Basic of Optical Microscope

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.


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.


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.


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.