Microscopy Principles, Light and Electron Microscopes, and Fluorescence

Immersion Oil: Used with a $100\text{x}$ objective lens to displace air between the glass specimen and the lens. It shares the same refractive index as glass, preventing light refraction to maximize light collection and resolution.
Contrast: The ability to visually differentiate a specimen from its background.
Bright-Field Microscopy: Illuminates the field evenly, displaying a dark specimen against a bright background.
Dark-Field Microscopy: Uses a specialized mechanism to direct light at an angle, displaying a illuminated specimen against a dark background.
Phase-Contrast Microscopy: Amplifies subtle differences in refractive indices between the specimen and background, causing cells and dense structures to appear darker.
Differential Interference Contrast (DIC) Microscopy: Splits light into two beams and recombines them using refractive indices to create detailed, three-dimensional images of intracellular structures.

Compound Light Microscope: Uses visible light passed through a specimen and a series of magnifying glass lenses.
- Ocular Lens (Eyepiece): Magnification rating is printed on the housing (e.g., $\text{WF}10\text{x}$ for wide-field $10\text{x}$ magnification).
- Objective Lenses: Standard configurations include $4\text{x}$ , $10\text{x}$ , $40\text{x}$ , and $100\text{x}$ objectives.
- Stage Control: Coarse focus knobs adjust the vertical position of the stage.
- Cost: Lab-grade compound microscopes (such as those used at Malcolm X College) cost approximately $\$25,000$ . Specialized individual objective lenses can cost over $\$1,000$ .
Foldscope:
- A portable compound microscope constructed from cardboard and glass lenses.
- Features a focus bridge, stage navigation, and phone camera attachment.
- Production cost is between $\$1.25$ and $\$2.25$ ; complete kits sell for $\$15$ to $\$40$ .

Mechanism: Specimen molecules absorb light at one wavelength and emit light at a lower/longer wavelength.
Epifluorescence: Projects ultraviolet light directly onto the top of the specimen rather than passing through it.
Fluorophores: Green fluorescent protein (GFP), originally purified from jellyfish, is used to tag target cellular molecules.
Operational Condition: Must be operated in complete darkness to prevent environmental ambient light from bleaching the fluorescent dyes.

Magnification & Resolving Power: Magnifies objects up to $100,000\text{x}$ with a resolving power of $0.3\,\text{nm}$ (compared to $0.2\,\mu\text{m}$ for standard light microscopes).
Image Generation: Uses electromagnets to focus electron beams. Output is recorded as black-and-white electron micrographs on fluorescent screens, which can be artificially colorized.
Transmission Electron Microscope (TEM):
- Passes electrons through a specimen to reveal internal structural details.
- Specimens are prepared via thin sectioning or freeze etching.
Scanning Electron Microscope (SEM):
- Scans electron beams over the specimen surface to generate detailed three-dimensional topographical views.

Question: Is special preparation required when observing non-fluorescent specimens under a fluorescent microscope?
Response: Non-fluorescent specimens must be tagged with fluorescent dyes prior to viewing. Imaging must be conducted in pitch-black conditions to avoid accidental bleaching of the sample by natural light.
Question: What operational precautions are necessary when working with high-magnification immersion oil lenses?
Response: When using a $100\text{x}$ objective with immersion oil, stage movements must be carefully controlled to prevent breaking the slide. Immersion oil must be wiped completely off the lens before switching back to lower objectives like $4\text{x}$ to prevent oil contamination across lenses.