Unit 5: Techniques in cell biology

Microscopy

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye.

Principles of Light Microscopy (LM)

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.

Phase Contrast Microscopy

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.

Fluorescence Microscopy

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).

Principles of Electron Microscopy (EM)

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.

Comparison: TEM vs. SEM

Principles of Chromatographic Techniques

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.

Paper Chromatography

• Stationary Phase: A strip of cellulose paper (with water bound to it).
• Mobile Phase: A solvent that moves up the paper by capillary action.
• Principle: Separation is based on partitioning and solubility. Components that are more soluble in the mobile phase and interact less with the stationary phase travel further up the paper.
• Analysis: Results are analyzed using the Rf (retardation factor) value. Rf = (distance travelled by spot) / (distance travelled by solvent).

TLC (Thin Layer Chromatography)

This is a more advanced version of paper chromatography. It's faster and provides better separation.

• Stationary Phase: A thin layer of an adsorbent (like silica gel or alumina) coated onto a glass or plastic plate.
• Mobile Phase: A solvent, just like in paper chromatography.
• Principle: Separation is based on adsorption. Components "stick" to the stationary phase with varying strengths.

Column Chromatography

This method is used to separate and purify larger quantities of a mixture.

• Stationary Phase: The adsorbent (like silica gel) is packed into a vertical glass column.
• Mobile Phase: The sample mixture is added to the top, and a solvent (the eluent) is allowed to flow through the column (often by gravity).
• Principle: Different components travel down the column at different rates and are collected in separate "fractions" (test tubes) as they exit the bottom.

HPLC (High-Performance Liquid Chromatography)

HPLC is a highly improved form of column chromatography. It is a very common and powerful analytical technique.

• Principle: Instead of gravity, high pressure (pumps) are used to force the mobile phase through a column packed with very fine stationary phase particles.
• Advantages: This high pressure results in much faster separation times and much higher resolution (better, cleaner separation) than traditional column chromatography.

Autoradiography and its Applications

Autoradiography is a technique used to visualize the location of a radioactive substance within a sample.

Basic Principle:

1. Labeling: A sample (like a cell or tissue) is "fed" a molecule containing a radioisotope (e.g., 3H-thymidine to label DNA, or 35S-methionine to label proteins). The cell incorporates this "hot" molecule.
2. Exposure: The sample is placed in the dark against a piece of photographic film or emulsion.
3. Development: The radiation emitted by the radioisotope exposes the film, creating a black silver grain (a "picture") exactly where the radioactive molecule is located.

Applications:

• Pulse-Chase Experiments: Famously used to trace the pathway of protein synthesis (the "secretory pathway"). Cells are "pulsed" with radioactive amino acids (to label new proteins) and then "chased" with non-radioactive ones. By taking samples at different times, researchers could see the label move from the ER → Golgi → Vesicles.
• DNA Sequencing: Used in the original Sanger sequencing method to visualize the DNA fragments.
• Metabolic Studies: Tracing the path of a molecule (e.g., carbon in photosynthesis).

Centrifugation

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.

Differential Centrifugation

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:

1. Low Speed Spin: Pellets the largest components, such as whole cells, nuclei, and cytoskeleton.
2. Medium Speed Spin: The supernatant from step 1 is spun faster. This pellets medium-sized components, like mitochondria, chloroplasts, lysosomes, and peroxisomes.
3. High Speed Spin: The supernatant from step 2 is spun even faster, pelleting microsomes (fragments of ER and Golgi) and small vesicles.
4. Very High Speed (Ultracentrifugation): The supernatant from step 3 is spun at extremely high speeds, pelleting the smallest components, such as ribosomes, large viruses, and macromolecules.