figure 7-108 molecular biology of the cell (© garland science 2008)
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Figure 7-108 Molecular Biology of the Cell (© Garland Science 2008)
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Resolution of biological objects
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Capturing and interpreting light images
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Different methods to visualize cellular morphology and objects
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Light and fluorescence microscopy
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Detection methods for subcellular structures
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Methods to better resolve objects in 3D
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Relative sizesOn the microscopic toMacroscopic scale
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Cellular proteins can be visualized inReal time in living cells
S. CEREVISCIAE - BAKERS YEAST
GOLGIGREEN AND RED FLUORESCENT PROTEINS
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How Flourescence works
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A.VICTORIA (AKA - GLOWING JELLYFISH)Isolate protein - green fluorescent protein (GFP)Abs. Max = 488nm
Now can fuse protein of interest with GFP
GFP Getmeoutaherenow
5’ 3’UTRUTRAUG UAG
UAAUGA
DNA plasmid
GFPGetmeoutaherenow
Making Fluorescent Cells
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Ta da…..The Brainbow
Making Fluorescent Cells
GFP is a Beta-can-helices
-sheets
chromophore
CeruleanGFP Banana Orange…….
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d = 0.61 n sin
Resolving power
D = minimum distance of 2 points = wavelengthn = numerical aperture = angle of cone of light (1/2)
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Methods to increase contrastSpecific stains
Light microscope comparisons
Bright field
Phase contrast
NomarskiCFP
YFPaxons
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Out of focus light can be removed by computers
Raw Deconvolved
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Other uses of Fluorescent proteins
FRET - fluorescence resonance energy transfer-can detect changes in interactions-donor FL energy reduced while acceptor increased
Bi-molecular complementation
Caged proteins
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Light microscope
Electron microscope
Electron microscopy
1nm resolution
Negative stain Shadow casting
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Sample preparation is time consuming
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Onion root cellFreeze-fractured
Ciliary axoneme - deep etch
Insect head - SEM - bacteriophage
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