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