Longwood Small Animal Imaging Facility

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The SAIF Series Bioluminescence (I of VI)

• Joanne T. Vannah
• Longwood Small Animal Imaging Facility

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What is bioluminescence?

• bio life/living matter
• lumin to fill with light
• essence process that changes a state
• Bioluminescence is the conversion of chemical
• energy into light within a living organism

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Optical imaging

• Optical imaging uses the properties of light
  emitted by an organism as a result of either
  exogenous stimulation (fluorescence) or a
  byproduct of an internal biological process
  (bioluminescence) in the visible light range.

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Visible light in the electromagnetic spectrum
Modified from http//www.sprawls.org/ppmi2/
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Bioluminescent organisms

• Bioluminescence is primarily found in a wide
  array of marine organisms
• In the absence of solar energy, marine species
  evolved a way to emit light
• Most marine bioluminescent organisms emit light
  in the range of 440-480 nm, which is conducive to
  optical transmission through seawater
• Select insects, bacteria, and wood fungus also
  express bioluminescent properties

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Bioluminescent organismsFirefly and sea pansy

• Photinus pyralis Renilla
  reniformis

Modified from http//www.everythingabout.net/arti
cles/biology/animals/arthropods/insects/beetles/fi
refly/index.shtml http//www.jaxshells.org/1113b
b.htm
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Chemical reaction to produce light
oxidation-reduction, exergonic reactionluciferin/
coelenterazine oxidized by luciferase, which is
reduced

• Firefly (Photinus pyralis)

? Successful experiment depends on
luciferase concentration, luciferin
concentration, O2, ATP, Mg2
Successful experiment depends on luciferase
concentration, coelenterazine concentration, O2
?
Sea Pansy (Renilla reniformis)
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Coelenterazine emission spectrum
Modified from www.biotium.com
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Luciferin emission spectra
Green line illustrates peak emission for
luciferin. Red line indicates tissue transmission
at 1 cm deep (gt 700nm)
Modified from www.xenogen.com
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Bioluminescent imaging strengths

• Very effective labeling technique gene
  expression results in production of luciferase
• Cell tracking applications amount of light is
  proportional to number of cells
• Functional applications light is produced in
  response to a stimulus
• Extremely low background compared to other in
  vivo methods (i.e., fluorescence)
• Non-invasive subject is anesthetized

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Combine promoter sequence with luciferase
expression
Modified from www.promega.com
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Designing a bioluminescent in vivo experiment

• Combine a promoter sequence with the expression
  of luciferase to quantify promoter activity in
  vivo
• Methods of transducing cells vector (e.g.,
  virus, plasmid), electroporation
• Stably integrate promoter luciferase in cells
  in tissue of interest

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Measuring BLI with the Xenogen IVIS 50 (Lumina)
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IVIS imaging chamber

Illuminator lights are used to visualize subject.
Lights are off during bioluminescent imaging
Modified from www.xenogen.com
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Standard image is a composite of two
Modified from www.xenogen.com
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Route from signal source to signal detection
Absorption and scatter are encountered through
the biological tissue.
Modified from www.xenogen.com
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Controlling light

• Exposure time luminescent signal is
  proportional to exposure time
• Longer exposures, increase signal
• Shorter exposure, increases throughput
• Min max (0.5 ms 5 minutes)
• F-stop (lens aperture) controls the amount of
  light reaching the detector

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System control panel
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Binning (resolution vs sensitivity)
Binning 1, 2 4 (small, medium and large
respectively)
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Units of surface radiance photons/s/sr/cm²
Modified from Living Image Manual, Xenogen
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Image acquired
Modified from Living Image Manual, Xenogen
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BLI applications

• Developmental studies
• Tumor growth
• Characterization of the spread of infectious
  diseases
• Gene expression (transcription and regulation)
• Protein-protein interactions

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Pros and cons of BLI

• Pros
• In vivo gene expression analysis
• Low background noise
• Longitudinal imaging
• Cons
• Inadvertent genetic alterations
• Attenuation of light through tissue
• O2 (/- ATP, Mg 2) required for Firefly
  luciferase substrate chemical reaction

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Summary

• Bioluminescence is produced from the chemical
  reaction between a substrate and a Luciferase
  enzyme
• BLI is low noise and high sensitivity medium for
  measuring gene expression in vivo
• Disadvantages of BLI include unintended genetic
  alterations and dependence on substrate
  ingredients
• The Xenogen IVIS 50 is a state of the art
  instrument for BLI