Overview
This lecture introduces microscopy, focusing on types of light microscopes (bright field, compound, and stereo), principles of magnification and resolution, sample preparation, and key applications.
Introduction to Microscopy
- A microscope is an instrument that magnifies objects too small for the naked eye.
- Microscopes use lenses and light to reveal fine details of small specimens.
- Time-lapse imaging captures images at intervals, allowing observation of dynamic biological processes.
Magnification vs. Resolution
- Magnification enlarges the specimen's image, but resolution measures the ability to distinguish two close points.
- Higher resolution is achieved with shorter wavelengths of light, as described by the Rayleigh Criterion.
- Typical resolution limits: 300 nm (dry lens), 200 nm (immersion lens).
Types of Light Microscopy
- Light microscopy includes bright field, dark field, fluorescence, and more advanced electron microscopes.
- Bright field microscopy uses transmission of white light to produce contrast.
- Bright field microscopes: simple (single lens), compound (multiple lenses), stereo (3D imaging).
Bright Field Microscopy: Principles and Parts
- Uses a light source, condenser, specimen stage, objective lens, and eyepiece.
- Contrast arises from differences in absorption or scattering within the specimen.
- Compound microscopes have multiple objective lenses (e.g., 4x, 10x, 40x, 100x) and adjustable diaphragms for optimal illumination.
- Proper specimen illumination (Kohler illumination) is crucial for clear images.
Sample Preparation and Staining
- Sample prep involves fixation (preserving structure), permeabilization, and mounting on slides with a cover slip.
- Microtomes and cryostats cut thin specimen sections for viewing.
- Staining enhances contrast; common stains include hematoxylin & eosin, Giemsa, and crystal violet.
Objective Lenses and Numerical Aperture
- Numerical aperture (NA) determines light-gathering/resolving power; higher NA gives better resolution.
- NA depends on refractive index of imaging medium and angle of accepted light.
- Shorter wavelength light and higher NA improve microscope resolution.
Compound vs. Stereo Microscopes
- Compound microscopes: high magnification, observe thin samples, use transmitted light.
- Stereo microscopes: low magnification, 3D view, suitable for large specimens, do not require fixation/staining.
Applications
- Bright field: cell/tissue observation, microorganism identification, blood analysis, education, quality control, environmental studies, agricultural and plant sciences.
- Stereo microscopes: dissection, entomology, botany, paleontology, industrial quality control, gemology.
Key Terms & Definitions
- Microscope — Instrument for magnifying small specimens.
- Magnification — Enlargement of an image.
- Resolution — Ability to distinguish two close points as separate.
- Bright Field Microscopy — Standard light microscopy method using transmitted white light.
- Numerical Aperture (NA) — Measure of objective lens's light-gathering ability.
- Fixation — Preserving tissue structure before microscopy.
- Microtome — Device for slicing thin sections of specimens.
- Kohler Illumination — Technique for even, optimal specimen lighting.
Action Items / Next Steps
- Review diagrams of compound/stereo microscopes and their light paths.
- Prepare specimen slides using fixation and staining protocols.
- Read about dark field microscopy for the next session.