Light Microscopy Techniques Overview

Light Microscopy Techniques

• Basic Light Microscopy: Light passes through the specimen, magnified by the eyepiece and objective lens. Resolving power is 0.2µm, meaning objects smaller than this cannot be distinguished. Two objects closer than 0.2µm together cannot be resolved.

Fluorescence Microscopy

• Uses fluorescent compounds to stain cells. Irradiating with UV light causes certain substances to emit light at longer wavelengths (fluorescence).
• Allows for visualization of specific cellular components by selecting dyes that bind to those components.

Phase-Contrast Microscopy

• Does not require staining, instead it measures the time light takes to pass through an object.
• Enables visualization of living cells.
• A variation called differential interference microscopy provides a more 3-dimensional view.

Confocal Microscopy

• Reduces light exposure to the sample, producing sharper images.
• Uses a focused light source (e.g., laser) to illuminate specific planes of the specimen, then stitching the images together.

Polarizing Microscopy

• Uses polarized light (vibrating in a single direction).
• Macromolecules between the light source and lens are visible, while other structures appear dark.

Electron Microscopy

• General Principle: Images are generated based on the interaction of electron beams with the specimen.

Transmission Electron Microscopy (TEM)

• Resolution: Achieves a resolution around 3 nm, enabling magnification up to 40,000x for isolated macromolecules/particles; and 120,000x for thin sections.
• Process: Electrons emitted from a cathode, focused, and pass through a specimen; image forms from varying electron transmission (white, black, and gray areas).
• Contrast Enhancement: Heavy metal ions in fixatives/dehydrators improve contrast.
• Preparation: Requires very thin sections. Cryofracture and freeze-etching methods allow TEM analysis without fixation, often used for membrane structure study. A specimen is frozen in liquid nitrogen, and then fractured, to reveal internal structures.

Scanning Electron Microscopy (SEM)

• Process: Electrons do not pass through the specimen, meaning it is not viewed directly. The surface is coated with a heavy metal (e.g., gold) which reflects the electrons, these reflected electrons are then used to generate a 3D image.
• Images: Results in 3-dimensional, black and white images.