This phenomenon can be applications/ed to shorten (increase) the wavelength (frequency) of the laser light. SHG has many features such that surface state can be studied by seeing SHG intensity which depends on fine structures of the materials, and 3D conformation of the materials can be studied because the SHG is observable only in the illuminated part.

CARS microscope

CARS is a Raman scattering which generates high frequency light rather than incident light. CARS microscope can image the fluorescent biological sample without any dyeing. And it is also possible to know 3D conformation same as SHG microscope.

Electron microscope

Transmission electron microscope (TEM)

Using not light but electron, TEM is the microscope which defeats the limit of spatial resolution in principle. Although electron beam is used for illumination and electron lens which can bend electron beam by electromagnet is used instead of optical lens, a principle of detect image of TEM is same as optical microscope.

Scanning electron microscope (SEM)

Although SEM uses electrons, a principle of detecting an image of SEM is different from the optical microscope in principle. It is more similar to Scanning probe microscope (SPM). By tracing the sample surface with electron beam, which is very narrow sting (probe), an image can be made from the information obtained during the tracing. At first, secondary electrons are observed in SEM. Recently, reflection electron and X-ray, which give a surface composition, can be observed, so that SEM has also been developed for analysis equipment.

Scanning probe microscope


Scanning tunneling microscope (STM)

Using sting as probe, STM measures distance by tunneling effect between sample and sting. As sample is scanned by piezo device precisely, the sting traces over the sample with keeping a certain distance to the sample surface. The position of sample and sting are reconstructed by computer.

Atomic force microscope (AFM)

STM measures tunneling effect, so that it can be utilized for only conductive samples. But AFM can be utilized for insulating sample and sample put under water. Probe is sting as same as STM. By detecting the sting moving by atomic force when the sting sets very near to the sample, the distance in height direction is recorded. As sample scanning is controled precisely by piezo device, the position of sample and sting are reconstructed by compluter same as STM.

Observation with Optical Microscope

This section introduces typical Observation with Optical Microscope in order to learn more about the microscope. 

Bright-field Observation

 This is the most popular way and everyone experiences this way at school by using microscope. The sample is visible in the bright background when observation of transparent. 
 This is the observation which can obtain the contrast of the image by illuminating the sample uniformly and measuring the difference in the reflectance and transmittance. This is the same as we see things every day. 
 But in case of biological, many samples are transparent and mostly colorless. When we see by the method of transparent observation, we can’t obtain a clear contrast. For this reason, method for staining to color the sample was developed. 
 In transparent observation, the illuminating device is set on the opposite side of objective lens, and we adjust the illumination light to enter the fully objective lens. For this reason, the background of the field of view looks bright. When we observed in reflected light such as metallurgical microscopes, epi-illumination that uses the objective lens as a lens for illuminating is performed.


Dark-field Observation

Bright-field Observation is the way to improve unclear, transparent and colorless samples by changing the lighting method. Broadly speaking, it is the way of observation to shed obliquely from the front when the lighting was bright-field. 
 Place the ring-shaped slip to hide the center in front of the illumination lens. And illumination light is only from the peripheral portion, and oblique light irradiates a sample. In the absence of the sample, illumination light does not enter the objective lens, and the background is dark. In the presence of the sample, the direction of the illumination light is changed by the refraction, and the light enters the objective lens. Then on the part of the sample looks bright. There is a possibility that the sample which is hard to see in bright-field can be seen with contrast. So it is not necessary to stain the sample. The resolution does not mean better, but in such cases there is a very small object on the surface of uniform marginal resolution, which might be easy to find.

Phase-Contrast observation

 It is devised to see the transparent sample without staining of. It is also said that it is a further evolution of Dark-field Observation. 
 In transparent observation, the light phase is different between the light pass through the sample and did not pass through. This is because the speed of lighting transmitted is changing by material. (affected by diffraction and refraction). But phase difference caused by presence or absence of materials can’t detect by the human eye. This is the method to convert phase difference to contrast utlizaing of the nature of light inference.
Different type illumination and special objective lens are used for this method. For the illumination, illumination light is irradiated on ring-shape area on the sampoel with ring-slit. The objective lens has the phase ring at the pupil.
Light passing trough the sample (phase material or different refractive index) will be affect by the material. Its phase will be shifted with less than quater wave, and its propagation direction will be changed so that the light does not go through the phase material in the objective lens. If the light does not go throug the sample, the light not goes through the phase ring and its phase is shifted in opposite way with phase shift with the sample. The phase difference between the light from the sample and not from the sample gives the interference at the camera and causes the contrast.

