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WK3 Cell Bio

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winniesmith's version from 2017-01-26 19:20

Section 1

Question Answer
What did the development of the light microscope lead too?the discovery of cells. Robert Cooke coined term 'cell' after observation of cork. 1670's- sperm,rbc,bacteria etc observed.
Cell theory (1838) proposed by sch&sch resulted from what and showed whatResulted from studies of plants and animal cells usiing microscopes, found that cell are not formed de novo but arise from divisions of pre existing cells.
Magnification of contemporary light microscopes1000x
what can light microscopes observemost cells between 1-100um and some organelles
What is 'resolution'the ability to distinguish objects separated by small distances.
How is the limit of resolution determinedn x the wavelength of visible light (λ) / Numerical aperture (NA) (the light gathering power of the lens.
Wavelength of visible light(λ) is fixed at approx 0.5um
NA can be envisioned asthe size of the cone of light that enters the lens (NA= η sinα (where η =refactive index of medium))
what is the value of η for air1.0
what is the (max) value of η using oil immersion lens1.4
what is max value for α90 degrees, at which sinα=1.
What is the max possible value for NA1.4 (as 1.4 sin90 and sin90=1)
who is a wollyyou are
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Section 2

Question Answer
Types of microscope many haha
Bright-feield microscopylight passes directly through cell, often preserved with fixatives and stained with dyes. Dead cells only.
differential interference-contrast and Phase-contrast microscopyconvert variations in density or thickness to differences in contrast that can be seen in the final image.
how can video cameras & computers be used in phase-contrast/differential interferece-contrast(dicm) m.?Can be used for image analysis and processing to enhance the contrast of images. Video enhanced dicm allows visualization oof movement of organalled along microtubules (cytoskeletal protein filaments with a diameter of 0.025um)
Fluorescence microscopyused for molecular analysis. Fluorescent dye is attched to a molecule of interest in fixed or living cells. Dye molecules absorb light at one wavelength and emit light at different.
How does fluoresence microscopy workFlouresence detected by illuminating specimen with wavelength of light that excites the Fdye. Filters used to detect specific wavelength light that dye emits.
What is green fluorescent protein (GFP)from jellyfish, can be fused to any protein using standard methods of recombinant DNA. Tagged protein expressed in cells & detected by Fmicroscopy (no need to stain/fix cells)
What is fluorescence recovery after photobleaching (FRAP)Used to study rate of protein movement in living cells (recovery time=movement). Region of cell with GFP-labeled protein is bleached by high intensity light. F recovers as GFP molecules move into bleached regions.
What is F resonance energy transfer (FRET)Used to study interactions between proteins in a cell. 2 proteins are coupled to different F dyes. Light emitted bu one GFP variant excites 2nd.
How can images from F microscopy be improvedBy image deconvolution: A computer analyzes images from different depths of focus & generates a sharper image from them.
What does confocal microscopy do?increases contrast & detail by analyzing F from single point. Small point of light from laser is focused on specimen at particular depth. Emitted F light is collected by detector such as a video cam.
How does confocal microscopy work?Emitted light must pass through a pin-hole/confocal aperture. Only light emitted from the plane of focus is able to reach the detector.Scanning across the specimen generates a 2D image of the plane of focus. A series of images can be used to reconstruct a 3D image.
Multi-photo exciation microscopyexcitation of a F dye requires 2 or more photons, which occurs at the point upon which the laser beam is focused. Localization of excitation minmizes damage to specimen, allowing 3D imagine of living cells.
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Section 3

Question Answer
Resolution of electron microscope0.2nm (because of short wavelength of electrons).
What is the resoultion for biological samples under electron m?1-2nm due to lack of contrast
Transmission electron mSpecimens are fixed and stained with salts of heavy metals, which provide contrast by scattering electrons. A beam of electrons is passed through the specimen and forms an image on a F screen. Specimens can be prepared by either positive or negative staining
What does electron tomography dogenerates 3D images by computer analysis of multiple 2D images ontained over a range of viewing directions.
What is metal shadowing used forto visualize the surface of subcellular structures or macromolecules.
How is metal shadowing donespecimen is sprayed with a thin layer of metal such as platinum, from an angle, which results in a shadowing effect.
What is freese fracturing specimens are frozen in liquid nitrogen and then fractured with a knife blade. The specimen is then shadowed with platinum. (can split lipid bilyar revealing interior faces of cell memb.)
What does scanning electron microscopy doprovides a 3D image of cells.
How does scanning electron microscopy workThe electron beam does not pass through the specimen. The surface of the cell is coated with a heavy metal, and a beam of electrons is used to scan across the specimen.
How is subcellular fractionation (organelle isolation) achievedby differential centrifugation (developed 1940s/50s)- separate cell components on the basis of size and density.
How are the components fragmented Plasma membranes and endoplasmic reticulum are fragmented by sonication, grinding, or high-speed blending. The suspension is fractionated in an ultracentrifuge, which spins at very high speeds. Larger, more dense organelles sediment at lower speeds; small organelles at high speeds.
What is density-gradient centrifugation organelles are separated by sedimentation through a gradient of a dense substance, such as sucrose.
What is velocity centrifugationstarting material is layered on top of the sucrose gradient. Particles of different sizes sediment through the gradient at different rates.
What is equilibrium centrifugationin density gradients is used to separate subcellular components on the basis of their buoyant density. particles are centrifuged until they reach an equilibrium position at which their buoyant density is equal to that of the surrounding sucrose or cesium chloride solution.
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