Microscopes on the Cutting Edge
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 objective lens. Because both illumination and image are focused together, the microscope is described as confocalmicroscope. 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 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 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.
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 detect image of SEM is different from 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.