Biotechnology & Microscopy

Cheatsheet Content

1. Chromatography Column Chromatography Principle: Separates mixtures based on differential partitioning between a stationary phase (solid adsorbent packed in a column) and a mobile phase (liquid solvent). Process: Packing the Column: Stationary phase (e.g., silica gel, alumina) is packed into a glass column. Sample Application: Mixture is loaded onto the top of the column. Elution: Mobile phase is passed through the column, carrying components at different rates based on their affinity for stationary vs. mobile phase. Collection: Separated components are collected as fractions at the column outlet. Applications: Purification of compounds, separation of isomers, analysis of complex mixtures. Ion Exchange Chromatography (IEC) Principle: Separates molecules based on their net surface charge. Utilizes a stationary phase with charged functional groups that bind oppositely charged molecules. Types: Cation Exchangers: Negatively charged stationary phase, binds positively charged molecules (cations). Anion Exchangers: Positively charged stationary phase, binds negatively charged molecules (anions). Process: Equilibration: Column is equilibrated with a buffer to set the initial charge state. Sample Loading: Sample is applied; target molecules bind to the resin. Washing: Unbound molecules are washed away. Elution: Bound molecules are eluted by changing the buffer's pH or salt concentration, which alters the charge of the molecules or competes with their binding. Applications: Purification of proteins, amino acids, nucleic acids, and other charged biomolecules. 2. Microscopy Role of Iris Diaphragm & Condenser in Microscope Condenser: Focuses light from the illuminator onto the sample. Optimizes illumination for high-resolution imaging. Aperture: Controls the numerical aperture (NA) of the illumination, influencing resolution and contrast. Iris Diaphragm: Located within the condenser. Adjusts the amount of light passing through the specimen and controls the angle of the light rays. Small opening: Increases contrast, increases depth of field, decreases resolution. Large opening: Decreases contrast, decreases depth of field, increases resolution. Bright Field vs. Dark Field Microscopy Feature Bright Field Microscopy Dark Field Microscopy Illumination Light passes directly through the specimen. Light is directed obliquely; only scattered light enters the objective. Specimen Appearance Dark specimen against a bright background. Bright specimen against a dark background. Contrast Low contrast for unstained, transparent specimens. High contrast for unstained, living specimens. Resolution Limited by the wavelength of light. Similar to bright field, but enhanced visibility of small structures. Applications Viewing stained cells, tissue sections. Viewing living, unstained bacteria, flagella, spirochetes. TEM vs. SEM Feature Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM) Principle Electrons pass through a very thin specimen. Electrons scan the surface of a specimen. Image Type 2D internal structure (ultrastructure). 3D surface topography. Resolution Very high (nanometer level), higher than SEM. High (nanometer level), lower than TEM. Specimen Prep Ultra-thin sections, heavy metal staining, vacuum. Coating with conductive material (e.g., gold), vacuum. Magnification Up to 1,000,000x or more. Up to 100,000x or more. Information Internal structure, crystal defects, chemical composition. Surface morphology, texture, elemental composition. Applications Cell biology, materials science (internal structure). Surface analysis, forensics, entomology (surface details). Transmission Electron Microscope (TEM) - Principles and Processes Principle: A beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through. An image is formed from the electrons that are transmitted through the specimen, magnified and focused by electromagnetic lenses onto a fluorescent screen or a digital detector. Key Components: Electron Gun: Generates a beam of high-energy electrons (e.g., tungsten filament, LaB$_6$ crystal). Condenser Lenses: Focus the electron beam onto the specimen. Specimen Stage: Holds the ultra-thin specimen in a vacuum. Objective Lens: Forms the first magnified image. Intermediate & Projector Lenses: Further magnify the image and project it onto the screen. Vacuum System: Maintains high vacuum to prevent electron scattering by air molecules. Detector: Fluorescent screen (for viewing) or digital camera (for recording). Processes: Electron Generation: Electrons are emitted from the electron gun. Beam Focusing: Condenser lenses focus the electron beam onto a small, coherent spot on the specimen. Specimen Interaction: Electrons pass through the specimen. Some are scattered (due to interactions with atomic nuclei or electron shells), others pass through unscattered. Image Formation: Unscattered and forward-scattered electrons are collected by the objective lens to form an image. Scattered electrons are often blocked by an aperture, contributing to contrast. Magnification: The image is successively magnified by intermediate and projector lenses. Detection: The final magnified image is projected onto a fluorescent screen or captured by a digital camera. Specimen Preparation: Requires meticulous preparation, including fixation, dehydration, embedding in resin, and ultra-thin sectioning (50-100 nm). Often stained with heavy metals (e.g., uranyl acetate, lead citrate) to enhance contrast. 