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

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

3. 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

4. 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

6. acridine orange fluoresces when bound to DNA and RNA
fluorescence exhibits different wavelengths
lmax DNA ~ 530 nm
lmax RNA ~ 640 nm

7. filters can distinguish fluorochromes
dual-labeling experiments

8. 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

10. 8 consecutive 0.5 mm optical sections

12. Flow Cytometer qualitative and quantitative data on individual cells
rapidly analyze > 10,000 cells

13. DNA synthesis
nuclear division
cytokinesis

14. Flow Cytometer

16. Fluorescence Activated Cell Sorter (FACS)