Solvent Effects

1. nonequilibrium excited state Solvent Effects Increased viscosity can increase luminescence intensity. H-bonding and dipole interactions with the solvent contribute to the Stokes shift. Ashutosh Sharma and Stephen Schulman, Fluorescence Spectroscopy, John Wiley & Sons, New York, 1999.

2. Solvent Polarity Increasing solvent polarity usually causes a red-shift in fluorescence. http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.html

3. Solvent Polarity Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999.

4. Temperature Increasing temperature increases ks Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999.

5. Decreasing temperature can induce a blue-shift in fluorescence. Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999.

6. Shpol’skii Spectroscopy • Analytical potential of fluorescence spectroscopy often limited by unresolved band structure (5-50 nm) • homogeneous band broadening – depends directly on radiative deactivation properties of the excited state (usually 10-3 nm) • inhomogeneous band broadening – various analyte microenvironments yields continuum of bands (usually few nm) • Solution: Incorporate molecules in rigid matrix at low temperature to minimize broadening • Result: Very narrow luminescence spectra with each band representing different substitution sites in the host crystalline matrix

7. Shpol’skii Spectroscopy • Requirements: • T < 77K with rapid freezing rate • Matrix with dimension match • Low analyte concentration • Instrumentation: • Xe lamp excitation • Cryogenerator with sample cell • High resolution monochromator with PMT Analytes: polycyclic aromatic compounds in environmental, toxicological, or geochemical systems Garrigues and Budzinski, Trends in Analytical Chemistry, 14 (5), 1995, pages 231-239.

8. Epi-Fluorescence Microscopy • Light Source - Mercury or xenon lamp (external to reduce thermal effects) • Dichroic mirror reflects one range of wavelengths and allows another range to pass. • Barrier filter eliminates all but fluorescent light. http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorosources.html

9. Fluorescence Microscopy Need 3 filters: Exciter Filters Barrier Filters Dichromatic Beamsplitters http://microscope.fsu.edu/primer/techniques/fluorescence/filters.html

10. Are you getting the concept? You plan to excite catecholamine with the 406 nm line from a Hg lamp and measure fluorescence emitted at 470 ± 15 nm. Choose the filter cube you would buy to do this. Sketch the transmission profiles for the three optics. http://microscope.fsu.edu/primer/techniques/fluorescence/fluorotable3.html

11. Fluorescence Microscopy Objectives Image intensity is a function of the objective numerical aperture and magnification: Fabricated with low fluorescence glass/quartz with anti- reflection coatings http://micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.html

12. Fluorescence Microscopy Detectors No spatial resolution required: PMT or photodiode Spatial resolution required: CCD http://micro.magnet.fsu.edu/primer/digitalimaging/digitalimagingdetectors.html

13. Special Fluorescence Techniques LIF TIRF http://microscopy.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html