How Fluorescence Microscopy differs from Confocal Laser Microscopy
Fluorescence microscope
1. A fluorescence microscope uses ultraviolet light as a light source to illuminate the object being inspected to make it emit fluorescence, and then observe the shape and location of the object under the microscope. Fluorescence microscopy is used to study the absorption and transport of substances within cells, as well as the distribution and positioning of chemical substances. Some substances in cells, such as chlorophyll, can fluoresce after being irradiated by ultraviolet rays; some substances themselves cannot fluoresce, but if they are stained with fluorescent dyes or fluorescent antibodies, they can fluoresce after irradiation with ultraviolet rays. Fluorescence microscopy is the One of the tools for qualitative and quantitative research on such substances.
2. Principle of fluorescence microscope:
(A) Light source: The light source radiates light of various wavelengths (from ultraviolet to infrared).
(B) Excitation filter light source: transmits light of a specific wavelength that can cause the specimen to fluoresce, while blocking light that is useless for stimulating fluorescence.
(C) Fluorescent specimens: Generally stained with fluorescent pigments.
(D) Blocking filter: Blocks the excitation light that is not absorbed by the specimen and selectively transmits fluorescence. Some wavelengths in the fluorescence are also selectively transmitted. A microscope that uses ultraviolet light as a light source to cause the illuminated object to fluoresce. The electron microscope was first assembled in 1931 in Berlin, Germany by Knorr and Hallowska. This microscope uses a high-speed electron beam instead of a light beam. Since the wavelength of electron flow is much shorter than that of light, the magnification of the electron microscope can reach 800,000 times, and the minimum resolution limit is 0.2 nanometers. The scanning electron microscope, which began to be used in 1963, allows people to see the tiny structures on the surface of objects.
3. Application scope: Used to enlarge images of small objects. Generally used in the observation of biology, medicine, microscopic particles, etc.
confocal microscope
1. The confocal microscope adds a semi-reflective semi-lens to the optical path of the reflected light, which refracts the reflected light that has passed through the lens to other directions. There is a baffle with a pinhole at its focus, and the pinhole is located At the focal point, behind the baffle, is a photomultiplier tube. It can be imagined that the reflected light before and after the detection light focus passes through this confocal system and cannot be focused on the small hole, and will be blocked by the baffle. So what the photometer measures is the intensity of reflected light at the focus.
2. Principle: Traditional optical microscopes use field light sources, and the image of each point on the specimen will be interfered by diffraction or scattered light from adjacent points; laser scanning confocal microscopes use laser beams to form point light sources through illuminating pinholes to illuminate the interior of the specimen. Each point of the focal plane is scanned, and the illuminated point on the specimen is imaged at the detection pinhole, which is received point by point or line by line by the photomultiplier tube (PMT) or cold coupling device (cCCD) behind the detection pinhole, and is quickly A fluorescent image forms on the computer monitor screen. The illumination pinhole and the detection pinhole are conjugate relative to the objective lens focal plane. Points on the focal plane are focused on the illumination pinhole and the emission pinhole at the same time. Points outside the focal plane will not be imaged at the detection pinhole. This is obtained Confocal images are optical cross-sections of specimens, overcoming the shortcomings of blurry images in ordinary microscopes.
3. Application fields: Involving medicine, animal and plant scientific research, biochemistry, bacteriology, cell biology, tissue embryology, food science, genetics, pharmacology, physiology, optics, pathology, botany, neuroscience, marine biology, and materials science , electronic science, mechanics, petroleum geology, mineralogy.
