Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye.
A light microscope uses visible light and a system of lenses to magnify images of small samples. Light passes through the specimen and is bent by the lenses (refraction) to create a magnified image.
Limitations: Its resolution (ability to distinguish two close points) is limited by the wavelength of light. It's generally used for viewing whole cells or tissues.
This is an advanced type of light microscopy that enhances contrast in transparent and unstained specimens (like living cells).
Principle: It converts differences in refractive index (how much light bends) into differences in brightness (contrast). Parts of the cell with different densities (like the nucleus vs. cytoplasm) will appear darker or lighter, making them visible without staining, which would kill the cell.
This technique uses fluorescence to generate an image. A sample is tagged with a fluorescent dye (fluorochrome) that absorbs light at one wavelength (e.g., blue) and emits it at a longer wavelength (e.g., green).
The microscope illuminates the sample with the excitation wavelength and uses filters to detect only the emitted wavelength. This allows for the precise visualization of specific molecules or structures (e.g., tagging a specific protein or the cytoskeleton).
Electron microscopy uses a beam of accelerated electrons instead of light. Electrons have a much shorter wavelength than light, which gives EM a much higher magnification and resolution, allowing for visualization of ultrastructure (e.g., ribosomes, membranes).
Limitation: Specimens must be viewed in a vacuum and are always dead.
| Feature | TEM (Transmission Electron Microscopy) | SEM (Scanning Electron Microscopy) |
|---|---|---|
| Electron Beam | Passes through a very thin specimen slice. | Scans the surface of the specimen. |
| Image Formed | A 2D, flat image of the internal structure. | A 3D image of the surface topography. |
| Main Use | Viewing organelles, membranes, viruses. | Viewing cell surfaces, pollen grains, insects. |
Chromatography is a powerful laboratory technique used to separate the components of a mixture.
Principle: All chromatography methods have a stationary phase (a solid or liquid) and a mobile phase (a liquid or gas). The mixture is dissolved in the mobile phase, which then flows past or through the stationary phase. Separation occurs because different components of the mixture interact differently with the stationary phase and travel at different speeds.
This is a more advanced version of paper chromatography. It's faster and provides better separation.
This method is used to separate and purify larger quantities of a mixture.
HPLC is a highly improved form of column chromatography. It is a very common and powerful analytical technique.
Autoradiography is a technique used to visualize the location of a radioactive substance within a sample.
Basic Principle:
Applications:
Centrifugation is a technique that uses centrifugal force (rapid spinning) to separate particles from a solution according to their size, shape, density, and the viscosity of the medium.
How it works: A centrifuge spins sample tubes at high speeds. This generates a force that causes denser and/or larger particles to move to the bottom of the tube faster, forming a pellet. The remaining liquid is called the supernatant.
This is a common method used to fractionate (separate) cell organelles. The cell sample (homogenate) is subjected to progressively higher speeds of centrifugation.
Typical Steps:
Order: Nuclei → Mitochondria/Chloroplasts → ER/Golgi fragments → Ribosomes