Microscopy Lecture

Microscopy is a technique used to obtain magnified images of small objects that cannot be seen with the naked eye.

Resolving Power: The resolving power of the light microscope under ideal conditions is about half the wavelength of the light being used.
- Definition: Resolving power is the minimum distance that must separate two point sources of light for them to be seen as distinct images.
- For yellow light with a wavelength of $0.4 \, ext{μm}$ , the smallest separable diameters are about $0.2 \, ext{μm}$ , equivalent to one-third the width of a typical prokaryotic cell.
Useful Magnification: The useful magnification of a microscope refers to the magnification necessary to make visible the smallest resolvable particles.
Commonly used types of light microscopes in microbiology include:
- Bright-field
- Dark-field
- Phase-contrast
- Fluorescence
- Confocal

The bright-field microscope is the most commonly used in microbiological work and consists of two series of lenses:
- Objective Lens: Primary lens that magnifies the image.
- Ocular/Eyepiece Lens: Further magnifies the image rendered by the objective lens.
Generally employs a $X100$ objective lens with a $10 \times$ ocular lens, allowing for a total magnification of up to $1,000 \times$ .
Visualization: Particles as small as $0.2 \, ext{μm}$ in diameter become visible as they are magnified to about $0.2 \, ext{mm}$ (or $200 \, ext{μm}$ ).
- Beyond this magnification, resolution of detail does not improve, and the visible area of the specimen decreases.
Samples are typically stained with dyes to enhance contrast between cells or organelles and the surrounding medium, making them more visible.

Metal Body: Composed of a base and an arm where all components are attached.
Light Source: Located in the base, may be a mirror or an electric illuminator.
Focusing Mechanisms: Includes fine and coarse adjustment knobs on the arm, allowing for precise focusing of the image by moving either the stage or the nosepiece.
Stage: Positioned midway up the arm, holds microscope slides with simple slide clips or mechanical stage clips.
- A mechanical stage enables smooth movement of the slide during observation.
Condenser: Located within or beneath the stage to focus a cone of light onto the specimen. Its height can be adjusted in advanced models.
Nosepiece: Holds 3-5 objective lenses with different magnifications, which can be rotated to either position.
- Advanced microscopes may have binocular eyepieces for both eyes.
Parfocal Design: Ideally, a microscope should be parfocal, allowing the image to remain in focus when objectives are changed.
Magnification Calculation: Total magnification is calculated by multiplying the objective magnification and eyepiece magnification.
- Example: A $45\times$ objective combined with a $10\times$ eyepiece results in a $450\times$ total magnification.

Specimen preparation can sometimes lead to loss or distortion of cellular components.
To preserve cellular integrity, microscopists may examine live cells without fixing or freezing. Only certain light microscopes with special optical systems are suitable for this.

Dark-Field Microscope: This microscope modifies the lighting system to illuminate the specimen from the sides only via a specialized condenser that blocks direct light rays.
- Effect: Creates a "dark field" with bright specimens against a dark background, useful for observing live cells and structures such as Treponema pallidum, a spirochete that causes syphilis and is smaller than $0.2 \, ext{μm}$ .
Phase Contrast Microscope: Utilizes refractive index variations to produce contrast in transparent specimens.
- Developed to enhance contrast differences without staining cells, thus allowing for visualization of live specimens.
- Works by altering the phase of light waves passing through different parts of the cell, creating an image based on interference effects.
- Differential Interference Contrast (DIC) microscopy offers enhanced image resolution through polarization methods.

Fluorescence Microscopy: Currently the most widely used contrast technique. Employs fluorescent dyes to visualize specific molecules such as proteins within cells.
- Fluorescent molecules absorb light at one wavelength and emit at a longer wavelength, glowing against a dark background when illuminated accordingly.
- Techniques such as epifluorescence light microscopy illuminate the sample from above and use barriers/filters to focus only emitted wavelengths.
- Notable Dyes: Fluorescein (excitation at 496 nm, emission at 518 nm).
- Emission filters ensure that only signals from the specific fluorochrome reach the detector.

Multiple fluorescent-probe microscopy allows the comparison of different molecules in the same cell by coupling antibodies to different fluorochromes, visualized by switching filter sets.
Recent advancements include stable inorganic fluorochromes, such as quantum dots, which have longer-lasting fluorescence and can track cellular components over extended periods.

Upright Microscopes and Inverted Microscopes: Designed for different sample preparations and ease of manipulation. Universal for observing various sample types, especially live cultures.
Dissecting Microscopes: Essential for manual inspection, counting, and selecting small specimens, particularly from agar plates or tissue samples.

Electron Microscopy (EM): Used for high-resolution imaging, revealing fine cellular structures. Includes:
- Transmission Electron Microscope (TEM): Passes electrons through a specimen, stained with electron-dense material, providing detailed imagery of internal structures.
- Scanning Electron Microscope (SEM): Scans the surface of specimens and provides depth perception and surface detail images.
Electron Gun: The source of electrons in EM, operating under high voltage to produce a focused beam. Requires a vacuum to prevent scattering by air molecules.
- The resolution limit approaches 1 nm, far exceeding light microscopy and allowing examination of structures only visible at this microscopic level.
Conclusion: Microscopy encompasses various techniques and tools that enhance our ability to visualize and analyze biological samples, each suited for different purposes based on the specimen and resolution requirements.