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Understanding cell biology with microscopy
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- 2. Dr. Vividha Raunekar Types ofMicroscopes: Principles, Applications, Advantages, and Disadvantages 1.Light Microscope (Optical Microscope) 1. Principle: Uses visible light and optical lenses to magnify specimens. 2. Applications: Used in biology, pathology, and microbiology to observe cells, tissues, and microorganisms. 3. Advantages: 1.Relatively inexpensive. 2.Easy to use. 3.Can observe live specimens. 4. Disadvantages: 1.Limited resolution (~200 nm). 2.Limited magnification (up to ~1500x). •Working: •A light source emits light, which passes through the condenser lens and illuminates the specimen. •The light passing through the specimen is refracted and magnified by the objective lens to form an intermediate image. •The intermediate image is further magnified by the eyepiece lens to produce the final image visible to the observer. •Light microscopes use stains to improve contrast between cellular components. •Key Components: Light source, condenser, objective lens, eyepiece lens, and stage.
- 3. Dr. Vividha Raunekar •Fluorescence Microscope •Principle:Uses high-intensity light to excite fluorescent molecules in the sample, which then emit light at a longer wavelength. •Applications: Visualizing specific proteins, organelles, or cellular structures using fluorescent tags. •Advantages: •High specificity with fluorescent labeling. •Can study live cells using live-cell dyes. •Disadvantages: •Fluorophores may photobleach over time. •Expensive equipment and reagents. 2. Working: 2.A high-intensity light source (e.g., mercury or xenon lamp, LED, or laser) excites fluorophores in the sample. 3.Fluorophores absorb light at a specific wavelength and emit light at a longer wavelength. 4.The emitted fluorescence passes through an emission filter, blocking the excitation light and allowing only the fluorescent signal to reach the detector or eyepiece. 5.Dyes or tags like GFP (green fluorescent protein) can highlight specific structures or molecules. 3. Key Components: Light source, excitation and emission filters, dichroic mirror, objective lens, and detector (e.g., camera).
- 4. Dr. Vividha Raunekar Confocal LaserScanning Microscope (CLSM) •Principle: Uses lasers and pinhole apertures to achieve optical sectioning, producing sharp, 3D images. •Applications: Studying 3D structures, biofilms, and intracellular localization of molecules. •Advantages: • High resolution and contrast. • Ability to obtain 3D reconstructions. •Disadvantages: • Expensive. • Time-consuming image acquisition. • Limited to fluorescently labeled samples. •Working: •A laser provides a focused beam of light that scans the specimen point-by-point in a raster pattern. •Light emitted from the fluorophores is collected, and a pinhole blocks out-of-focus light, improving resolution. •Multiple optical sections at different depths are captured and combined to create a 3D reconstruction. •Key Components: Laser, scanning mirrors, pinhole aperture, objective lens, photodetector, and computer for image processing.
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- 6. Dr. Vividha Raunekar Electron Microscopes(EM) •a. Transmission Electron Microscope (TEM) • Principle: Uses a beam of electrons transmitted through an ultra-thin sample to create an image. • Applications: Visualizing cellular organelles, viruses, and macromolecules. • Advantages: • Extremely high resolution (up to 0.1 nm). • Allows detailed ultrastructural studies. • Disadvantages: • Requires thin-sectioned, fixed samples. • Expensive and complex to operate. • Cannot observe live specimens. 4. Working: 4.A tungsten filament or electron gun generates an electron beam. 5.The beam is accelerated and focused onto a thin sample by electromagnetic lenses. 6.Electrons passing through the sample are scattered depending on the density and atomic composition of the sample. 7.A detector (e.g., phosphorescent screen or camera) captures the transmitted electrons, forming a highly detailed image. 5. Key Components: Electron gun, condenser lens, objective lens, projector lens, vacuum chamber, and detector.