3. Protein Separation Electrophoresis for Proteins with Similar Molecular Weights Problem: Separating two proteins with similar molecular weights. Standard SDS-PAGE might not resolve them well if their sizes are too close. Solution: Techniques that separate based on properties other than just size, or provide finer resolution for size. Recommended Techniques: Isoelectric Focusing (IEF): Principle: Separates proteins based on their isoelectric point (pI), the pH at which a protein has no net charge. Process: Proteins migrate through a pH gradient in an electric field until they reach the pH where their net charge is zero (their pI), at which point they stop migrating. Advantage: Highly resolving for proteins with even very small differences in pI, regardless of molecular weight. Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE): Principle: Combines IEF (1st dimension, separation by pI) with SDS-PAGE (2nd dimension, separation by molecular weight). Process: After IEF, the gel strip is placed atop an SDS-PAGE gel, and separation by size occurs. Advantage: Extremely high resolution, allowing separation of thousands of proteins, even those with similar molecular weights but different pI values. Native-PAGE (Polyacrylamide Gel Electrophoresis): Principle: Separates proteins based on their charge, size, and shape in their native, folded state (without SDS). Advantage: Can separate proteins with similar molecular weights if they have different charges or shapes. Maintains protein activity. Limitation: Resolution might be lower than 2D-PAGE if differences are subtle. High-Resolution SDS-PAGE (e.g., using a very long gel or specialized gradient gels): While SDS-PAGE primarily separates by size, using longer gels or specific gradient gels (e.g., 10-20% gradient) can sometimes provide sufficient resolution to separate proteins with very close molecular weights, provided there is a slight difference. Conclusion: For optimal separation of proteins with similar molecular weights, Isoelectric Focusing (IEF) or, even better, Two-Dimensional PAGE (2D-PAGE) would be the most effective techniques because they leverage differences in charge (pI) in addition to or instead of just size. 4. Bioinstrumentation Importance in Research & Development, Pharmaceutical, and Biotechnology Industries Definition: Bioinstrumentation involves the application of engineering principles and techniques to design and develop devices and methods for measuring, monitoring, and manipulating biological systems. Importance: Enhanced Measurement and Monitoring: Provides precise and accurate data from biological samples (e.g., pH meters, spectrophotometers, biosensors, DNA sequencers). Enables real-time monitoring of physiological parameters (e.g., ECG, EEG, blood glucose monitors). Accelerated Research & Development: Drug Discovery: High-throughput screening (HTS) systems automate the testing of thousands of compounds, significantly speeding up the identification of potential drug candidates. Genomics & Proteomics: DNA sequencers, PCR machines, and mass spectrometers are crucial for understanding genetic information and protein functions, driving basic research and target identification. Cell Culture: Bioreactors and cell culture monitoring systems optimize conditions for cell growth and product yield. Quality Control and Process Optimization in Pharmaceutical Production: Ensures consistency and purity of pharmaceutical products (e.g., chromatography systems, spectroscopy for impurity detection). Monitors fermentation processes in real-time to maximize yield and quality of biopharmaceuticals. Advancements in Diagnostics: Development of rapid diagnostic tests (RDTs) and point-of-care devices for quick and accurate disease detection (e.g., lateral flow assays, portable immunoassay readers). Automated clinical analyzers reduce human error and increase throughput in medical laboratories. Therapeutic Applications: Design of medical devices for treatment (e.g., pacemakers, insulin pumps, dialysis machines). Enables precision medicine by providing tools for personalized diagnostics and therapy. Biotechnology Innovation: Essential for genetic engineering (e.g., gene editing tools, electrophoresis for DNA separation). Facilitates the production of biofuels, enzymes, and other biotechnological products. Reproducibility and Standardization: Automated systems reduce variability and improve the reproducibility of experiments and manufacturing processes. Helps in adhering to regulatory standards and ensuring product safety and efficacy. Conclusion: Bioinstrumentation is the backbone of modern biological and medical sciences, indispensable for innovation, efficiency, and quality across research, pharmaceutical, and biotechnology sectors.