1. Shpol’skii Spectroscopy Analytical potential of fluorescence spectroscopy often limited by unresolved band structure (5-50 nm)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)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)inhomogeneous band broadening – various analyte microenvironments yields continuum of bands (usually few nm) Solution: Incorporate molecules in rigid matrix at low temperature to minimize broadeningSolution: 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 matrixResult: Very narrow luminescence spectra with each band representing different substitution sites in the host crystalline matrix 1

2. Shpol’skii Spectroscopy Requirements: 1.T < 77K with rapid freezing rate 2.Matrix with dimension match 3.Low analyte concentration Instrumentation: 1.Xe lamp excitation 2.Cryogenerator with sample cell 3.High resolution monochromator with PMT Analytes: polycyclic aromatic compounds in environmental, toxicological, or geochemical systems 2 Garrigues and Budzinski, Trends in Analytical Chemistry, 14 (5), 1995, pages 231-239.

3. 3 Shpol’skii Spectroscopy

4. Fluorescence Microscopy Need 3 filters: Exciter Filters Barrier Filters Dichromatic Beamsplitters http://microscope.fsu.edu/primer/techniques/fluorescence/filters.html 4

5. 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 5

6. 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 6

7. Fluorescence Microscopy Detectors No spatial resolution required: PMT or photodiode Spatial resolution required: CCD http://micro.magnet.fsu.edu/primer/digitalimaging/digitalimagingdetectors.html 7

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

9. Special Fluorescence TechniquesTIRF http://microscopy.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html 9 Langmuir 2009, 25, 2563-2566

10. Fluorescence Resonance Energy Transfer (FRET) 10

11. Photoactivated Localization Microscopy http://www.hhmi.org/bulletin/nov2006/upfront/image.html Left: Viewing a mitochondrion using conventional diffraction-limited microscopy offers a resolution (200 nanometers) barely sufficient to visualize the mitochondrial internal membranes. Right: Viewing the same mitochondrion by imaging sparsely activated fluorescent molecules one at a time—using PALM— provides much better resolution (20 nanometers), producing a detailed picture of the mitochondrion’s internal membranes. http://www.hhmi.org/news/palm20060810.html