Longwood SAIF Series: Multi and HyperSpectral Fluorescence II of VI

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Longwood SAIF SeriesMulti- and Hyper-Spectral
Fluorescence (II of VI)

• Joanne T. Vannah
• Longwood Small Animal Imaging Facility
• Beth Israel Deaconess Medical Center
• Boston, MA

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Fluorescence

• The property of matter to absorb a photon at a
  certain wavelength and re-emit the photon at a
  lower energy state (longer wavelength)
• The shift in wavelengths that occurs during the
  fluorescence process (from higher to lower
  energy) is known as Stokes shift

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Three stages in fluorescence

• Excitation of molecule, fluorophore, by an
  incoming photon (10-15s)
• Loss of energy (10-12s)
• Emission of a longer wavelength photon and return
  of molecule to ground state (10-9s)

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Jablonski diagram
With permission from Single Molecule Optics,
MoNOS, Universiteit Leiden http//www.monos.leide
nuniv.nl/smo/index.html?basics/photophysics.htm
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Three parameters to describe fluorescence

• Extinction coefficient
• Direct measurement of the fluorophores ability
  to absorb light
• High extinction coefficients increase the
  probability of fluorescence emission
• Quantum yield
• Ratio of photons emitted over photons absorbed
• Fluorescence lifetime
• The time during which the initial fluorescence
  intensity of the fluorophore decays to 1/e of of
  its initial intensity

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Three measurements to describe optical properties
in tissues

• Each tissue has distinctive transmission,
  absorption, and anisotropy
• µA Absorption coefficient (cm-1)
• µs Scattering coefficient (cm-1)
• s Anisotropy (i.e. polarization)
• Total attenuation of light µ T µS µA

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Absorption coefficient , µA

• Factors that contribute to the absorption
  coefficient, µA , are listed below and are
  wavelength dependent
• Oxyhemoglobin
• Deoxyhemoglobin
• Lipid
• Tissue pigments
• Water (gt 1000 nm)

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Scatter coefficient, µs

• These factors listed below contribute to scatter
  and are wavelength dependent
• Collagen fibrils
• Nuclei and cellular organelles
• Refractive index boundaries

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Photobleaching

• Fluorophore has permanently lost its ability to
  produce fluorescence
• Occurs because of photon-induced chemical damage
• Limits to the number of excitation/emission
  cycles differs depending on the fluorophore

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Multi-spectral fluorescence

• All fluorescent materials produce a unique
  spectral emission
• Allows the imaging of multiple spectral bands
  simultaneously
• Allows the emitted fluorescence over a range of
  wavelengths and records the intensity over this
  range to form an emission spectrum

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Hyper-spectral fluorescence

• Also referred to as spectral unmixing
• Allows processing of multi-spectral data to
  determine pure spectra of individual components
• Distinguishes between background/autofluorescence
  and signal of interest
• Thereby improves signal-to-background ratio
  300-fold

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CRI Maestro Multi-Spectral Fluorescence (model
500)
www.cri-inc.com
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Hardware components

• High resolution, scientific grade CCD
• Excitation bandpass filters
• Solid-state liquid crystal tunable filter
• Longpass (emission) filters
• Light tight enclosure
• Light source Cermax 300 Watt Xenon lamp, 5600K
  illumination
• Wavelength range from 500-950 nm

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Software components

• Acquire panel
• Controls needed to form new spectral images and
  image cubes
• Spectral processing panel
• Unmix cubes (either manually or automatically)
• Measure panel
• Find, measure, and adjust regions in images
• Display panel
• Change current display settings of images

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Acquiring an image cube

• Exposure time and wavelength range (filters) are
  selected
• Light from the Xenon light source passes through
  the excitation filter to the subject and
  re-emitted through the emission filter, through
  the liquid crystal tunable filter and longpass
  emission filter
• Each spectral is imaged in 3 components
• x, y, and ? where ? is the wavelength

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Liquid crystal tunable filter
The Maestro LCTF has an OD 3 therefore a
longpass filter is used to further process ?
http//www.sprawls.org/ppmi2/
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Longpass vs bandpass

• Because the Maestro LCTF has an OD 3, a
  longpass emission filter is used
• Longpass filters allows transmission of long
  wavelength radiation while blocking short
  wavelengths
• A bandpass filter allows transmission of
  frequencies within a certain range and rejects
  frequencies outside that range

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Rats labeled with fluorescent dyes
Two 300 g rats labeled with 3 fluorescent dyes
www.cri-inc.com
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Skin (background) signal
,
www.cri-inc.com
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Food signal
www.cri-inc.com
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TRITC signal
www.cri.inc-com

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Cy 3.5 Signal
www.cri-inc.com
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FITC signal
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Unmixed image of 3 dyes

• FITC, TRITC, and Cy 3.5, plus skin and food
  signals

www.cri-inc.com
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Multi- and hyper-spectral fluorescence
applications

• Tumor models
• Drug pharmacokinetics
• Angiogenesis markers
• Molecular targeting agents
• Brain slices
• Pre-histologic selection

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Multi- and hyper-spectral fluorescence

• Pros
• Multiple, simultaneous imaging of fluorophores
  and segregation into pure spectra
• Highly sensitive and fast
• Cons
• Limited by time (fluorescent state is short
  photobleaching)
• An exogenous light source required

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Summary

• Multi-spectral fluorescence permits the
  simultaneous imaging of a range of spectra
• Hyper-spectral fluorescence is the the unmixing
  from multi-spectral fluorescence
• Multi- and hyper-spectral fluorescence reduce
  background and increase the ability to visualize
  pure signals
• The CRI Maestro Multi-Spectral Fluorescence
  System can precisely and accurately quantify
  fluorescence data