Electron Microscopy Techniques

• Determine the truth (T) or falsehood (F) of statements about electron microscopy and related techniques.

Light Microscopy Basics

• Common histological slides are not observed using an inverted microscope
• The eyepiece is indeed an optical component of a light microscope
• Coarse focus should not be used for sharpening at the highest magnifications
• The condenser is an illuminating and optical component of a light microscope

Light Microscopy - Resolution and Usage

• The resolving power of a light microscope is limited by the wavelength of photons of light in the visible spectrum
• Inverted microscopes are suitable for observing cell cultures
• Immersion objectives are used to observe blood smears
• The resolving power of a light microscope is not 0.2 nm
• Inverted microscopes have the light source and condenser positioned above the sample, with objective lenses below the stage, allowing observation from the bottom.

Microscope Stage and Components

• A microscope stage can be moved in two parallel planes
• A fully open iris aperture decreases, not increases, the contrast of structures on a histological slide
• Inverted microscopes feature a condenser above and objectives below the slide stage
• Lenses in a light microscope are found in objectives and eyepieces, not only objectives

Magnification and Illumination

• Begin microscopy at the lowest magnification and move up
• Total magnification is calculated by multiplying the magnification of the eyepiece and objective lens
• The arm and focus knobs (fine and coarse) are mechanical parts of a microscope
• Common light microscopy uses visible light, not ultraviolet light

Observation Methods

• Brightfield microscopy is the most commonly used method for observing common histological slides
• Binocular light microscopes contain two tubes and two eyepieces
• Microscopy begins with the stage in the lowest position
• The eye views the final image produced by the objective and eyepiece, not the primary image shown by the eyepiece

Electron Microscopy - Resolution and Advantages

• The resolving power of an electron microscope is 0.05 - 0.2 nm
• Electron microscopes offer higher resolution than light microscopes, due to the significantly shorter electron wavelengths compared to visible light photons

Electron Microscopy - Types and Applications

• Scanning electron microscopes (SEM) can visualize a sample's surface, such as microvilli
• Inner structures of cilia axonemes cannot be observed with a scanning electron microscope, it requires a TEM

Electron Microscopy - Lenses and Image

• Electromagnets function as lenses in electron microscopes
• Electron microscope images are observed in shades of gray
• The maximum useful magnification of an electron microscope is far greater than one thousand times
• Images from electron microscopes are called electron micrographs

Electron Microscopy - Sample Preparation

• Accelerated electrons do not pass through ground glass lenses
• Coating with a precious metal like gold is desirable for conductivity when observing samples under high vacuum in a scanning electron microscope
• The resolving power of a scanning electron microscope is indeed approximately 1-10 nm
• Slides need special preparation for electron microscopy, not similar staining to light microscopy

Electron Microscopy - Components and Inventors

• In electron microscopes, electrons are emitted from the cathode and accelerated by high voltage applied to the anode
• Transmission electron microscopes (TEM) observe the ultrastructure of cells
• Recorded images from electron microscopes are called electron micrographs
• Electron microscopy was invented by Ernst Ruska and Max Knoll, not Antonie van Leeuwenhoek

Electron Microscopy - Sections and Principles

• Ultrathin sections are required for transmission electron microscopy
• Objectives and eyepieces in light microscopes are optical lenses but in electron microscopes use electromagnets
• Transmission electron microscopy relies on the transmission of electrons through the sample, not reflection
• Chromatin structure is typically observed with transmission electron microscopy (TEM) rather than scanning (SEM)

Special Microscopic Techniques - Dark-field and Polarization

• Dark-field observation is not a routine/common technique for tissue microscopy
• Light rays that do not interact with the specimen are excluded from image formation in dark-field observation
• Polarization microscopy uses the ability of some tissues to alter the plane of polarization of light
• Polarizing microscopy facilitates studying the orientation of hydroxyapatite crystals in bone matrix

