Comprehensive Notes on Microscopy: Light and Electron Techniques

Fundamentals of Microscopy

• Microscopy is defined simply as the use of a microscope.
• It is the scientific discipline focused on using microscopes to achieve a magnified view of objects that are otherwise invisible to the naked or bare eyes.
• As a scientific tool, microscopy is essential in fields such as biological sciences, biology, nanotechnology, and biotechnology.
• It is considered obligatory in the branch of microbiology for the observation of microbial cells.

Comparison of Light and Electron Microscopy

• There are two fundamentally different types of microscopes: the light microscope and the electron microscope.
• Light Microscope (LM) Characteristics:
  • Principle: Uses visible light and glass optical lenses to magnify specimens.
  • Resolution: Approximately $200\,nm$ (limited by the wavelength of light) or a resolution limit of $0.2\,\mu m$.
  • Magnification: Capable of magnifying images up to a maximum of approximately $1000\text{--}1500\times$.
  • Sample Preparation: Relatively simple process; live cells can be observed.
  • Applications: Used for studying cell morphology, tissues, and microorganisms in biological laboratories.
• Electron Microscope (EM) Characteristics:
  • Principle: Uses a beam of accelerated electrons instead of light; images are formed by the interaction between electrons and the specimen. It uses electromagnetic lenses to focus the electron beam.
  • Resolution: Up to $0.1\,nm$ or a resolution limit of $1\,nm$. The higher resolution is due to the shorter wavelength of electrons.
  • Magnification: Capable of magnifying to a maximum of approximately $200,000\times$ or up to $2,000,000\times$.
  • Sample Preparation: Complex process involving fixation, dehydration, and coating. Only dead (non-living) specimens can be observed.
  • Applications: Used for detailed studies of cell ultrastructure, viruses, and nanomaterials.
• Comparative Estimates of Utility:
  • Resolution is a more reliable estimate of a microscope's utility than magnification. Resolution is the ability to distinguish between two closely spaced points in a specimen.
  • Standard light microscopes have a lateral resolution limit of approximately $0.5\,\text{micrometers (mm)}$ for routine analysis.
  • Light microscopes allow for the viewing of both living and dead specimens, often in real color.
  • Electron microscopes only allow the viewing of dead specimens, and the images are never in real color.

Detailed Components and Paths of Microscopy Systems

• Path of Light in a Compound Light Microscope:
  • Light source (lamp) $\rightarrow$ Condenser lens (glass) $\rightarrow$ Specimen (mounted on a glass slide with a coverslip) $\rightarrow$ Objective lens (glass) $\rightarrow$ Eyepiece lens $\rightarrow$ Detector (eye or digital camera).
• Path of Electrons in a Transmission Electron Microscope (TEM):
  • Electron gun $\rightarrow$ Condenser lens (electromagnetic) $\rightarrow$ Specimen (placed on a copper or gold electron microscope grid) $\rightarrow$ Objective lens (electromagnetic) $\rightarrow$ Projector lens (electromagnetic) $\rightarrow$ Detector (phosphorescent viewing screen or digital camera).

Applications of Microscopes

• Cell Structure Observation: Microscopes facilitate the visualization of detailed cell structures and organelles, such as the nucleus and mitochondria, to help understand cellular function.
• Microorganism Identification: They are used to observe bacteria, viruses, and fungi that are invisible to the naked eye, which is critical for microbiology and infection studies.
• Histological Studies: Microscopy allows for the examination of tissue sections (e.g., muscle, liver, brain) for understanding anatomy and diagnosing diseases.
• Cell Division and Genetics: Microscopes are used to study mitosis, meiosis, and chromosome behavior during genetic research and education.

Types of Optical Microscopes

• Optical microscopes are generally classified based on the types of lenses used.
• Simple Microscope:
  • Consists of a single glass lens mounted in a metal frame (similar to a magnifying glass).
  • The specimen requires very little preparation and is held close to the eye.
  • Focusing is achieved by moving the lens and specimen relative to one another.
  • Light source is usually the sun or ambient indoor light; the detector is the human eye; recording is done via hand drawings or anecdotes.
  • Used for basic magnification in soil study (pedology), schools, and dermatology.
  • Magnifying power is up to $300\times$.
  • Typically uses a concave mirror.
• Compound Microscope:
  • Employs more than one lens (eyepiece and multiple objective lenses) to achieve higher orders of magnification.
  • Uses an artificial light source (illuminator) and a condenser lens to focus light onto the specimen.
  • The objective lens picks up transmitted light to create a primary image inside the body tube.
  • Includes both coarse and fine adjustment screws for focusing.
  • Used for advanced analysis of microorganisms and in-depth cellular study to identify bacterial infections.
  • Magnifying power is up to $2000\times$.
  • Mirror type is concave on one side and plain on the other.

Components and Functions of a Microscope

• Base: The bottom region made of heavy metal that supports the microscope and attaches it to the working surface.
• Eyepiece Lens: Located at the top, facilitating sample visualization; typically contributes $10\times$ or $15\times$ magnification.
• Eyepiece Tube: Mediates the connection between the eyepiece and the objective lens.
• Revolving Nosepiece (Turret): A rotating part containing two or more objective lenses of different magnifying powers.
• Objective Lenses: Typically set at $4\times$, $10\times$, or $40\times$ magnification. When a $10\times$ eyepiece lens is coordinated with a $10\times$ objective lens, a total magnification of $1000\times$ is achieved.
• Arm: Facilitates the holding of the microscope and connects the tube to the base.
• Specimen Stage: The area where slides are placed, held by stage clips to avoid slipping.
• Stage Holder Clamp: Holds the slide in place on the stage.
• Safety Rack Stop: Prevents contact between high-power objective lenses and the slide to avoid cranking or breaking.
• Diaphragm or Iris: A rotating disc below the stage with different-sized holes to adjust the intensity of light reaching the slide.
• Focus Knobs:
  • Coarse Focus Knob: Moves the stage quickly up and down for focusing with low-power objectives.
  • Fine Focus Knob: Used for minute, short-distance movements of the body tube to bring the image into sharp clarity.
• Illumination Mirror: Reflects light toward the specimen.

