Difference between fluorescence microscope and laser confocal microscope
fluorescent microscope
1. The fluorescence microscope uses ultraviolet light as the light source, which is used to irradiate the detected object to make it emit fluorescence, and then observe the shape and position of the object under the microscope. Fluorescence microscope is used to study the absorption, transportation, distribution and location of chemical substances in cells. Some substances in cells, such as chlorophyll, can fluoresce after being irradiated by ultraviolet rays; Other substances can not fluoresce by themselves, but they can fluoresce after being dyed with fluorescent dyes or fluorescent antibodies and irradiated by ultraviolet rays. Fluorescence microscope is one of the tools for qualitative and quantitative research on these substances.
2, fluorescence microscope principle:
(a) Light source: The light source radiates light of various wavelengths (from ultraviolet to infrared).
(b) Excitation filter light source: transmitting light with a specific wavelength that can make the sample fluoresce, while blocking light that is useless for excitation fluorescence.
(c) Fluorescent specimens: generally stained with fluorescent pigments.
(d) Blocking filter: blocking excitation light not absorbed by the specimen to selectively transmit fluorescence, and some wavelengths in the fluorescence are also selectively transmitted. A microscope that uses ultraviolet light as the light source to make the irradiated object emit fluorescence. The electron microscope was first assembled by Knohl and Ha Roska in Berlin in 1931. This microscope uses a high-speed electron beam instead of a light beam. Because the wavelength of electron flow is much shorter than that of light wave, the magnification of electron microscope can reach 800 thousand times, and the minimum resolution limit is 0.2 nanometer. The scanning electron microscope, which began to be used in 1963, can make people see the tiny structures on the surface of objects.
3. Scope of application: used to enlarge the image of tiny objects. It is generally applied to the observation of biology, medicine and microscopic particles.
Confocal microscope
1. Confocal microscope adds a semi-reflective semi-lens on the optical path of reflected light, which refracts the reflected light that has passed through the lens to other directions. At its focus, there is a baffle with a pinhole, and the pinhole is located at the focus. Behind the baffle is a photomultiplier tube. It can be imagined that the reflected light before and after the focus of the detection light passes through this confocal system, and will not be focused on the small hole, but will be blocked by the baffle. So the photometer measures the intensity of reflected light at the focus.
2. Principle: The traditional optical microscope uses the field light source, and the image of each point on the specimen will be interfered by the diffraction or scattered light of the adjacent points; The laser scanning confocal microscope scans every point of the focal plane in the specimen by using the point light source formed by the laser beam passing through the illumination pinhole. The irradiated point on the specimen is imaged at the detection pinhole, which is received point by point or line by point by photomultiplier tube (PMT) or cold-coupled device (cCCD) after detecting the pinhole, and a fluorescent image is quickly formed on the computer monitor screen. The illumination pinhole and the detection pinhole are conjugate with respect to the focal plane of the objective lens, and the points on the focal plane focus on the illumination pinhole and the emission pinhole at the same time, and the points outside the focal plane will not be imaged at the detection pinhole, so that the confocal image obtained is the optical cross section of the specimen, which overcomes the defect of blurred image of the common microscope.
3. Application fields: involving medicine, animal and plant research, biochemistry, bacteriology, cell biology, tissue and embryo, food science, genetics, pharmacology, physiology, optics, pathology, botany, neuroscience, marine biology, materials science, electronic science, mechanics, petroleum geology and mineralogy.
