Light Microscopy Techniques

Light Microscopy Techniques

• Basic Light Microscopy: Light passes through a specimen, magnified by the eyepiece and objective lenses. Resolving power is limited to 0.2 µm, meaning objects smaller than this are indistinguishable.

Fluorescence Microscopy

• Fluorescence Principle: Certain substances emit longer wavelengths of light (fluorescence) when irradiated with light of a specific wavelength.
• Visualization Enhancement: Dyeing cells with fluorescent compounds and illuminating them with UV light improves visualization compared to basic light microscopy.
• Targeted Imaging: Specific dyes can bind to particular cellular components, allowing targeted visualization of those parts.

Phase-Contrast Microscopy

• No Staining Required: This method visualizes differences in the time light takes to pass through a specimen to visualize it, without staining.
• Live Cell Imaging: Allows observation of living cells.
• 3-D Enhancement: A variant, differential interference microscopy, produces a more three-dimensional image.

Confocal Microscopy

• Reduced Light Exposure: Reduces light exposure to the sample for clearer images.
• Laser Focusing: Uses a focused laser beam to illuminate the sample.
• Computer Stitching: Images from different focal planes are stitched together by a computer to create a composite image.

Polarizing Microscopy

• Polarized Light: Uses light that vibrates in a single direction.
• Macromolecule Visibility: Macromolecules located between the light source and lens are visible, while other structures appear dark.

Electron Microscopy

Transmission Electron Microscopy (TEM)

• High Resolution: Offers a resolution around 3 nm and magnification up to 40,000 times for isolated materials, and up to 120,000 for thin sections.
• Electron Beam Focusing: Electrons emitted from a cathode are focused through a hole.
• Image Formation: Image shows areas as white, black, or gray based on electron penetration.
• Contrast Enhancement: Heavy metal compounds can be added to enhance contrast.
• Sample Preparation: Requires very thin sections.
• Specialized Techniques: Cryofracture and freeze etching allow TEM study without fixation, providing details on membrane structure.

Scanning Electron Microscopy (SEM)

• Surface Imaging: Electrons do not pass through the specimen; instead, the sample's surface is coated with a heavy metal (often gold).
• Reflected Electron Detection: Reflected electrons are detected to create a 3-D black and white image of the sample's surface.