Return to ground state results in emission of radiation (fluorochrome).

Absorption of photon elevates chromophore to excited state.

Absorbance (solid) and Fluorescence (dashed) Spectra of Tryptophan

• enzyme assays• nucleic acids

• measurement• detection (gels)

• microscopy• flow cytometry

Applications of Fluorescence

substrate product*

Detection Limits for Nucleic Acids

• UV absorbance 1 g/ml• ethidium bromide 10 ng/ml

Fluorescent Microscopy• label cells with fluorescent probe• illuminate with UV light• examine epifluorescence

ethidium bromide (DNA/RNA)

DAPI (only DNA) rhodamine 123 (mitochondria)

C5-CMB-ceramide (Golgi) free Ca2+ indicatorspH indicatorsmembrane potential indicators

antibodies

acridine orange• fluoresces when bound to

DNA and RNA• fluorescence exhibits

different wavelengths max DNA ~ 530 nm max RNA ~ 640 nm

• filters can distinguish fluorochromes• dual-labeling experiments

Confocal Laser Scanning Microscopy

• minimizes the light from outside the plane of focus• apertures (or ‘pinholes’)• objective lens = condenser

• wide field vs. scanning (x, y dimensions)• excitation with lasers

• movable stage allows ‘optical’ sections (z dimension)• generate 3-D images

8 consecutive 0.5 m optical sections

Fluorometry vs Microscopy

quantitative data average of mixture

qualitative data quantitative data

more difficult individual cells

Flow Cytometer

• qualitative and quantitative data on individual cells• rapidly analyze > 10,000 cells

DNA synthesis

nuclear division

cytokinesis

Flow Cytometer

Fluorescence Activated Cell Sorter (FACS)