Lecture 3 Microscopy Xray 2023.pdf

Research Techniques II: Microscopy and Cell Imaging Seeing at the cellular and subcellular level (continued...) 1 Electron Microscopy • • • • • Technique with a long history. We will discuss two major types: 1) Transmission electron microscope (TEM). 2) Scanning electron microscope (SEM). Use...

Research Techniques II: Microscopy and Cell Imaging Seeing at the cellular and subcellular level (continued...) 1 Electron Microscopy • • • • • Technique with a long history. We will discuss two major types: 1) Transmission electron microscope (TEM). 2) Scanning electron microscope (SEM). Uses bombardment of electrons rather than light. Advantage: Resolution 2,000´ that of LM. 0.1 nm = 1.0 Å (Ångströms). Lower resolution in some biological preparations (2 nm). Disadvantage: Very time-consuming preparation. But for some experiments, this is the only appropriate technique. 2 Transmission EM • General configuration analogous to LM. • Electrons emitted at filament or cathode. • Accelerated by high voltage (105 V) in a vacuum. • Magnetic coils focus electron beam like a lens. • Sample may be stained, producing “electron dense” images. 3 Preparation for TEM • Lengthy procedure that takes days to weeks. • Tissue must be fixed in glutaraldehyde. • Addition of OsO4 increases electron density. • Dehydration and infiltration with a plastic resin gives extra support. • Ultrathin sections (50 – 100 nm) must be cut with a diamond knife. • Sections cannot be handled directly. • Placed on copper grids. 4 TEM Micrograph A mitochondrion A mitochondrion in serial sections 5 Immunogold EM 6 Scanning Electron Microscope • • • • • Produces 3D images of surface structures. Used to study whole cells and tissues, rather than intracellular structures. Principles of preparation and operation similar to TEM Cells/tissues coated with heavy metal. Scattered electrons from the specimen surface are collected. 7 SEM Micrographs Insect head 2.0 mm T4 Bacteriophage 0.1 µm Figure 18.19. Karp. 2008 Cell and Molecular Biology. 8 EM and LM Compared SEM DIC TEM Stereocilia of hair cell from frog inner ear. 9 Ion Imaging • Changes in intracellular ion concentrations (e.g. Ca2+ and H+) are physiologically important. • Ion-selective indicators emit light depending on local ion concentrations. • These reveal rapid intracellular dynamics. 10 Ca2+ Imaging • Intracellular Ca2+ is low. • Bioluminescent aequorin injected into a fish egg reveals Ca2+ wave propagated during fertilization. • Other synthesized indicator molecules produce signal (e.g. Fura-2, Fluo-4) Ca2+ wave fert. 11 Ca2+ Imaging • Wavelengths correlated with ionic binding and Ca2+ concentration. • Dyes can be injected, or “AM” analogues can be used to cross cell membrane. = acetoxymethyl ester Ca2+ sig glia 12 X-ray Crystallography ‘Seeing’ at the molecular level 13 X-ray Crystallography • • • • • • • Structure - function Macromolecules Atomic resolution X-rays (0.1 nm) Crystallized proteins Bombardment and diffraction. e.g. Interference patterns Problems: • Years ago was very time-consuming (first structure...22 yrs!). • Large amount of material required. • Insoluble protein crystallization (e.g. membranes)? Becker. 2006 World of the Cell 14 X-ray Crystallography diffracted beam Synchrotron The structure of one molecule is derived from crystal diffraction pattern. The crystal contains many copies of the same molecule 15 X-ray Crystallography Protein folding Groups of atoms Individual atoms Electron density Karp. 2008. Cell and Molecular Biology 16 X-ray Crystallography • Diffraction pattern • Space-filling model of DNA double helix. From Becker. 2006 World of the Cell Fig. 4-5A. Alberts et al. Francis Crick James Watson Maurice Wilkins Rosalind Franklin Cobb M and Comfort N. (2023). Nature. 616:657-660. 17 X-ray Crystallography • Structure of K+ ion channel by X-ray crystallography. • Monoclonal antibodies (green) used to “hold” protein. Nobel Prize 2003 3.2 Å resolution Roderick MacKinnon Jiang et al. 2003. Nature 423:33-41. 18 Protein structure from AI • Today, protein 3D structure can be predicted by computation and artificial intelligence: • https://alphafold.ebi.ac.uk/ 19 Things to Consider... 1. 2. Think about applications in Cell Biology in which specific techniques of microscopy are most appropriate. For example, would you use an electron microscope to study intracellular Ca2+ dynamics? What are the major differences between each technique that we’ve discussed? For example, use of light or electrons, linear vs. non-linear, advantages and disadvantages. 20