Microscopy Techniques

Microscopy Techniques

• Bright Field Microscopy is the oldest, most common technique, used in pathology to examine fixed, stained tissue samples after fixation, dehydration, infiltration, sectioning, and staining.
• Paraffin sections are fixed, dehydrated, wax-embedded samples for permanent use, while cryosections are rapidly frozen and sliced, often for quick cancer diagnosis like in Mohs surgery.
• Phase Contrast Microscopy enhances contrast in live cell imaging without staining, useful for cell culture biologists observing cell structures.
• Differential Interference Contrast (DIC) Microscopy creates 3D images of live cells, beneficial in electrophysiology for monitoring neuron activity.
• Dark Field Microscopy features high contrast with a dark background and bright specimens, used to visualize microorganisms.
• Polarizing Light Microscopy uses polarized light to detect highly ordered structures like microtubules and muscle fibers.
• Confocal Microscopy utilizes lasers and pinholes to eliminate out-of-focus light for sharp 3D images; point scanning offers high resolution but is slow, while spinning disk is faster and better for live cells.
• Fluorescence Microscopy employs fluorescent dyes or proteins to highlight specific molecules.
• Vital Fluorescent Dyes track cell activity, indicating calcium levels and mitochondrial health.
• FRAP (Fluorescence Recovery After Photobleaching) tracks membrane fluidity by observing recovery after bleaching a section of the cell membrane.
• FRET (Förster Resonance Energy Transfer) measures protein-protein interactions based on energy transfer between fluorescent molecules.
• FRET Biosensor is a modified FRET technique used to detect cellular changes in real-time.
• TIRF (Total Internal Reflection Fluorescence Microscopy) focuses on molecules near the coverslip, suitable for imaging cell membranes.
• TEM (Transmission Electron Microscopy) offers high resolution for visualizing internal cell structures, while HVEM (High Voltage Electron Microscopy) is used for thicker biological samples.
• SEM (Scanning Electron Microscopy) produces 3D surface images.
• Electron Tomography creates 3D reconstructions from multiple TEM images.
• AFM (Atomic Force Microscopy) measures forces between molecules for surface analysis.
• STM (Scanning Tunneling Microscopy) examines atoms on surfaces with ultra-high resolution.
• Laser-Capture Microdissection Microscopy uses a laser to isolate specific cells from tissue samples.
• Super-Resolution Microscopy breaks the diffraction limit of light microscopy for nanometer-level imaging.
• Deconvolution Microscopy uses computational methods to sharpen blurry images from fluorescence microscopy.

Cell & Molecular Techniques

• ELISA (Enzyme-Linked Immunosorbent Assay) quantifies proteins using antibodies and color change detection.
• Western Blot detects specific proteins using gel electrophoresis and antibodies.
• Flow Cytometry / FACS (Fluorescence-Activated Cell Sorting) identifies and sorts cells based on fluorescence markers.
• Immunocytochemistry (ICC) employs fluorescent antibodies to locate proteins in cells.
• Microspectrofluorometry & Plate Reading Spectrofluorometer measures fluorescence in samples, which is useful for quantifying cell activity.
• Autoradiography utilizes radioactive labels to track cellular processes such as DNA replication.
• FISH (Fluorescence In Situ Hybridization) detects specific DNA or RNA sequences in cells using fluorescent probes.
• Patch Clamp measures ion channel activity through intracellular injection techniques.
• Microinjection introduces substances directly into cells.
• Electroporation introduces DNA using electric pulses.
• Liposomes/Viral Transfection delivers genetic material using lipids or viruses.

Cell Separation & Purification

• Differential Centrifugation separates cell parts by size and density.
• Equilibrium Density (Rate Zonal) Centrifugation separates molecules according to their density.
• Ion Exchange chromatography separates proteins by charge.
• Gel Filtration chromatography separates proteins by size.
• Affinity Chromatography uses specific binding interactions, such as insulin receptors binding to insulin-coated beads.
• Native Gel Electrophoresis preserves protein structure.
• SDS-PAGE denatures proteins for improved separation.
• Isoelectric Focusing separates proteins by pH.
• 2D Gel Electrophoresis combines Isoelectric focusing with SDS-PAGE.

Biotechnology & Tissue Engineering

• GFP, YFP, CFP (Fluorescent Proteins) are used as reporter molecules to track gene expression.
• Scaffolds are biodegradable structures that support cell growth in tissue engineering and regenerative medicine.
• Organoids are miniature, organ-like structures grown in vitro.
• Bioprinting / 3D Printing uses cells and biomaterials to construct tissues.
• Decellularization removes cells from tissues/organs, leaving only the extracellular matrix for transplantation.