Shpol’skii Spectroscopy•Analytical potential of fluorescence spectroscopy often limited by unresolved Analytical potential of fluorescence spectroscopy often limited by unresolved band structure (5-50 nm)band structure (5-50 nm)

•homogeneous band broadening homogeneous band broadening – depends directly on radiative deactivation – depends directly on radiative deactivation properties of the excited state (usually 10properties of the excited state (usually 10-3-3 nm) nm)•inhomogeneous band broadening inhomogeneous band broadening – various analyte microenvironments – various analyte microenvironments yields continuum of bands (usually few nm)yields continuum of bands (usually few nm)•Solution: Incorporate molecules in rigid matrix at low temperature to Solution: Incorporate molecules in rigid matrix at low temperature to minimize broadeningminimize broadening

•Result: Very narrow luminescence spectra with each band representing different Result: Very narrow luminescence spectra with each band representing different substitution sites in the host crystalline matrixsubstitution sites in the host crystalline matrix

1

Shpol’skii SpectroscopyRequirements:Requirements:1.1. T < 77K with rapid freezing rateT < 77K with rapid freezing rate2.2. Matrix with dimension matchMatrix with dimension match3.3. Low analyte concentrationLow analyte concentration

Instrumentation:Instrumentation:1.1. Xe lamp excitationXe lamp excitation2.2. Cryogenerator with sample cellCryogenerator with sample cell3.3. High resolution monochromator with PMTHigh resolution monochromator with PMT

Analytes:Analytes: polycyclic aromatic compounds in polycyclic aromatic compounds in environmental, toxicological, or geochemical environmental, toxicological, or geochemical systemssystems

2Garrigues and Budzinski, Garrigues and Budzinski, Trends in Analytical ChemistryTrends in Analytical Chemistry, 14 (5), 1995, pages , 14 (5), 1995, pages 231-239.231-239.

3Garrigues and Budzinski, Garrigues and Budzinski, Trends in Analytical ChemistryTrends in Analytical Chemistry, 14 (5), 1995, pages , 14 (5), 1995, pages 231-239.231-239.

Shpol’skii Spectroscopy

Fluorescence Microscopy

Need 3 filters:Need 3 filters:Exciter FiltersExciter FiltersBarrier FiltersBarrier FiltersDichromatic BeamsplittersDichromatic Beamsplitters

http://microscope.fsu.edu/primer/techniques/fluorescence/filters.htmlhttp://microscope.fsu.edu/primer/techniques/fluorescence/filters.html4

Are you getting the concept?You plan to excite catecholamine with the 406 nm line fromYou plan to excite catecholamine with the 406 nm line froma Hg lamp and measure fluorescence emitted at 470 a Hg lamp and measure fluorescence emitted at 470 ± 15± 15nm. Choose the filter cube you would buy to do this.nm. Choose the filter cube you would buy to do this.Sketch the transmission profiles for the three optics.Sketch the transmission profiles for the three optics.

http://microscope.fsu.edu/primer/techniques/fluorescence/fluorotable3.htmlhttp://microscope.fsu.edu/primer/techniques/fluorescence/fluorotable3.html5

Fluorescence Microscopy Objectives

Image intensity is a function of the objective numericalImage intensity is a function of the objective numericalaperture and magnification: aperture and magnification:

2

4

)(

)( mag

NAI obj

Fabricated with low fluorescence glass/quartz with anti-Fabricated with low fluorescence glass/quartz with anti-reflection coatingsreflection coatings

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.htmlhttp://micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.html 6

Fluorescence Microscopy Detectors

No spatial resolution required: PMT or photodiodeNo spatial resolution required: PMT or photodiodeSpatial resolution required: CCDSpatial resolution required: CCD

http://micro.magnet.fsu.edu/primer/digitalimaging/digitalimagingdetectors.htmlhttp://micro.magnet.fsu.edu/primer/digitalimaging/digitalimagingdetectors.html 7

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 Dichroic mirror reflects one range of wavelengths and allows another range to

pass. pass.• Barrier filter eliminates all but fluorescent light.Barrier filter eliminates all but fluorescent light.

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorosources.htmlhttp://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorosources.html8

http://web.uvic.ca/ail/techniques/epi-fluor.jpg

Special Fluorescence Techniques

TIRFTIRF

http://microscopy.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.htmlhttp://microscopy.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html9

Langmuir 2009, 25, 2563-2566

Fluorescence Resonance Energy Transfer (FRET)

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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