- 7. Dr. Vividha Raunekar Myelinated cells andSchwann cell
- 8. Dr. Vividha Raunekar •Scanning ElectronMicroscope (SEM) •Principle: Uses a focused beam of electrons that scans the surface of a specimen, with secondary electrons emitted to create a 3D image. •Applications: Imaging surface details of cells, tissues, and materials. •Advantages: •Produces detailed 3D images. •High depth of field. •Disadvantages: •Cannot visualize internal structures. •Requires a vacuum environment and conductive coating for non-metallic samples. •Working: •A focused beam of electrons scans the surface of the sample. •Electrons interact with the surface atoms, producing secondary electrons, backscattered electrons, and characteristic X-rays. •Secondary electrons are collected by a detector to generate a 3D image of the sample’s surface topography. •The sample must often be coated with a thin layer of conductive material (e.g., gold). •Key Components: Electron gun, condenser lens, scanning coils, vacuum chamber, and detectors.
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- 10. Dr. Vividha Raunekar Atomic ForceMicroscope (AFM) •Principle: Measures surface topography using a sharp probe that scans the sample, detecting forces between the probe and sample surface. •Applications: Imaging molecular structures, nanoparticles, and surface roughness. •Advantages: • High-resolution imaging of surfaces. • Can operate in air, vacuum, or liquid environments. •Disadvantages: • Limited to surface imaging. • Slow scanning speed. •Working: •A sharp probe attached to a cantilever scans the surface of a sample. •The probe experiences forces (e.g., van der Waals forces, electrostatic forces) from the sample surface. •These forces cause the cantilever to deflect, and the deflection is measured using a laser beam reflected off the back of the cantilever. •Data from the deflection is used to generate a topographical map of the sample surface. •Key Components: Probe, cantilever, laser, photodetector, and piezoelectric scanner.
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- 12. Dr. Vividha Raunekar Phase ContrastMicroscope •Principle: Converts phase shifts in light passing through a transparent specimen into differences in intensity. •Applications: Observing live, unstained cells (e.g., bacteria, organelles). •Advantages: • Allows observation of live cells without staining. • Simple to use. •Disadvantages: • Limited to thin samples. • Halo artifacts may reduce image clarity. •Working: •Light passing through different parts of a transparent specimen experiences phase shifts due to variations in optical density. •A phase plate in the microscope converts these phase shifts into intensity differences, making otherwise invisible structures visible. •Ideal for observing live, unstained specimens. •Key Components: Light source, condenser with phase annulus, phase plate, objective lens.
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- 14. Dr. Vividha Raunekar Polarizing Microscope •Principle:Utilizes polarized light to enhance contrast in birefringent materials. •Applications: Studying crystals, minerals, and fibers. •Advantages: • Reveals optical properties of anisotropic materials. •Disadvantages: • Limited to specific samples. • Requires polarizing filters. •Working: •A polarizer filters light to oscillate in a single plane. •When this light interacts with birefringent materials in the sample, it is split into two rays with different velocities. •These rays interfere, creating contrast and revealing the material's optical properties. •Key Components: Light source, polarizer, analyzer, and rotating stage.
- 15. Dr. Vividha Raunekar Dark-Field Microscope •Principle:Illuminates the specimen with light scattered by the object, making it appear bright against a dark background. •Applications: Observing live, unstained specimens like spirochetes or motile cells. •Advantages: • Enhances contrast in transparent specimens. • Suitable for live cells. •Disadvantages: • Low light levels can reduce image clarity. • Requires careful alignment. •Working: •A dark-field condenser directs light at an oblique angle onto the sample. •Only scattered light from the specimen enters the objective lens, making the specimen appear bright against a dark background. •Key Components: Light source, dark-field condenser, and objective lens
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- 17. Dr. Vividha Raunekar Super-Resolution Microscopes(e.g., STED, PALM, STORM) •Principle: Use specialized techniques to surpass the diffraction limit of light microscopy. •Applications: Visualizing molecular interactions, protein localization, and nanostructures. •Advantages: • Ultra-high resolution (<50 nm). • Ideal for studying nanoscale biology. •Disadvantages: • Requires advanced techniques and expensive equipment. • Sample preparation can be complex. •Working: •Various techniques surpass the diffraction limit of light microscopy: •STED (Stimulated Emission Depletion): Uses a depletion laser to restrict fluorescence to a small region. •PALM/STORM (Photoactivated Localization Microscopy/Stochastic Optical Reconstruction Microscopy): Activates individual fluorophores stochastically and determines their precise positions to build high-resolution images. •These methods rely on advanced optics, computational analysis, and high-quality fluorophores. •Key Components: High-precision lasers, optical systems, fluorophores, and image processing software.
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