Differences between fluorescence microscopy and confocal microscopy
fluorescence microscope
1. Fluorescence microscope uses ultraviolet light as a light source to irradiate the object being inspected, 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, transport, distribution, and localization of substances within cells. Some substances in cells, such as chlorophyll, can fluoresce when exposed to ultraviolet radiation; There are also some substances that cannot emit fluorescence themselves, but can also emit fluorescence if 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 such 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 cause fluorescence in the specimen, while blocking light that is useless for exciting fluorescence.
(C) Fluorescent specimens: usually stained with fluorescent pigments.
(D) Blocking filter: It selectively transmits fluorescence by blocking excitation light that is not absorbed by the specimen, and some wavelengths in the fluorescence are also selectively transmitted. A microscope that uses ultraviolet light as a light source to make the illuminated object emit fluorescence. The electron microscope was first assembled by Knorr and Haruska in Berlin, Germany in 1931. This microscope uses high-speed electron beams instead of light beams. Due to the much shorter wavelength of electron flow compared to light waves, the magnification of an electron microscope can reach 800000 times, with a minimum resolution limit of 0.2 nanometers. The scanning electron microscope, which was first 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 observations of biology, medicine, microscopic particles, etc.
confocal microscope
1. Confocal microscope adds a semi reflective half lens to the reflected light path, which deflects the reflected light that has passed through the lens in other directions. At its focal point, there is a baffle with a pinhole located at the focal point, and behind the baffle is a photomultiplier tube. It can be imagined that the reflected light before and after the detection light focus cannot be focused on the small hole through this confocal system and will be blocked by the baffle. So the photometer measures the reflected light intensity at the focal point.
2. Principle: Traditional optical microscopes use field light sources, and the image of each point on the specimen is affected by diffraction or scattered light from neighboring points; Laser scanning confocal microscope uses a laser beam to illuminate a pinhole and form a point light source to scan every point on 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 cold coupling device (cCCD) after detecting the pinhole, quickly forming a fluorescence image on the computer monitor screen. The illumination pinhole and the detection pinhole are conjugate 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. The resulting confocal image is the optical cross-section of the specimen, overcoming the disadvantage of blurred images in general microscopes.
