Comparison of confocal microscope and ordinary optical microscope
Microscope is the main tool for observing cells. Depending on the light source, it can be divided into two categories: optical microscope and electron microscope. The former uses visible light (ultraviolet microscope to ultraviolet light) as the light source, while the latter uses an electron beam as the light source. Ordinary optical microscope and laser confocal microscope belong to the same optical microscope.
I. General Optical Microscope
Ordinary biological microscope consists of three parts, namely: ① illumination system, including light source and condenser; ② optical magnification system, consisting of objectives and eyepieces, is the main body of the microscope, in order to eliminate spherical aberration and chromatic aberration, the eyepieces and objectives are composed of a complex lens group; ③ mechanical device, used to fix the material and observation of convenience.
Microscope object is clear not only determined by the magnification, but also with the microscope's resolving power (resolution), the resolving power refers to the microscope (or the human eye from the target 25cm) can distinguish the object zui small intervals of the ability to distinguish the size of the resolving power is determined by the wavelength of the light and the mirror mouth rate and the refractive index of the medium, expressed in the formula:
R = 0.61λ /N.A. N.A. = nsinα/2 where: n = the refractive index of the medium; α = the mirror mouth angle (specimen on the objective lens mouth of the angle of tension), N.A. = mirror mouth rate (numeric aperture). The angle of the mouth of the mirror is always to be less than 180?, so the zui value of sina/2 is necessarily less than 1.
The refractive index of glass used in the production of optical lenses is 1.65 ~ 1.78, the refractive index of the medium used the closer to the glass the better. For dry objective lens, the medium for air, mirror mouth rate is generally 0.05 ~ 0.95; oil lens with cedar oil as the medium, mirror mouth rate can be close to 1.5.
Ordinary light wavelength of 400~700nm, so the microscope resolving power value will not be less than 0.2μm, the human eye's resolving power is 0.2mm, so the general design of the microscope zui magnification is usually 1000X.
Second, laser confocal scanning microscope
Laser confocal scanning microscope (laser confocal scanning microscope), with a laser as a scanning light source, point by point, line by line, surface by surface rapid scanning imaging, scanning laser and fluorescence collection of a common objective, objective scanning laser focusing point, but also instantaneous imaging of the object point. Due to the shorter wavelength of the laser beam, the beam is very fine, so the confocal laser scanning microscope has a high resolution, about 3 times that of an ordinary optical microscope. The system is focused once and the scan is limited to one plane of the sample. When the focusing depth is not the same, the images of different depth levels of the sample can be obtained, and these image information are stored in the computer, through computer analysis and simulation, it can show the three-dimensional structure of the cell sample.
