Microscope Types and Parts

Types of Microscopes

• Light microscopes utilize visible light and optical lenses to magnify tissues, ideal for routine histology.
• Electron microscopes employ electron beams, providing higher resolution for observing cell membranes and organelles.
• Staining and microscopy are essential for detailed tissue analysis, helping identify structural abnormalities, diagnose diseases, and conduct medical research.

Light Microscope (Bright-Field)

• The condenser narrows the light beam, reducing scattering.
• The diaphragm controls and regulates the electricity.
• Eyepiece is that lens through which the user looks.
• Interpupillary Adjustment adjusts the distance between the two eyepieces.
• Binocular Tubes hold eyepieces and allow for alignment.
• Head connects eyepieces to the microscope.
• Beamsplitter splits the light for different optical paths.
• Measuring Graticule is a scale inside the eyepiece for measurements.
• Revolving Nosepiece holds multiple objective lenses for rotation.
• Objective Lenses magnify specimens at different levels.
• Specimen Holder secures the slide.
• Mechanical Stage moves the slide.
• Condenser focuses light onto the specimen.
• Field Lens & Diaphragm controls and directs light.
• Collector Lens focuses light from the lamp.
• Tungsten Halogen Lamp is the light source.
• Base provides the foundation.
• Stand supports the structure.
• On/Off Switch & Illumination Intensity Control adjusts brightness.
• X-Y Translation Mechanism allows precise stage movement.
• Bright-field microscopy is the most common and cheapest light microscopy.
• Stained tissue is examined with ordinary light passing through the preparation.
• The microscope includes an optical system and mechanisms to move and focus the specimen.
• A condenser collects and focuses light, illuminating the tissue slide.
• Objective lenses enlarge and project the image toward the eyepiece.
• Eyepieces (oculars) magnify the image another 10X, yielding total magnifications of 40X, 100X, or 400X.

Fluorescence Microscopy

• Fluorescence occurs when cellular substances are irradiated by light of a proper wavelength, emitting light with a longer wavelength.
• In fluorescence microscopy, tissue sections are irradiated with ultraviolet (UV) light.
• The emission is in the visible portion of the spectrum.
• Fluorescent substances appear bright on a dark background.
• The instrument has a UV or other light source and filters to select wavelengths emitted by the substances.
• The primary antibody has limited fluorophore compared to the secondary antibody.
• The secondary antibody loads more fluorophore from the protein, amplifying the signal for easier visualization.
• Cultured cells are grown in dishes (incubator), and viewed to visualize immunostained sections.
• Filamentous proteins concentrate under the plasma lamina.
• Cell’s life in cultures depends on their social interactions and communication.

Phase-Contrast Microscopy

• For studying unstained cells and tissue sections (colorless; similar optical densities).
• It has a lens system that produces visible images from transparent objects.
• It is used with living, cultured cells.
• Light changes speed when passing through cellular and extracellular structures with different refractive indices, causing them to appear lighter or darker in relation to each other.

Electron Microscope

• Interaction of tissue with a beam of electrons - reflected, absorbed, or passed through.
• TEM involves the electron beam passing through the tissue.
• Very high magnification achieved.
• Very thin sections, 40-90 nm, are needed.
• The electron beam interacts with tissue, producing black, white, and shades of gray images.
• SEM involves the electron beam not passing through the tissue.
• The surface of cells and tissue is coated with heavy metals (gold) which reflect the electrons.
• SEM then produces 3D images that record the specimen topography.

Resolution

• Resolution defines how finely detailed the images are.
• Resolving power is the smallest distance between two structures at which they can be seen as separate objects.
• The maximal resolving power of a light microscope is approximately 0.2 µm, which allows one to see clear images magnified 1000-1500 times.
• Objects smaller than 0.2 µm (like single ribosomes or cytoplasmic microfilaments) cannot be distinguished.
• A microscope's resolving power determines the quality of the image, clarity, and richness of detail.
• Resolving power depends mainly on the quality of its objective lens.
• Magnification is of value only when accompanied by high resolution.
• Resolving of TEM is 3 nm because the electron wavelength is shorter than that of light.