BIOL 2020 - Lecture 2: Microscopy

Light microscope key parts

Eyepiece (ocular lens), objective lens, specimen stage, condenser, and light source — light passes through the specimen and is focused by lenses to magnify the image.

Light microscopy resolution

About 250 nm — limited by the wavelength of visible light; can image live cells and use fluorescent tags to highlight specific structures.

Fluorescence microscopy

Uses fluorescent molecules (dyes or GFP-tagged proteins) that absorb light at one wavelength and emit at another, allowing specific structures to be visualized against a dark background.

Electron microscopy resolution

About 0.1 nm — far higher resolution than light microscopy because electron wavelengths are much shorter, but requires fixed (dead), non-living samples in a vacuum.

Transmission electron microscopy (TEM)

Beams electrons through a thin, fixed sample to reveal internal ultrastructure (e.g., organelle membranes) at very high resolution.

Scanning electron microscopy (SEM)

Bounces electrons off the surface of a fixed sample to produce detailed 3D-looking images of surface structure.

Why cells are small

Small size keeps a high surface-area-to-volume ratio, which is needed for efficient diffusion of nutrients, gases, and waste across the plasma membrane; as a cell grows, volume increases faster than surface area, limiting diffusion efficiency.

Surface-area-to-volume ratio

Surface area scales with the square of linear dimension while volume scales with the cube, so larger cells have proportionally less membrane surface per unit of volume, constraining cell size.

Diffusion and cell size

Diffusion is only efficient over short distances, so cells must stay small (or become highly folded/compartmentalized) to allow molecules to reach all parts of the cell quickly enough.

Staining/dyes in microscopy

Chemical stains bind specific cell components (e.g., DNA, proteins) to increase contrast and make structures visible under light microscopy.

Live-cell vs fixed-cell imaging

Light microscopy can observe living cells over time (dynamic processes); electron microscopy requires fixation and vacuum conditions, so only dead/preserved samples can be imaged.

Magnification vs resolution

Magnification is how much larger an image appears; resolution is the ability to distinguish two close objects as separate — higher resolution (smaller number) reveals finer detail regardless of magnification.