Figure 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 sizes On the microscopic to Macroscopic scale
Cellular proteins can be visualized in Real time in living cells S. CEREVISCIAE - BAKERS YEAST GOLGI GREEN 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’ UTR AUG UAG UAA UGA DNA plasmid GFP Getmeoutaherenow Making Fluorescent Cells
Ta da…..The Brainbow Making Fluorescent Cells GFP is a Beta-can -helices -sheets chromophore Cerulean GFP Banana Orange…….
d = 0.61 n sin Resolving power D = minimum distance of 2 points = wavelength n = numerical aperture = angle of cone of light (1/2)
Methods to increase contrast Specific stains Light microscope comparisons Bright field Phase contrast Nomarski CFP YFP axons
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 cell Freeze-fractured Ciliary axoneme - deep etch Insect head - SEM - bacteriophage