Differential interference contrast observation

 This method is similar to Phase-Contrast method in terms of utilizing interference. The different point is that the phase shift is caused by the illumination light path difference between neighbor.
With this method, Nomarski prism is used. After through the prism, the illumination light is splited in two ways with slightly different angle. Each light has different status of polarization (orthogonal). These two lights are focused into neighbor the sample with the objective lens. The distance between two focus is only half spot to one spot. After these two lights pass through the sample, these lights are coupled to one light with another Nomarski prism. If one light pass through the sample of different refractive index, the coupled light intensity will be affected by the interference and as the results, the contrast are give by the interference.

Polarization observation

  For anisotropic sample, polarization observation is effective. In polarizaion observation, polarized light is illuminated on sample and the light passing trough the sample is observed though polarizer. When this polarizer is rotated, the sample color will be changed.

Fluorescence observation

 This is the method to see the stained sample with fluorescent dye. Typically the UV or blue light is irradiated on the stained sample. The light will excite the fluorescent dye. The fluorescent light from the dye will be observed easily.

Optical microscope terms

Here, terms are explained in order to get a better understanding of microscope terms that we often see or hear.

Magnification

If a 10 micron sample looks 10 mm, the magnification will be 1000 times.
If it is in the picture, it will be clear because you can hit the ruler. A 10 μm object that is 10 mm in the picture is 1000 times larger.
The magnification during observation is the magnification of the microscope by multiplying the magnification of the objective lens and the magnification of the eyepiece. This magnification is calculated correctly as long as you use an objective and an eyepiece that meets the microscope. (It is designed that way.)
There are microscopes of 160 mm and 210 mm in length depending on the manufacturer, depending on the manufacturer. The eyepiece and the objective lens are made to fit this distance. The magnification is in this design specification, so if you use an objective lens with a different setting, the magnification will be different. Other two lenses are designed so that the best image and performance can be obtained, so it is not recommended to mix and use lenses from various manufacturers for unknown reasons.
Magnification simply means how much you stretch, so it is not a measure of whether the image is sharp or not. Even if you stretch a mountain landscape photograph taken with a digital camera, you will not see the leaves that grow on the mountain, it will be blurry and you won’t know what it is.

Immersion lens

It is usually an objective lens with a line under the notation of magnification. It is also known as “OIL”.
This lens is filled with an oil with a glass-like refractive index between the lens and the sample to eliminate the effects of air and lens refraction. Therefore, if you do not use oil, performance can not be fully achieved.
With ordinary lenses, the medium that passes light changes in two places: lens (glass) → air → cover glass, and refraction occurs. Oils and immersion oils used in oil immersion lenses have a refractive index that matches that of glass, so refraction does not occur. It is a feeling that the sample is taken into the glass. This leads to an increase in the numerical aperture, which in turn leads to an increase in resolution.
Because of their high performance, oil immersion lenses are also being applied to semiconductor exposure equipment. In addition, even with lenses that do not use light, such as electron microscopes, electron lenses that function similarly are called oil immersion lenses.


Numerical aperture(NA)

The numerical aperture determines the resolution. Assuming that the angle of the cone that is fully extended to the objective lens from the focal point of the objective lens is θ, the NA and numerical aperture are represented by n sin θ. Here, n is the refractive index of the substance between the objective lens and the sample.
Intuitively, it is how wide you can take in the light from the sample. The lens also shows the numerical aperture, which is about 0.95 for high-performance objectives.
Since the refractive index of air is 1, NA does not exceed 1. Conversely, if n is larger than one, a larger numerical aperture can be obtained. The oil immersion lens has a larger NA by filling an oil or the like with a higher refractive index than air between the objective lens and the sample. The immersion lens has a numerical aperture greater than 1 and in some cases 1.40. The fact that the numerical aperture is 1 or more means that θ is 90 ° and NA = 1. Therefore, intuitively, it is a sense that light reflected in the opposite direction to the objective lens is also collected.
The limit of resolution is wavelength / 2NA in air, and the larger the numerical aperture, the smaller the resolution, which means that you can see the finer things. In addition, the lens with a large NA will necessarily have a short working distance.

