Figure 7-108 Molecular Biology of the Cell (© Garland Science 2008)
Resolution of biological objects
Capturing and interpreting light images
Different methods to visualize cellular morphology and objects
Light and fluorescence microscopy
Detection methods for subcellular structures
Methods to better resolve objects in 3D
Relative sizesOn the microscopic toMacroscopic scale
Cellular proteins can be visualized inReal time in living cells
S. CEREVISCIAE - BAKERS YEAST
GOLGIGREEN AND RED FLUORESCENT PROTEINS
How Flourescence works
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
Ta da…..The Brainbow
Making Fluorescent Cells
GFP is a Beta-can-helices
-sheets
chromophore
CeruleanGFP Banana Orange…….
d = 0.61 n sin
Resolving power
D = minimum distance of 2 points = wavelengthn = numerical aperture = angle of cone of light (1/2)
Methods to increase contrastSpecific stains
Light microscope comparisons
Bright field
Phase contrast
NomarskiCFP
YFPaxons
Out of focus light can be removed by computers
Raw Deconvolved
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
Light microscope
Electron microscope
Electron microscopy
1nm resolution
Negative stain Shadow casting
Sample preparation is time consuming
Onion root cellFreeze-fractured
Ciliary axoneme - deep etch
Insect head - SEM - bacteriophage
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