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34. Summary. Cells follow rules of chemistry; Water is the most abundant substance proteins constitutes most of a cell’s dry mass; Four major classes of small organic molecules make macromolecules; Living cells undergo metabolism; A reaction will happen if it can result in lower

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Summary

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  1. 34 Summary Cells follow rules of chemistry; Water is the most abundant substance proteins constitutes most of a cell’s dry mass; Four major classes of small organic molecules make macromolecules; Living cells undergo metabolism; A reaction will happen if it can result in lower free energy in the system; Proteins and protein complexes execute almost all cell functions.

  2. Lecture 3 Microscopy

  3. Birth of Cell Biology 1838 Schleiden and Schwann “cell doctrine”

  4. A typical cell is 10-20 um Light microscope sees 0.2 um 1 mm=10-3 m 1 um=10-6 m 1 nm=10-9 m 1 A=Angstrom =10-10 m

  5. Resolution is limited by the wavelength of radiation Nondestructive Live imaging Challenge: sample preparation Computerized 3-D reconstruction

  6. Four types of light microscopy Normaski DIC Bright-field Phase-contrast Dark-field

  7. Images can be digitally enhanced Eyes have limitation in seeing dim signals and resolve bright images Camera (the same kind as in night surveillance) 2. Digitally extract info (contrast) Improves interpretation, resolution (0.025 um), but appears 0.2 um (can’t tell whether it is single or double MT)

  8. Sample preparation: sectioning

  9. Sample preparation: staining to visualize cellular contents Hematoxylin Eosin: HE

  10. Fluorescence microscope

  11. Fluorescent dyes

  12. Immunofluorescence imaging does not show actual sizes

  13. 3-D imaging (esp fluorescence) Image deconvolution: a computational approach to remove blur from a stack of images taken in different focal planes (digital technique) Needs a fast computer, less bleaching, very sensitive

  14. 3-D imaging (esp fluorescence) Confocal microscope produces optical sections by excluding out-of-focus light (analog technique) Expensive scope, more bleaching, limitations in depth

  15. Two photon

  16. Fast electrons has short waves: 100,000V and 0.004 nm In theory 0.002 nm resolution: 10,000 of light microscope In practice, 0.1 nM (1A) Difficulties: Specimen preparation Contrast Radiation damage Therefore, effective 2 nM (20 A) 100 better than light microscope Gold atoms (bright spots) 0.2 nm apart

  17. Transmission EM (TEM)

  18. Immunogold EM

  19. 3D reconstruction Distorions of immuno EM: Large depth of field deep structures in the same layer Ab and Gold-Ab don’t penetrate too deep Label before imbedding

  20. Scanning EM for surface imaging (SEM) Smaller cheaper 10 nM resolution

  21. Imaging surface DIC TEM SEM

  22. Metal shadowing (platinum) and observe under TEM Individual marcomolecules can been seen

  23. Freeze-fracture electron microscopy Intramembrane particles (large TM proteins) Chloroplast

  24. Freeze-etch electron microscopy Interior of cells Protein filaments in muscle cells Crack the frozen block, lower ice level by subliming ice In a vacuum (freeze drying), shadow the exposed parts of cells and observe replica

  25. Negative staining of actin filaments Helical chain of acitin monomers 8 nm Diameter

  26. EM tomography for 3-D reconstruction TEM Hepatitis B virus See details of macro- molecular Complexes!!! Resolution rivals X-ray crytallography 0.3 nm for crystalline arrays 0.8 nm for single particle reconstruction (subunits, domains, 2nd structures)

  27. Calcium imaging Ion-sensitive indicators Brighter better resolution 0.5-10 um Not bright Fluorescent Ca indicator:fura2 Aequorin, a luminescent protein

  28. Introducing membrane-impermeable substance into a cell

  29. Caged molecules Ca++ can be caged too photolysis Tubulin labeled With caged fluoresent dyes

  30. Jellyfish GFP Live imaging Cajal bodies

  31. Pulse-chase experiments using radioisotopes

  32. Summary Cell doctrine; Two major types of microscopes: light and electron; Limitation of resolution: wavelength of radiation; Advantage and disadvantage of light and electron MS Different types of light microscopes: bright field, phase contrast, DIC, dark field,fluorescent, confocal Image processing: digital enhancement Two major types of EM: TEM and SEM Additional tricks: shadowing, freeze-fracture, freeze etching, negative staining, tomography; Live imaging, calcium indicators, caged compounds, GFP, pulse chasing

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