NSC 837 - Lecutre 6 - photobleaching, autofluorescence labeling, live cell imaging, colocalization

fading/photobleaching

irreversible decomposition of a fluorescent molecule

primarily due to the effects of oxygen free-radicals

caused by both the high power, focused beam of confocal system long exposures to the lower intensity mercury lamp systems

if sample is photobleached won’t be able to look at it again

what is fading/photobleaching affected by

intensity of light, duration of light, wavelength of light, pH of embeddng medium (fixed samples)

what can slow the fading process

antifade reagents

difficult for live cells (vitamin c instead)

autofluorescnce

endogenous fluorescence: compounds nnaturally present in cells or tisssue (ex fatty acids, b vitamins, nadh)

fixation-induced fluorescence often the result of aldehyde fixation— glutaraldehyde (worse) to formaldehyde

how to reduce background autofluorescence for fixed cells

image-iT FX signal enhancer - reduces background from non-specific staining by secondary conjugates or antibodies

how to reduce background autofluorescence for live cells

backdrop background suppressor - supressses background in live cells. apply 2 drops per ml directly to live cells

antibodies

5 classes (IgG, IgM, IgA, IgD, IgE), composed of 2 heavy chaing and 2 light chains

antibodies and imaging

can use to look for component in cell (protein)

usually need to fix sample to use an antibody, it is too big to fit in cell.

nanobodies

isolated from llamas. The consist of a heavy chain and on variable region. It is smaller and can get into smaller regions that regular antibodies can not fit into.

advantages of nanobodies

smalle

enhanced tissue pentration

more easily penetrates small cavities

high affinity and increased specificty

more stable to extreme temps and pH

indirect immunoflourescence

1) add primary (non-fluorescent) antibody to cells tissue

2) incubate

3) wash away excess antibody

4) add secondary antibody with fluorochrome to cells/tissue already labeled with primary

5) incubate

6) wash away excess antibody

controls

1) image fully stained specimen - optimize settings and don’t change

2) image specimen labeled with primary alone -autofluorescence control

3) image specimen labeled with secondary alon (nonspecific binding of secondary antibodies)

fluorescent proteins

GFP is the main one and is a type of fusion protein

fusion proteins

gene of florescent protein is attached to gene of protein of interest

clickable proteins

uses a small sequene that can be encoded with the protein of interest to creat a dusion protein and then snaps an organic dye molecule that reacts (snaps into place)with the target sequnce through a chemical reaction.