What is the difference between fluorescence and laser confocal microscopy?
fluorescence microscope
1. 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 position of the object under the microscope. Fluorescence microscopy is used to study the absorption, transportation, distribution, and localization of substances within cells. Some substances in cells, such as chlorophyll, can emit fluorescence after being exposed to ultraviolet radiation; Some substances themselves may not emit fluorescence, but if stained with fluorescent dyes or fluorescent antibodies, they can also emit fluorescence under ultraviolet radiation. 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 specimens: generally stained with fluorescent pigments.
(D) Blocking filter: selectively transmits fluorescence by blocking excitation light 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 light source to emit fluorescence from the irradiated object. The electron microscope was first assembled by Knorr and Harroska in Berlin, Germany in 1931. This type of microscope uses a high-speed electron beam instead of a beam of light. 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 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 for observation of biology, medicine, microscopic particles, etc.
confocal microscope
1. A confocal microscope adds a semi reflective lens to the reflected light path, which bends the reflected light that has already passed through the lens in 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 the photometer measures the intensity of reflected light at the focal point.
2. Principle: Traditional optical microscopes use field light sources, and the image of each point on the specimen will be affected by diffraction or scattered light from neighboring points; Laser scanning confocal microscope uses a laser beam to form a point light source 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 detection pinhole, and is received point by point or line by a photomultiplier tube (PMT) or a thermoelectric coupling device (cCCD) after the detection 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 will not be imaged at the detection pinhole. This results in a confocal image that represents the optical cross-section of the specimen, overcoming the drawback of blurred images in conventional microscopy.
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.
