The principles of fluorescence microscopy and laser confocal microscopy
fluorescence microscope
1. A fluorescence microscope is a device that uses ultraviolet light as a light source to illuminate the object being tested, causing it to emit fluorescence, and then observe the shape and location of the object under the microscope. Fluorescence microscopy is used to study the absorption, transportation, distribution, and localization of intracellular substances. Some substances in cells, such as chlorophyll, can emit fluorescence after being exposed to ultraviolet radiation; Although some substances themselves cannot emit fluorescence, they can also emit fluorescence after being stained with fluorescent dyes or fluorescent antibodies and irradiated with ultraviolet light. Fluorescence microscopy is one of the tools for qualitative and quantitative research on these substances.
2. Principle of fluorescence microscope:
(A) Light source: The light source emits light of various wavelengths (from ultraviolet to infrared).
(B) Excitation filter light source: Transmitting light of a specific wavelength that can produce fluorescence in the specimen, while blocking light that is useless for excitation fluorescence.
(C) Fluorescent specimen: Generally stained with fluorescent pigment.
(D) Blocking filter: selectively transmits fluorescence by blocking excitation that has not been absorbed by the specimen, and some wavelengths are also selectively transmitted in fluorescence. A microscope that uses ultraviolet light as a source of light to emit fluorescence from irradiated objects. The electron microscope was first assembled by Knorr and Harroska in Berlin, Germany in 1931. This type of microscope uses high-speed electron beams instead of light beams. Due to the much shorter wavelength of the electron flow compared to light waves, the magnification of the electron microscope can reach 800000 times, with a minimum resolution limit of 0.2 nanometers. The scanning electron microscope, which began using it in 1963, allows people to see the tiny structures on the surface of objects.
3. Application scope: Used to magnify images of small objects. Generally used for observation of biology, medicine, microscopic particles, etc.
confocal microscope
1. A confocal microscope adds a semi reflective semi lens to the reflected light path, which bends the reflected light that has already passed through the lens towards other directions. There is a baffle with a pinhole at its focal point, and the small hole 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 focal point cannot be focused on the small hole through this confocal system, and will be blocked by the baffle. So what the photometer measures is the intensity of reflected light at the focal point.
2. Principle: Traditional optical microscopes use a field light source, and the image of each point on the specimen is affected by diffraction or scattered light from adjacent points; The laser scanning confocal microscope uses a point light source formed by a laser beam passing through an illuminated pinhole to scan every point in the focal plane of the specimen. The illuminated point on the specimen is imaged at the probe pinhole, and is received point by point or line by the photomultiplier tube (PMT) or thermoelectric coupling device (cCCD) after the probe pinhole, quickly forming a fluorescent image on the computer monitor screen. The illumination pinhole and the detection pinhole are conjugated relative to the focal plane of the objective lens. The points on the focal plane are simultaneously focused on the illumination pinhole and the emission pinhole, and points outside the focal plane are not imaged at the detection pinhole. This results in a confocal image that is the optical cross-section of the specimen, overcoming the drawback of blurring in general microscope images.
3. Application fields: involving 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, and mineralogy.
