Observation with Optical Microscope
This section introduces typical Observation with Optical Microscope in order to learn more about the microscope.
|・Bright-field Observation||It is the most popular way.|
|・Dark-field Observation||Although it is a common way, is there any advantages?|
|・Phase-Difference Observation||Method to enhance contrast of transparent sample without staining|
|・Differential interference observation||It is the way to emphasize the subtle difference in height of the surface.|
|・Polarization observation||It is the way to observe the particular sample.|
|・Fluorescence observation||It takes a fluorescent dye and a special microscope.|
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.
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.
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 inteference. The different point is that the phase shift is caused by the illumination ligth path difference between neibour neighbor.
With this method, Nomarski prizm is used. After through the prizm, 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 neibor area on 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.
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.
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.