1590, Janssen, Netherlands, the first primitive microscope 1610, Galileo, Italy, compound microscope with objective lens, eyepieces and tube 1611, Kepler, basic principles of microscopy In 1625, Fabre proposed the term microscope In 1665, the British Robert. Hooke built a microscope capable of magnifying 140 times In 1674, Anthony of the Netherlands. Fan. Leeuwenhoek, microscope with 270x magnification 1684, Dutch Huygens, double lens eyepiece - Huygens eyepiece In the mid-19th century, Ernst. Abbe proposed a perfect theory of microscopy 1902, Ives, modern binoculars In the 1930s, the first electron microscope was born

Resolution: the minimum distance that can distinguish two similar points (the higher the resolution, the stronger the imaging capability of the microscope) Main indicators to measure the imaging capabilities of microscopes Calculation formula: α: half angle of the opening angle of the specimen to the objective lens, λ: wavelength of the illumination source n: The refractive index of the medium between the condenser and the objective lens (air is about 1, the lens oil of the oil immersion lens is 1.3~1.5, the cedar oil is 1.5)

Microscopic structure (microscopic structure) The resolution of ordinary optical microscopes is about 0.2μm, cell nuclei, chromosomes, chloroplasts Cell structures beyond the resolution level of optical microscopes are collectively called ultrastructure and submicroscopic structures.

Magnification: Maximum magnification = human eye resolution / light microscope resolution = ~100μ m / ~0.2μ m ~500 times Magnifications exceeding the above will not increase the resolution and are considered infinite magnifications.

specimen preparation techniques

Sample making process

Phase contrast microscope: Principle: Using the diffraction and interference effects of light, the optical path difference (phase difference) of the light waves passing through different areas of the specimen is It becomes an amplitude difference (difference between light and dark), which makes the various structures in living cells present a clearly visible light and dark contrast; special structures: annular aperture, phase plate; Purpose: Observe colorless, transparent, living cell structures

Dark field microscope: Principle: scattered light imaging; Features: High resolution; Disadvantages: Only the outline of the object can be observed, and the internal cell structure cannot be distinguished.

Fluorescence microscope—a color image with strong contrast; principle: irradiating an object with ultraviolet light to cause the fluorescent material it carries to emit fluorescence; Purpose: Qualitative, quantitative and localized research on fluorescently labeled substances in tissue cells, observation of slices, or real-time observation of molecules in living cells; Features: The light source does not directly illuminate, but excites energy, requiring two special filters

Confocal laser scanning microscope—high-definition color three-dimensional images, reaching the theoretical value of light microscope resolution; purpose: non-damaging optical sectioning of cells or tissue planes—cell CT

Super-resolution optical microscope super-resolution microscope—nanoscale; super-resolution microscopy technology; advantages: non-contact, no damage, observable internal structure; use: living cells or tissues, imaging of deep internal three-dimensional structures

Inverted microscope—in vivo observation

Polarizing microscope, in medicine, is used to identify bones, teeth, cholesterol, nerve fibers, tumor cells, striated muscles, hair, etc.; protein fibers, spindles, collagen, chromosomes, etc. in cells also have birefringence.

Differential interference contrast microscopy can display three-dimensional images of structures

electron microscope

In solid tissue: mechanical dispersion, enzymatic hydrolysis, laser capture microdissection technology

In liquid environment: centrifugation, flow cytometry, cell electrophoresis, immunomagnetic bead method

definition

Classification

condition

Features

Cells derived from malignant tumor tissue can be reproduced and passaged indefinitely in vitro, such as: HeLa cell line

Use cell cloning to further improve the uniformity of cell lines, that is, isolate single cells and proliferate them to form a cell population.

Also known as cell hybridization, it refers to the process in which two or more cells synthesize one cell.

Separation steps: ① To separate cells from tissues, mechanical separation or digestion can be used; ② To separate and purify a single type of cells, density gradient centrifugation or flow cytometry is generally used; ③ Break the cells and prepare cell homogenate. You can use homogenization method or ultrasonic disruption method; ④ Use different methods to separate and purify specific cell components or organelles according to the purpose of the experiment.

1. Homogenization method: large sample processing capacity, high efficiency, fast speed, and less damage to biological macromolecules 2. Chemical lysis method: Use chemical reagents such as surfactants (SDS, TritonX-100, etc.), chelating agents (EDTA, etc.), fat-soluble solvents (acetone, chloroform, toluene) to lyse the cell membrane. 3. Repeated freezing and thawing method: It is more effective to release intracellular products that exist around the cytoplasm and close to the cell membrane. 4. Ultrasonic crushing method

Differential centrifugation: continuously increase the centrifugation speed and time Density gradient centrifugation: Use media (cesium chloride, sucrose, polysucrose solution) to form a continuous or discontinuous density gradient in the centrifuge tube

Use chromatography to purify proteins; use electrophoresis to analyze and identify proteins

Differential centrifugal precipitation is the main method for nucleic acid separation and purification; gel electrophoresis is the main method for nucleic acid identification.

Main process