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

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Presentation on theme: "Return to ground state results in emission of radiation (fluorochrome). Absorption of photon elevates chromophore to excited state."— Presentation transcript:

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

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

3 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

4 Fluorescent Microscopy label cells with fluorescent probe illuminate with UV light examine epifluorescence ethidium bromide (DNA/RNA) DAPI (only DNA) rhodamine 123 (mitochondria) C 5 -CMB-ceramide (Golgi) free Ca 2+ indicators pH indicators membrane potential indicators antibodies

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

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10 8 consecutive 0.5 m optical sections

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

13 DNA synthesis nuclear division cytokinesis

14 Flow Cytometer

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16 Fluorescence Activated Cell Sorter (FACS)


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