 

Resolution

It is a numerical value that indicates how small things can be seen. The magnification can be increased by increasing it, but if the resolution is not sufficient, it will be blurred when expanded. This is why if you enlarge the image of your camera phone, it will become blurry.
It is also called resolution, but with a microscope, the resolution is the smallest distance between two lines (points) that can be discerned by the device. A resolution of 1 micron means that two lines spaced 1 micron apart look like two lines. The smaller the distance, the higher the resolution and the higher the resolution.
The number of pixels is also a number that represents another feature of resolution. Even with a 5 million-pixel digital camera, if you don’t have the resolution, you can only get blurred photos. However, usually you should have a high resolution lens with a large number of pixels.
When the slit of the interval d can be determined at the last minute, its resolution is d. The light passing through the slit will be split off as it gets smaller. The light that has passed straight through (0th order) is the light that is always visible, even if the distance is changed. If at least the lowest-order diffracted light (± 1st order) does not enter the lens, it is not clear that it is a slit. The angle θ of diffracted light at this time is d sin θ = λ, where λ is the wavelength. This angle is the same as the definition of the numerical aperture NA, so d = λ / NA. As the wavelength is smaller and the numerical aperture NA is larger, d is smaller, and it can be understood that higher resolution can be obtained.
The above explanation is for the case where the light strikes vertically, but in fact the light strikes also from an angle, so it can be seen that the way of lighting also affects the resolution. Ideally, if the illumination is done with the same lens, twice the resolution is obtained, so the resolution limit is λ / 2NA.

Aberration

Thin lenses or lenses with long focal lengths do not refract as much and the light is almost in focus, but generally it is out of ideal focus. This shift is called aberration, which causes the image to blur. Aberrations are caused by the fact that light is refracted and the shape of the lens itself.
Chromatic aberration: When sunlight (white light) is passed through a prism and white light passes through glass as the light is split into iridescent colors, the angle of refraction differs depending on the wavelength (color), and the passage path is different. You If you let go through the lens, the color will be faint.
Spherical aberration: The surface of an ordinary lens is spherical, and collimated light entering the periphery and collimated light entering near the center do not focus at the same point. It is a parabolic mirror that the focus is tied to one point. Therefore, for CD and DVD optical pickups, an aspheric lens is used whose curved surface is designed to minimize this distortion. In a microscope, it is a lens with high refractive index, and the curvature is reduced, and the curvature per sheet is reduced by using two lenses to reduce the influence.
Other than this, coma aberration in which the image in the peripheral part is blurred, astigmatism generated in a ray from the outside of the optical axis, curvature of field which can make the image into a plane and curved, and straight lines in the periphery of the screen There are distortions etc. which are bent in the mold.

Aberrations are designed to be small using several lenses, such as changing the material of the glass and combining and canceling lenses of opposite nature. Depending on the type of this correction, microscope objectives include lenses such as “Achromat,” “Apo Chromat,” and “Plan Apo Chromat.”
Achromatic has chromatic aberration removed for only two colors (red and blue), so some colors will be blurred even if it is in focus. However, if you take monochrome photos with monochromatic light, that is enough. Apochromat is a lens that removes chromatic aberration of three colors, and Plan Achromat is a lens that corrects the curvature of field of achromatic. Plan lenses can capture the entire field of view without distortion using photographs or CCDs. And Plan Apochromat is a lens with the above two performances, and of course, it becomes an expensive lens.

Depth of focus

When focusing on the sample, an image is created on the focal plane. The distance that the image can tolerate (do not blur) when the focal plane is moved is called the depth of focus. Since the depth of focus is inversely proportional to the numerical aperture, we can not say that the resolution is high and the depth of focus is deep. Generally, the higher the magnification, the higher the numerical aperture, the higher the resolution and the shallower the depth of focus. In addition, the depth of focus in photography is only about half of that observed with the naked eye, and it is more difficult to focus in photography than that with the naked eye. The CCD further reduces the depth of focus, but you may focus on it while looking at the monitor, so it may not bother you.
On the other hand, when the sample is in focus, the distance that does not blur even if you move the sample is called the depth of field. Things within the depth of field will be visible at the same time. The depth of field is also inversely proportional to the numerical aperture.


Confocal microscopes have a smaller depth of field than conventional microscopes, but only in focus areas are bright. By using this to shift the sample in the direction of the optical axis (scan in the Z direction) and record only the maximum value of the brightness, you can in principle create an image with an infinite depth of field. Some recent image processing apparatuses select and combine only the images in focus to realize the same function.