Contrast Techniques in Light Microscopy

• Most cells and tissues are colorless, transparent, and lack contrast.
• Contrast can be introduced through optical means or by staining with dyes.
• Optical Contrast: Achieved by introducing elements (filters, colored glass, neutral density filters) into the light path or by adjusting light intensity and the condenser aperture.
• Brightfield Microscopy:
  • The most basic mode with a bright background.
  • Contrast is produced by the specimen's own color.
  • Used for pigmented tissues, histological sections, or cells stained with colorful dyes.
• Darkfield Microscopy:
  • Produces brightly illuminated objects against a black background.
  • A darkfield stop is placed in the condenser to create a hollow cone of light.
  • Traditionally used for viewing outlines of objects in liquid media, such as living spermatozoa, cells in tissue culture, or microorganisms.
  • Requires a specialized darkfield condenser and objective lens for high magnification.
• Phase Contrast Microscopy:
  • Used for viewing unstained living cells and cell organelle preparations for lysis.
  • Images differences in the refractive index; light passing through thicker parts of the cell is delayed relative to light passing through thinner cytoplasm.
  • Requires specialized phase condenser settings (Phase 1, 2, and 3) matched with соответствующий phase objective lenses.

Fluorescence Microscopy

• Current widely used contrast technique providing superior signal-to-noise ratios (typically white on black).
• Epifluorescence Light Microscopy: "Epi" means "from above"; the light source is above the sample, and the objective lens acts as both the condenser and objective.
• Light Sources: High-pressure mercury or xenon vapor lamps, lasers, and LED sources emitting from UV to red wavelengths.
• Mechanism:
  • A specific wavelength excites a fluorophore in the specimen.
  • The fluorophore emits light of a longer wavelength, which is then imaged.
  • Three Main Filters: Excitation filter (selects desired excitation wavelength), Dichromatic mirror (reflects excitation light toward sample, transmits longer wavelengths toward detector), and Barrier/Emission filter (blocks excitation light, only transmits signal from the fluorophore of interest).
• Example (Fluorescein): Excitation occurs at $488\,nm$; maximum emission occurs at $518\,nm$. An exciter filter allows $488\,nm$ light through, and the emission filter only allows $518\,nm$ light to reach the detector.
• Detection: Fluorescence is often too low for the human eye or outside the visible range; sensitive digital cameras like CCD (Charge-Coupled Device) or PMT (Photomultiplier Tube) are used.

Specimen Stains and Immunofluorescence

• Non-specific Stains: General protein stains like Coomassie blue or stains for specific organelles like the nucleus or mitochondria.
• Immunofluorescence Microscopy:
  • Maps the spatial distribution of macromolecules (proteins) using the specific binding of antibodies.
  • Indirect Immunofluorescence: A primary antibody binds to the protein of interest; then, a secondary antibody carrying a fluorescent tag binds to the primary antibody.

Electron Microscopy: TEM and SEM

• Electron Gun Principles:
  • Acts as the light source; a tungsten filament (or materials like lanthanum hexaboride) emits electrons when an accelerating voltage ($40,000\text{--}100,000\,V$) is applied between the cathode and anode.
  • Electromagnetic lenses focus the beam by changing the voltage across them.
• Transmission Electron Microscope (TEM):
  • Images electrons that pass through the specimen.
  • Specimens must be very thin ($50\text{--}100\,nm$) to allow electron penetration.
  • Requires a high vacuum to prevent electrons from being scattered by air.
  • The objective aperture cuts off large-angle scattered electrons to create amplitude contrast.
• Scanning Electron Microscope (SEM):
  • Images the surface of thick specimens by collecting reflected secondary electrons or back-scattered electrons.
  • The beam scans the specimen in a raster pattern (like a television tube).
  • Intensity measurement: high scattering at a point results in a bright spot on the screen; low scattering results in a dark spot.
• Advanced Voltage Modalities:
  • High-voltage electron microscope (HVEM): Uses $1,000,000\,V$ for thicker specimens.
  • Intermediate voltage electron microscope (IVEM): Uses approximately $400,000\,V$.

Specimen Preparation for Electron Microscopy

• Contrast in EM depends on the atomic number; higher atomic numbers lead to greater scattering.
• Heavy metals (Uranium, Lead, Osmium) are used as stains to add contrast; labeled structures appear "electron dense" (black).
• All water must be removed from biological specimens because the electron beam requires a vacuum.
• TEM Preparation Steps:
  1. Fixation in glutaraldehyde (cross-links proteins).
  2. Post-fixation/staining in osmium tetroxide (fixes lipid membranes).
  3. Dehydration through a series of alcohols.
  4. Embedding in plastic (e.g., EPON).
  5. Sectioning on an ultramicrotome with glass or diamond knives into ultrathin sections (approx. $60\,nm$).
  6. Assessment of thickness using interference colors on the water surface (silver/gold color indicates $60\,nm$).
  7. Mounting on copper or gold EM grids.
  8. Staining with heavy metals like uranyl acetate and lead citrate.
• SEM Preparation Steps:
  1. Fixation in glutaraldehyde.
  2. Dehydration through solvents.
  3. Critical point drying: Removes water instantly to avoid surface tension and drying artifacts.
  4. Mounting on a metal holder or stub.
  5. Coating with a thin layer of gold.