Explain working of confocal microscope.Long answer is needed
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Answer:
A laser is used to provide the excitation light (in order to get very high intensities). The laser light (blue) reflects off a dichroic mirror. From there, the laser hits two mirrors which are mounted on motors; these mirrors scan the laser across the sample. Dye in the sample fluoresces, and the emitted light (green) gets descanned by the same mirrors that are used to scan the excitation light (blue) from the laser. The emitted light passes through the dichroic and is focused onto the pinhole. The light that passes through the pinhole is measured by a detector, ie., a photomultiplier tube.
So, there never is a complete image of the sample -- at any given instant, only one point of the sample is observed. The detector is attached to a computer which builds up the image, one pixel at a time. In practice, this can be done perhaps 3 times a second, for a 512x512 pixel image. The limitation is in the scanning mirrors. Our confocal microscope (from Noran) uses a special Acoustic Optical Deflector in place of one of the mirrors, in order to speed up the scanning. This uses a high-frequency sound wave in a special crystal to create a diffraction grating, which deflects the laser light (actually, the first diffraction peak is used, with the zeroth-order peak being thrown away). By varying the frequency of the sound wave, the AOD changes the angle of the diffracted light, helping scan the sample quickly, allowing us to take 512x480 pixel images 30 times per second. If you want to look at a smaller field of view, our confocal microscope can go even faster (up to 480 frames per second, although I personally find that 240 frames per second is a good practical limit
How does a fluorescence microscope work?
In the picture above, we are supposing the excitation light needs to be violet, and the emitted light is red. The microscope uses a special dichroic mirror (or more properly, a "dichromatic mirror", although this term only seems to be used by purists). This mirror reflects light shorter than a certain wavelength, and passes light longer than that wavelength. Thus your eye only sees the emitted red light from the fluorescent dye, rather than seeing scattered purple light. The purple and red bars next to the dichroic mirror represent additional filters to help prevent the different wavelengths of light from going the wrong directions.
This particular style of fluorescence microscopy is known as epi-fluorescence, and uses the microscope objective to illuminate the sample (rather than illuminating the sample from the other side, which would be trans-fluorescence).
What's this got to do with confocal microscopy?
Imagine we have some lenses inside the microscope, that focus light from the focal point of one lens to another point. This is represented by the blue rays of light in the above picture. The red rays of light represent light from another point in the sample, which is not at the focal point of the lens, but which nonetheless get imaged by the lenses of the microscope. (Note that the red and blue rays in the picture are meant to distinguish the two sets of rays, but they aren't meant to be different wavelengths of light.) The image of the red point is not at the same location as the image of the blue point. (You may remember this from introductory optics, perhaps you have seen a formula such as 1/s + 1/s' = 1/f for locating the image formed by a lens. Points don't need to be at the focal point of the lens in order for the lens to form an image.)
So, we want to just look at the blue point, that is, the point directly at the focus of the lens. If we put a screen with a pinhole at the other side of the lens system, at the image of the blue point, then all of the light from the original blue point will pass through this pinhole. However, most of the light from the red point is still out of focus at this screen, and gets blocked by the pinhole.
This solves one of the problems of regular fluorescence microscopy. Normally, the sample is completely illuminated by the excitation light, so all of the sample is fluorescing at the same time. Of course, the highest intensity of the excitation light is at the focal point of the lens, but nonetheless, the other parts of the sample do get some of this light and they do fluoresce. This contributes to a background haze in the resulting image. Adding a pinhole/screen combination solves this problem. Because the focal point of the objective lens of the microscope forms an image where the pinhole is, these two points are known as "conjugate points" (or alternatively, the sample plane and the pinhole/screen are conjugate planes). The pinhole is conjugate to the focal point of the lens, thus it is a confocal pinhole.
How does a confocal microscope work?
A laser is used to provide the excitation light (in order to get very high intensities). The laser light (blue) reflects off a dichroic mirror. From there, the laser hits two mirrors which are mounted on motors; these mirrors scan the laser across the sample. Dye in the sample fluoresces, and the emitted light (green) gets descanned by the same mirrors that are used to scan the excitation light (blue) from the laser. The emitted light passes through the dichroic and is focused onto the pinhole. The light that passes through the pinhole is measured by a detector, ie., a photomultiplier tube.
So, there never is a complete image of the sample -- at any given instant, only one point of the sample is observed. The detector is attached to a computer which builds up the image, one pixel at a time. In practice, this can be done perhaps 3 times a second, for a 512x512 pixel image. The limitation is in the scanning mirrors. Our confocal microscope (from Noran) uses a special Acoustic Optical Deflector in place of one of the mirrors, in order to speed up the scanning. This uses a high-frequency sound wave in a special crystal to create a diffraction grating, which deflects the laser light (actually, the first diffraction peak is used, with the zeroth-order peak being thrown away). By varying the frequency of the sound wave, the AOD changes the angle of the diffracted light, helping scan the sample quickly, allowing us to take 512x480 pixel images 30 times per second. If you want to look at a smaller field of view, our confocal microscope can go even faster (up to 480 frames per second, although I personally find that 240 frames per second is a good practical limit).
I Hope its enough..