Special Microscopic Techniques - Phase Contrast

• Slides stained with hematoxylin and eosin are for brightfield microscopy, not polarizing microscopy
• Phase contrast microscopy enables visualization of living cells in cell culture
• Phase contrast microscopy does not require prior staining of the sample
• Phase contrast allows observation of living cells, not fixed slides impregnated with heavy metal salts

Special Microscopic Techniques - Fluorescence

• Fluorescence microscopy uses fluorophores, not the sample's ability to rotate polarized light
• Fluorochromes are excited by shorter wavelengths and emit light at longer wavelengths
• Hemoglobin does not exhibit intrinsic fluorescence
• Fluorescence microscopes are a type of light microscope

Special Microscopic Techniques - Confocal

• Confocal microscopy allows the reconstruction of three-dimensional images
• Confocal microscopes are a type of light microscope
• Confocal microscopy uses confocal apertures (pinholes) to focus on a specific plane within the sample
• Confocal microscopy is often used in conjunction with fluorescence microscopy

Special Microscopic Techniques - Dark-field and Phase Contrast

• Observation in dark field does not exploit natural fluorescence
• Light polarization is not the phenomenon of excitation and emission of light at different wavelengths
• Phase contrast is useful for observing vital spermatozoa
• Structures below the resolution of a light microscope cannot be seen in a dark field

Histologic Techniques - Fixation

• Removing tissue from a living donor is called a biopsy
• Tissue fixation hardens tissue to facilitate slicing, but it's not its primary function
• The most commonly used chemical fixative is 10% formalin (formol)
• Basophilic structures are chemically acidic and stain with basic dyes, such as DNA staining with hematoxylin

Histologic Techniques - Eosinophilia and Frozen Sections

• Eosinophilia indicates an affinity for acidic dyes, not alkaline dyes
• Frozen section methods are faster than paraffin section methods
• Paraffin section methods produce better image quality and permanent slides, unlike frozen sections
• Paraffin techniques are not routinely used in fast perioperative biopsy diagnostics

Histologic Techniques - Paraffin Section

• The paraffin section technique uses a chemical method of fixation
• A cryomicrotome is used to cut frozen blocks, not paraffin blocks
• Staining of paraffin sections is not possible immediately after cutting; the paraffin must be removed first
• Dehydration of the sample involves a series of alcohols in increasing concentration

Histologic Techniques - Silver Staining

• Argyrophilia and argentaphilia are related phenomena involving staining with silver salts
• Impregnation reveals structures by reducing metal salts on their surface
• Hematoxylin and eosin are not used for impregnation with metal salts
• Hematoxylin stains nuclei blue, not collagen fibers purple

Histologic Techniques - Staining and Sectioning

• Eosin stains the cytoplasm pink, not the nucleus
• Paraffin sections are cut with a microtome to a standard thickness of 2-7 micrometers
• Paraffin is used to embed specimens for solid slicing
• Xylene is used to clear the samples, making them transparent

Histochemistry - Definition and Examples

• Histochemistry involves staining of chemical substances in cells, not immunocomplexes
• Histochemistry is the staining of chemical substances, such as enzymes, in cells
• Glycogen can be histochemically proven using the PAS method
• Alkaline phosphatase can be demonstrated in epithelial cells, e.g., in the kidney, using histochemistry

Histochemistry - Details of Various Methods

• The PAS method combines Schiff's reagent and periodic acid
• Hematoxylin stains DNA, but is not a histochemical visualization technique
• Detection of alkaline phosphatase is classified as an enzyme histochemical method
• The PAS reaction is used to demonstrate carbohydrates, not substances of a fatty nature

Immunohistochemistry - Antibodies

• Immunohistochemistry demonstrates specific epitopes of antigens
• Diaminobenzidine (DAB) is a commonly used chromogen in immunohistochemistry
• Monoclonal antibodies are directed against single epitopes, not multiple epitopes
• Immunization produces both monoclonal and polyclonal, not exclusively monoclonal, antibodies