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)