How fluorescence microscopy differs from laser confocal microscopy
Fluorescence microscope
1, fluorescence microscope is to use ultraviolet light as a light source, used to irradiate the object to be examined, so that it emits fluorescence, and then observe the shape of the object and its location under the microscope. Fluorescence microscope is used to study the absorption, transportation, distribution and localization of chemical substances in cells. Some substances in the cell, such as chlorophyll, can fluoresce after irradiation by ultraviolet light; there are some substances that cannot fluoresce by themselves, but if stained with fluorescent dyes or fluorescent antibodies, they can also fluoresce after irradiation by ultraviolet light, and fluorescence microscopy is one of the tools for qualitative and quantitative research of this kind of substances.
2, fluorescence microscope principle:
(A) light source: the light source radiates various wavelengths of light (in the ultraviolet to infrared).
(B) excitation filter source: through the specimen can produce fluorescence of a specific wavelength of light, while blocking the excitation of fluorescence useless light.
(C) Fluorescent specimen: generally stained with a fluorochrome.
(D) Blocking filters: block out excitation light that is 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 a light source to make the irradiated object fluoresce. The electron microscope was first assembled in 1931 in Berlin, Germany by Knorr and Haroska. This microscope uses a high-speed electron beam instead of a light beam. Because the wavelength of the electron stream is much shorter than the light wave, so the magnification of the electron microscope can be up to 800,000 times, the resolution of the minimum limit of 0.2 nanometers. 1963 began to use the scanning electron microscope can be seen on the surface of the object's tiny structure.
3, the scope of application: used to magnify the image of tiny objects. Generally used in biology, medicine, microscopic particles and other observations.
Confocal microscope
1, confocal microscope in the reflected light on the road plus a half-reflective half-lens, will have been through the lens of the reflected light folded in other directions, in its focus on a baffle with a pinhole, the hole is located in the focus, behind the baffle is a photomultiplier tube. It can be imagined that the reflected light before and after the focal point of the detector light through this set of confocal system, will not be able to focus on the small hole, will be blocked by the baffle. So the photometer measures the intensity of the reflected light at the focal point.
2, principle: the traditional optical microscope uses a field light source, the image of each point on the specimen will be interfered with by the diffraction or scattering of light from neighboring points; laser scanning confocal microscope uses a laser beam through the illuminating pinhole to form a point source of light on the specimen in the focal plane of the scanning of each point on the specimen, the specimen is irradiated, in the detection of the pinhole at the imaging, by the detection of the pinhole after the photomultiplier tube (PMT) or the cold electrocoupling device (cCCD) point by point or point by point or point by point, the light intensity is measured by a photometer. cCCD) receives point by point or line by line, and rapidly forms a fluorescent image on the computer monitor screen. Illumination pinhole and detection pinhole relative to the focal plane of the objective lens is conjugate, the point on the focal plane at the same time focusing on the illumination pinhole and emission pinhole, the point outside the focal plane will not be in the detection pinhole at the imaging, so that the confocal image is the specimen of the optical cross-section, overcoming the shortcomings of the blurred images of ordinary microscopes.
3、Application fields: medicine, animal and plant research, biochemistry, bacteriology, cell biology, tissue embryology, food science, genetics, pharmacology, physiology, optics, pathology, botany, neuroscience, marine biology, materials science, electronic science, mechanics, petroleum geology, mineralogy.
