Laser Induced Fluorescence Spectroscopy LIFS

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Laser Induced Fluorescence  Spectroscopy (LIFS)

• Liang Gao
• Department of Bioengineering
• Rice University
• ELEC 568
• Final Presentation
• Fall 2007

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Outline

• Introduction to Laser Induced Fluorescence 
  Spectroscopy (LIFS)
• LIFS on Tissue Level
• Time-integrated spectrum
• Time-resolved spectrum
• LIFS on Molecular Level
• Fluorescent proteins
• Fluorescent contrast agents
• Imaging techniques

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Introduction

• LIFS is a spectroscopic method used for studying
  structure of molecules, detection of selective
  species, and flow visualization and measurements
• Excited with the help of a laser
• Wavelength is selected at species largest cross
  section
• In the order of few nanoseconds to microseconds,
  de-excite and emit light at a wavelength larger
  than the excitation wavelength
• Greater sensitivity achievable because of very
  low background of fluorescent signal compared to
  absorption measurements

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LIFS on Tissue Level

• Core Instruments
• Laser excitation source
• Focusing and collection optics (lenses/fiber
  optics)
• Spectrometer and a sensitive spectroscopic CCD
  detector
• Intensified Charge-Coupled Device (ICCD) is used
  for time-resolved LIFS

Olaf Koschützke, LOT-Oriel Gruppe Europa.
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Time-integrated spectrum

• Normal tissue, metaplasia, and high grade
  dysplasia
• 337nm and 405 nm laser excitation
• The patient had received ?-aminolevulinic acid
  (ALA) prior to the investigation
• ALA will be converted to the fluorescent tumour
  marker with emission peak at about 635 nm

Olaf Koschützke, LOT-Oriel Gruppe Europa.
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Time-resolved spectrum

• A basal cell carcinoma adjacent normal skin
• 405nm laser excitation
• Marker peaks 635nm, 705nm, longer lifetime
• Tissue auto fluorescence 490 nm, shorter
  lifetime

Olaf Koschützke, LOT-Oriel Gruppe Europa.
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• (a) Normal arterial wall (b) Type 2 lesion
• (c) type 4 lesion (d) type 5a lesion

Laura Marcu, 2001
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Energy transfer on molecular level

• Fluorescence
• Stokes shift
• Fluorescence resonance energy transfer (FRET)
• Photon Switch
• Free Radical Generation
• Oxygen Radical
• Phototoxicity
• Photobleaching
• Tumor treatment

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Green Fluorescent Protein (GFP)

• Originally isolated from the jellyfish
• Excitation Emission 395nm 475nm
• GFP has a unique can-like shape with a single
  alpha helical strand containing the fluorophore
  running through the center
• Cyclization of Ser65Tyr66Gly67 residuals leads
  to fluorophore formation

http//en.wikipedia.org/wiki/Green_Fluorescent_Pro
tein
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• Procedures for making GFP conjugates

Images Courtesy of Vandi Bharucha, Ph.D.,
Invitrogen Inc., Jennifer Lucitti, Ph.D., and Aya
Wada Ph.D., Baylor College of Medicine
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GFP derivatives

• Point mutations
• Spectral characteristics, photostablility,
  optical crosssection, quantum yield

Shaner NC, 2005
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HEK 293T cells

• Fluorescence resonance energy transfer (FRET)
• Genetically encoded
• GFP biosensor
• Calcium detection via Calmodulin
• 240 nM dissociation constant for calcium, range
  0.1-100 µM Ca2

Images Courtesy of Vandi Bharucha, Ph.D.,
Invitrogen Inc.
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• Adult stem cell from left atrial appendage of
  pig heart stimulated with 20 uM ATP

Images Courtesy of Vandi Bharucha, Ph.D.,
Invitrogen
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Optical Imaging Contrast Agents

• Organic fluorescent dyes
• Extensive p-conjugation
• Fluorescein isothiocyanate (FITC), reactive
  derivative of fluorescein
• Derivatives of Rhodamine, Coumarin and Cyanine
• Inorganic fluorescent dyes
• Quantum Dots
• Semiconductor nanostructure
• Good Tuneability
• 500-800nm Emission
• No photobleaching, high excitation
• cross-section

www.invitrogen.com
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Optical Imaging Contrast Agents-Contd.

• Conjugates
• Antibody
• Signal tag
• Direct injection
• Problems
• Too big
• High Background
• Affect functions of the target
• Photobleaching and Phototoxicity

Howarth M, 2005
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Images Courtesy of Tegy Vadakkan, Baylor College
of Medicine and Scott Clarke, Invitrogen.inc.
www.invitrogen.com
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Imaging techniques

• Confocal Laser-Scanning Microscopy
• Good optical section
• Problems
• Large excitation volume
• Significant photobleaching and phototoxitity
• Limited penetration depth
• Absorption of excitation energy throughout the
  beam path
• Scattering of both excitation and emission
  photons

MPE Tutorial, Coherent Inc, wwwo.coherent.com
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Imaging techniques-Contd.

• Multiphoton Laser-Scanning Microscopy
• Two-photon microscopy
• Mode-locked (pulsed) lasers
• Quasi-simultaneous 10E(-18) absorption of two
  photons from Near-IR laser
• Approximately one million time photon density is
  needed
• Absence of out-of focus absorption
• IR light undergoes less scattering

MPE Tutorial, Coherent Inc, wwwo.coherent.com
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Two-photon Laser-Scanning microscopy
MPE Tutorial, Coherent Inc, wwwo.coherent.com
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Reference

• Lucitti JL, et. al, Vascular remodeling of the
  mouse yolk sac requires hemodynamic force.
  Development. 2007 Sep134(18)3317-26.
• Shaner NC, Steinbach PA, Tsien RY. A guide to
  choosing fluorescent proteins. Nat Methods. 2005
  Dec2(12)905-9.
• Howarth M, Takao K, Hayashi Y, Ting AY. Targeting
  quantum dots to surface proteins in living cells
  with biotin ligase. Proc Natl Acad Sci U S A.
  2005 May 24102(21)7583-8
• Marcu L, et. al, Discrimination of Human Coronary
  Artery Atherosclerotic Lipid-Rich Lesions, by
  Time-Resolved Laser-Induced Fluorescence
  Spectroscopy, Arterioscler Thromb Vasc Biol. 2001
  Jul21(7)1244-50.
• Olaf Koschützke, LOT-Oriel Gruppe Europa. Laser
  Induced Fluorescence Spectroscopy (LIFS)
• www.invitrogen.com
• www.coherent.com
• http//en.wikipedia.org/wiki/