Shpolskii Spectroscopy

1
Shpolskii 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

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Shpolskii Spectroscopy

• Requirements
• T lt 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.
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Shpolskii Spectroscopy
Garrigues and Budzinski, Trends in Analytical
Chemistry, 14 (5), 1995, pages 231-239.
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Fluorescence Microscopy
Need 3 filters Exciter Filters Barrier
Filters Dichromatic Beamsplitters
http//microscope.fsu.edu/primer/techniques/fluore
scence/filters.html
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/fluore
scence/fluorotable3.html
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/fluo
rescence/anatomy/fluoromicroanatomy.html
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Fluorescence Microscopy Detectors
No spatial resolution required PMT or
photodiode Spatial resolution required CCD
http//micro.magnet.fsu.edu/primer/digitalimaging/
digitalimagingdetectors.html
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//web.uvic.ca/ail/techniques/epi-fluor.jpg
http//micro.magnet.fsu.edu/primer/techniques/fluo
rescence/fluorosources.html
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Special Fluorescence Techniques
TIRF
Langmuir 2009, 25, 2563-2566
http//microscopy.fsu.edu/primer/techniques/fluore
scence/tirf/tirfintro.html
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Fluorescence Resonance Energy Transfer (FRET)
11
Photoactivated Localization Microscopy
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
timeusing PALMprovides much better resolution
(20 nanometers), producing a detailed picture of
the mitochondrions internal membranes.
http//www.hhmi.org/news/palm20060810.html

• http//www.hhmi.org/bulletin/nov2006/upfront/image
  .html