Techniques in Cell and Molecular Biology

Chapter 18: Techniques in Cell and Molecular Biology

Microscopy

Looking at Cells

  • Light Microscope Components:

    • Ocular lens

    • Light beam

    • Objective lens

    • Specimen

    • Condenser lens

    • Light source

  • Light Microscope Description:

    • Utilizes glass lenses and visible light to form an image.

    • Resolution of approximately 0.2 ext{ } ext{μm}; 1000 times greater than the human eye.

    • Visualizes cell sizes, shapes, and some internal cell structures.

    • Internal structures are difficult to see under visible light; involves chemical treatment and staining with various dyes to enhance contrast.

    • Reference: LIFE: THE SCIENCE OF BIOLOGY 11e, Figure 5.3 (Part 1)

Specialized Light Microscopes

  • Techniques Developed to Observe Living Cells:

    • Phase-Contrast Microscopy:

    • Increases contrast by emphasizing differences in refractive index, enhancing light and dark regions.

    • Differential Interference Contrast (DIC) Microscopy:

    • Uses two beams of polarized light to create images that appear as if the cell casts a shadow.

    • Fluorescence Microscopy:

    • A method to visualize structures using fluorochromes that emit light when excited by UV rays.

    • Confocal Microscopy:

    • Produces sharp images of thin sections, utilizes laser and pinhole to eliminate out-of-focus light.

Bright-Field Microscopy

  • Characteristics:

    • Light passes through specimen; if no natural pigments exist, it presents low contrast.

    • Image detail may be enhanced by staining.

Staining and Contrast Enhancement

  • Stains Variety:

    • Stains chemically differ and bind to different cell materials. They significantly improve visualization in microscopy.

Fluorescence Microscopy Techniques

  • Immunofluorescence:

    • Uses fluorochrome-conjugated antibodies to study specific cellular components.

  • Green Fluorescent Protein (GFP):

    • Isolated from jellyfish Aequorea victoria by Osamu Shimomura in the 1960s.

    • GFP can be fused with target genes in transgenic model organisms for live-cell imaging and gene expression tracking.

    • Applications include studying protein localization in living cells, following dynamic neuronal interactions.

  • GFP Variants:

    • Developed by Roger Tsien; used to create brighter and color-shifted fluorescent proteins.

  • Impact of GFP:

    • Revolutionized molecular biology by enabling live-cell imaging.

    • Awarded the Nobel Prize in Chemistry in 2008 to Shimomura, Chalfie, and Tsien.

Examples of GFP Applications

  • GloFish:

    • Transgenic zebrafish containing red fluorescent protein useful in environmental testing.

  • GFP in Research:

    • Used to observe gene expression and protein interactions under various conditions.

Fluorescent In Situ Hybridization (FISH)

  • Technique Description:

    • Allows detection of specific nucleic acid sequences within fixed cells.

  • Application:

    • Gene mapping and visualization of chromosomal regions.

Immunostaining Techniques

  • Methods to Detect Proteins:

    • Uses primary antibodies binding to specific antigens, secondary antibodies for visualization.

RNA-FISH Techniques

  • RNA Detection Methodology:

    • Involves dissection, fixation, prehybridization, and imaging subsequent to hybridization with fluorescent probes.

Advanced Fluorescence Techniques

  • Fluorescence Resonance Energy Transfer (FRET):

    • Measures distances between molecules using different fluorescent labels to detect protein-protein interactions.

Confocal vs Regular Fluorescence Microscopy Comparison

  • Regular Fluorescence Microscopy:

    • Broad sample illumination, captures both in-focus and out-of-focus light; quicker but can cause blurred images.

  • Confocal Microscopy:

    • Laser focus on thin optical sections, produces clearer images, and can construct 3D representations.

Electron Microscopy (EM)

  • Types of Electron Microscopy:

    • Transmission Electron Microscopy (TEM):

    • Uses electrons transmitted through the specimen; higher magnification up to 1,000,000×.

    • Scanning Electron Microscopy (SEM):

    • Generates detailed 3D images from electrons reflected off the specimen's surface.

  • Freeze-Fracture Replication:

    • Technique involves freezing specimens, fracturing them, and visualizing their internal structures in TEM.

Atomic Force Microscopy (AFM)

  • Description:

    • High-resolution technique for obtaining topographical images of individual molecules.

  • Applications:

    • Real-time imaging and manipulation of macromolecules, receptor/ligand interactions measurement.

Cell Culture Techniques

  • Definition:

    • The practice of growing cells under controlled conditions for biological studies.

  • Types of Culture:

    • Primary culture (cells direct from organisms), Secondary culture (derived from a primary culture), Cell lines (immortal cells).

Studying Organelles and Cell Fractionation

  • Methods for Studying Organelles:

    • First using light microscopy, then electron microscopy, staining techniques targeted to identify organelle contents.

    • Cell Fractionation:

    • Separates organelles based on size/density via centrifugation.

Protein Purification Techniques

  • Process Description:

    • Involves removing contaminants to enhance specific protein activities.

  • Methods:

    • Liquid column chromatography; separates components via affinity to a material; High-performance liquid chromatography (HPLC) for resolution.