What are the main applications of laser scanning confocal microscopy technology?

Laser scanning focus scanning microscope is widely used and has attracted increasing attention in life science and medical research.

In situ identification of biological macromolecules in cells or tissues, observation of cell or subcellular morphological structure.

In situ detection of nucleic acids;

detection of proteins, antibodies, and other macromolecules;

detection of apoptosis;

observation and determination of organelles (mitochondria, lysosomes, endoplasmic reticulum and Golgi apparatus);

Detection of cell fusion;

observation of the cytoskeleton;

detection of intercellular junction communication;

Detection of fat within cells or tissues;

Three-dimensional reconstruction of cell or tissue structures, etc.

Histomorphological observations.

Research on the physiology, mechanism, and function of living organs, such as neurobiology and developmental biology.

Open state observation and functional study of microorganisms, bacteria, and viruses.

High-resolution imaging in interdisciplinary courses, such as pharmacology research, materials research, chemical raw materials, etc.

In the field of material production, the confocal laser scanning microscope based on laser confocal microscopy technology measures and analyzes the surface morphology characteristics of various products, components and materials, such as surface profile, surface defects, abrasion, corrosion, flatness, roughness, waviness, pore clearance, step height, bending deformation, and processing.

Based on laser confocal microscopy technology.

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The laser scanning confocal microscope uses the focused laser spot as the illumination source, and at the same time, a pinhole is introduced in front of the detector to filter the interference signal outside the focused spot, so the image signal-to-noise ratio is improved, and the lateral resolution can reach about 200nm.

Laser confocal microscopy (LSC) is a very common microscopy technique with high resolution. It uses laser beams to co-concentrate in the projected image for fast and autofocus, which can be used for microscopic imaging and quantitative analysis. Laser confocal microscopy can achieve faster observation of invisible tiny objects, and its resolution can reach the nanometer scale of the unit distribution pin, which can turn tiny objects into clearly visible ultrastructures.

For example, looking at the molecular mechanism of intracellular molecules at the molecular scale, it can be used to accurately locate intracellular proteins in cancer research, and can also comprehensively study the internal biological structure of microorganisms, so as to better understand the relationship between microorganisms.

The resolution of an LSC depends on the quality of its projection objective. In general, the smaller the refractive index difference between the projection objective and the air, the higher the resolution achieved. When the gap between them is less than 10, a resolution of 1nm can be reached, and even it can be broken through to achieve extremely high resolution.

The imaging resolution of laser confocal microscopy is used to obtain higher image clarity than traditional far-small techniques. Therefore, laser confocal microscopy is an important technique in microscopy, and its resolution plays an important role in improving the clarity of microscopic imaging.

Answer] The principle of :d laser scanning confocal microscope is to use the laser scanning beam to irradiate the point light source formed through the illuminated pinhole, scan each point of the focal plane in the specimen, and the irradiated point on the specimen is imaged at the detection pinhole. Most fluorescein requires short-wavelength excitation, and ultraviolet light is used as the excitation source.

Ordinary optical microscopes use visible light as the source of light talk.

One is the laser confocal microscope applied in biological and medical research, which can obtain fluorescence images of the internal microstructure of cells or tissues, and observe physiological signals such as Ca2+, pH, membrane potential and cell morphological changes at the subcellular level, which is a new generation of powerful research tools in the fields of morphology, molecular biology, neuroscience, pharmacology, genetics, etc.

Another laser confocal microscope is a detection instrument that uses confocal microscopy technology as the principle, combined with precision Z-direction scanning module, 3D modeling algorithm, etc., to scan the surface of the device and establish a 3D image of the surface, which is used to measure the surface of various precision instruments and materials at the micro and nano level. It mainly realizes 3D measurement of the surface topography of the device, and can measure and analyze the surface morphology characteristics of various products, components and materials such as surface profile, surface defects, wear, corrosion, flatness, roughness, waviness, porosity, step height, bending deformation, processing and other surface morphology characteristics of various products, components and materials.

Laser confocal scanning microscopy.

First, the principle is different.

1. Fluorescence microscope: It uses ultraviolet light as the light source to irradiate the object to be examined to make it fluoresce, and then observes the shape and location of the object under the microscope.

2. Laser confocal microscope: a laser scanning device is installed on the basis of fluorescence microscope imaging, and the fluorescent probe is excited by ultraviolet or visible light.

Second, the characteristics are different.

1. Fluorescence microscopy: used to study the absorption, transport, distribution and localization of chemical substances in cells, etc. Some substances in cells, such as chlorophyll, can fluoresce after being irradiated with ultraviolet light; Other substances do not fluoresce on their own, but they can fluoresce when irradiated with ultraviolet light after staining with fluorescent dyes or fluorescent antibodies.

2. Laser confocal microscopy: use computer for image processing, so as to obtain fluorescence images of the internal microstructure of cells or tissues, and observe physiological signals such as Ca2+, pH value, membrane potential and cell morphological changes at the subcellular level.

Third, the use is different.

1. Fluorescence microscopy: Fluorescence microscopy is a basic tool for immunofluorescence cytochemistry. It is composed of the main components such as the light source, the filter plate system and the optical system.

It is the fluorescence image of the specimen that is emitted by a certain wavelength of light and the emission of the specimen, which is magnified by the objective lens and eyepiece system to observe the specimen.

2. Laser confocal microscopy: Laser scanning confocal microscopy has been used in the study of cell morphological positioning, three-dimensional structure recombination, dynamic change process, etc., and provides practical research methods such as quantitative fluorescence measurement and quantitative image analysis, combined with other related biosilver deficiency technologies, and has been widely used in the fields of molecular cell biology such as morphology, physiology, immunology, and genetics.

The laser confocal microscope setup is to place two small holes on the conjugate surface of the focal plane of the object to be measured, one of which is placed in front of the light source and the other in front of the detector, as shown in the figure.

Schematic diagram of the optical path of a confocal microscope.

The resulting image is a result of light from one focal plane focused by a pinhole digital camera, and the complete 3D image is compiled using software through the accumulated sequence of images in different focal planes.

VT6000 based on confocal microscopy

Laser confocal microscopy shows clearer and finer morphological details and higher lateral resolution. This is because traditional optical microscopes are often equipped with low-sensitivity CCD cameras to acquire images, and for low-illumination light, such as fluorescence, it cannot be detected and hidden, while confocal microscope systems use a high-sensitivity photomultiplier tube, which can show high sensitivity to weak fluorescence signals, and can also eliminate background noise by narrowing the excitation range and using optical sectioning.

The laser confocal microscope is detached from the field light source of the traditional optical microscope and the local plane imaging mode, and the optical path diagram of the laser scanning confocal microscope uses the laser beam as the light source rolling, and the laser beam is reflected to the objective lens through the illumination pinhole, and is focused on the sample, and every point on the focal plane of the specimen is scanned.

Lasers can replace daylight and lights, etc. Lasers for cold light sources can increase resolution. Arguments.

The laser confocal microscope is detached from the field light source and local plane imaging mode of the traditional optical microscope, and the optical path diagram of the laser scanning confocal mirror uses the laser beam as the light source, and the laser beam is reflected to the objective lens through the illumination pinhole, and is focused on the sample, and each point on the focal plane of the specimen is scanned.

Lasers can replace daylight and lights, etc. Lasers for cold light sources can increase resolution.