Imaging Living Organs - Lecture 13 PDF

imaging living organs - lecture 13 Created @December 9, 2024 8:32 PM Class its whats the inside that counts Overview The lecture discusses advancements in microscopy since the Jansen...

imaging living organs - lecture 13 Created @December 9, 2024 8:32 PM Class its whats the inside that counts Overview The lecture discusses advancements in microscopy since the Jansen microscope. Focuses on fluorescence and electron microscopy. These advanced techniques allow detailed examination of biological specimens, offering higher sensitivity and resolution. Fluorescence Microscopy Key Concepts: 1. Visible Spectrum: Light detected by the human retina ranges from violet (~400 nm) to red (~700 nm). Fluorescence microscopy isolates specific wavelengths using filters. 2. How Fluorescence Works: Excitation Wavelength: Light of a specific wavelength excites the fluorescent molecule. Emission Wavelength: The molecule emits light at a longer (lower-energy) wavelength. E.g., Exciting with blue light (~480 nm) results in green emission (~520 nm). 3. Fluorescence Microscope Configuration: imaging living organs - lecture 13 1  Pre-treated specimen with fluorescent molecules. Excitation Filter: Allows only the desired excitation wavelength to pass (e.g., blue light). Dichroic Mirror: Reflects lower wavelengths (e.g., blue light). Allows higher wavelengths (e.g., green light) to pass through. Emission Filter: Blocks unwanted wavelengths to enhance contrast. Result: Fluorescently labeled structures visible against a dark background. 4. Applications: Identifying specific proteins or structures in cells. Multi-color labeling (e.g., combining different fluorescent markers for complex imaging). Green Fluorescent Protein (GFP): Originates from the bioluminescent jellyfish Aequorea victoria. Nobel Prize in Chemistry (2008): Shimomura, Chalfie, and Tsien. GFP Variants: Cyan (CFP), Yellow (YFP), Red (RFP). Uses: Studying gene expression and protein localization. Labeling cells or structures in live organisms (e.g., zebrafish, mice, pigs). Example Experiments: Nerve Regeneration: GFP-tagged nerve cells used to study spinal cord injury. Transgenic Animals: GFP used to monitor developmental and disease processes in zebrafish, mice, etc. Electron Microscopy Key Features: imaging living organs - lecture 13 2  Uses electrons instead of light for imaging. Provides much higher resolution than light microscopy. Requires vacuum environments and extensive specimen preparation. Types of Electron Microscopy: 1. Transmission Electron Microscopy (TEM): Electrons pass through a thin specimen. Dense areas block electrons (dark spots), while less dense areas allow electrons to pass (light spots). Applications: Visualizing organelles (e.g., nucleus, mitochondria), viruses. 2. Scanning Electron Microscopy (SEM): Electrons scatter off the specimen's surface. Detector captures scattered electrons to create a 3D surface image. Applications: Detailed surface structures (e.g., blood cells, insects). Comparison of TEM and SEM: Aspect TEM SEM Image Type 2D 3D Details Internal structures Surface structures Resolution ~0.1 nm ~10 nm Microscope Comparison Table Microscope Magnification Resolution Radiation Used Limitations Type Light ~400x ~200 nm White light Limited resolution Microscope Surface details SEM ~20,000x ~10 nm Electron beam only imaging living organs - lecture 13 3  2D images, TEM ~1,000,000x ~0.1 nm Electron beam complex prep Interactive Examples: SEM images (3D): Nerve cells, insects, Velcro hooks. TEM images (2D): Mitochondria, Golgi apparatus, viruses. Key Takeaways: Fluorescence microscopy excels in labeling and dynamic studies of cells. Electron microscopy provides unparalleled resolution for ultrastructural and surface details. Advancements in microscopy enable deeper exploration of biological processes and disease mechanisms. imaging living organs - lecture 13 4