Absorption of photon elevates chromophore to excited state. Return to ground state results in emission of radiation (fluorochrome).

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

Applications of Fluorescence enzyme assays nucleic acids measurement detection (gels) microscopy flow cytometry substrate  product* Detection Limits for Nucleic Acids UV absorbance 1 mg/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+ indicators pH indicators membrane potential indicators antibodies

acridine orange fluoresces when bound to DNA and RNA fluorescence exhibits different wavelengths lmax DNA ~ 530 nm lmax 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 mm optical sections

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)