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Questions and Answers
What is the primary application of silver salts in histology?
What is the maximum resolving power of a light microscope?
What type of microscopy uses a beam of electrons to produce an image?
What is the purpose of fluorescent stains in fluorescence microscopy?
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What is the typical magnification range of images produced by a light microscope?
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In fluorescence microscopy, what is the wavelength of the emitted electromagnetic wave?
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What is the purpose of dehydration in histological preparation?
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What is the typical thickness of sections cut for microscopy?
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What type of compounds do basic dyes behave like?
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What is the term for cell components that react with acidic dyes?
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What is the purpose of embedding tissue in a paraffin or plastic resin?
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What is the instrument used to slice the hardened block into extremely thin sections?
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What is the primary function of phase-contrast microscopy?
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What is the typical thickness of sections required for Transmission Electron Microscopy (TEM)?
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What is the purpose of coating a specimen with a metal in Scanning Electron Microscopy (SEM)?
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Which type of microscopy uses a beam of electrons instead of light photons?
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What is the advantage of using confocal microscopy?
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Which type of microscopy is suitable for studying unstained cells and tissue sections?
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Study Notes
Light Microscopy
- Uses ordinary beam of light for visualization
- Bright-Field Microscopy is widely used by students of histology
- Stained preparations are examined using ordinary light that passes through the specimen
- Maximal resolving power of the light microscope is approximately 0.2 μm
- Images can be magnified 1000-1500 times
Fluorescence Microscopy
- Uses a ray of certain wavelength to irradiate tissue sections, emitting an electromagnetic wave of a longer wavelength
- Fluorescent substances appear brilliant on a dark background
- Fluorescent compounds with affinity for specific cell macromolecules can be used as fluorescent stains
- Preparation of cryosections does not involve dehydration steps
Embedding and Sectioning
- Tissue is embedded in a paraffin or plastic resin
- The block is left overnight at room temperature to harden
- The resulting paraffin block is trimmed to expose the tissue for sectioning
- Sections are generally cut at 1-10 μm thickness
- Very thin sections are placed on glass slides and stained for light microscopy or on special grids for electron microscopic staining and examination
Staining
- Most cells and extracellular material are completely colorless, and require staining to be easily visible
- Dyes stain tissue components more or less selectively, with many behaving like acidic or basic compounds
- Basophilic components (with a net negative charge) react with basic dyes (usually blue), e.g. DNA, RNA, Glycosaminoglycans
- Acidophilic components (with a net positive charge) react with acidic dyes (usually red), e.g. proteins, collagen fibers, mitochondria
Confocal Microscopy
- Achieves high resolution and sharp focus by using a small point of high-intensity light, often from a laser
- Uses a plate with a pinhole aperture in front of the image detector
Phase-Contrast Microscopy
- Allows the study of unstained cells and tissue sections, which are usually transparent and colorless
- Uses a lens system that produces visible images from transparent objects
- Can be used with living, cultured cells
Polarizing Microscopy
- Allows the recognition of stained or unstained structures made of highly organized subunits
Electron Microscopy
- Uses a beam of electrons instead of light photons, with a shorter wavelength
- Transmission Electron Microscopy (TEM) permits resolution around 3 nm
- TEM allows magnifications of up to 400,000 times to be viewed in detail
- TEM normally requires very thin sections (40-90 nm)
- Scanning Electron Microscopy provides a high-resolution view of the surfaces of cells, tissues, and organs
- Specimen is first coated with a metal that reflects electrons
- Electron beam scans the specimen from end to end, producing a pseudo 3D image of the coated surface
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Description
Test your knowledge of microscopy techniques, including confocal microscopy, fluorescence microscopy, and phase-contrast microscopy. Learn how these techniques are used to study unstained cells and tissue sections.
