In Vivo Imaging: Xenogens IVIS 200 Series and Living Image Software

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In Vivo Imaging Xenogens IVIS 200 Series and
Living Image Software
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What will be covered?

• Introduction
• Science of In Vivo Imaging
• Xenogen IVIS 200 Series Hardware Overview
• Living Image Software Overview
• Fluorescence System
• Training
• Hands on Training

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Why Optical In Vivo Imaging?

• Powerful labeling technique - gene expression
  results in production of luciferase
• Tracer Applications Amount of light is
  proportional to number of cells
• Functional Applications Light is produced in
  response to a stimulus
• Non-invasive does not require subject to be
  euthanized
• Relatively simple instrumentation. Users can run
  it themselves a lab instrument, not an imaging
  center.

Science
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Tissue is not Transparent - Light Absorbance
Depends on Wavelength
Luciferase Emission Spectra and Tissue
Transmission
Science
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Photons Diffuse Through Tissue and the IVIS
Views this Signal on the Surface of the Subject
CCD

• Light traveling through tissue
• scatters many times creating a "fuzzy" image
  at the surface of the animal
• The IVIS views the diffuse image on the surface
  of the subject

Optics
Bioluminescent Source
Science
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IVIS Imaging System 200 Series Hardware

• Customized for in vivo imaging
• High sensitivity from 300-900 nm
• Large dynamic range
• Living Image software

Hardware
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IVIS 200 Imaging System Hardware
CCD camera
Filter Wheels
Lenses
Heated Sample Stage
Electronics
Chiller and Camera controller
Hardware
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Alignment Light Projector

• Allows rapid and reproducible positioning of
  subjects.
• Size changes with Field of View setting

Hardware
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Living Image Software

• Controls all settings in the IVIS system
• Provides advanced cataloging and browsing tools
• Provides analysis tools
• Instrument settings are analogous to photography
• Images are acquired in a two step process

Software
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Standard Images are Composed of Two
ImagesPhotographic Luminescent Overlay
Software Acquisition
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Camera and Lens Settings are Analogous to Those
Used in Standard Photography
CCD
Shutter

• Field of View (FOV) is dependent on the
  distance from the lens to the sample
• Light collected is proportional to how long the
  shutter is open (exposure time)
• Aperture (f/stop) controls the amount of light
  collected
• CCD resolution is referred to as Binning

Lens
Emission Filters
Aperture (f/stop)
A
B
C
D
E
Field of View
Software
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Setting Sensitivity Luminescent Signal Level

• The IVIS CCD camera has a signal range of 0 to
  65535 Analog to Digital Counts (216).
• Adjust camera settings to obtain a signal level
  of 600 to 60,000 counts.
• Settings that control signal level are
• Exposure time
• Binning (CCD Resolution)
• f/stop (Aperture)

Software
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Living Image Control Panel
Controls Sensitivity
Affects Sensitivity
Software
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Field-of-View
FOV E 25 cm
FOV D 19.5 cm
FOV B 6.5 cm
FOV A 4.0 cm
FOV C 13 cm
Software
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Exposure Time

• Signal level is proportional to
• exposure time
• Shorter exposure time improves throughput.
• Longer exposure time increases signal
• Min exposure time 0.5 second
• Max exposure time 5 minutes

2 sec f/1 small binning 5000 counts peak
10 sec f/1 small binning 25000 counts peak
Software Acquisition
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f/stop (lens aperture)

• f/stop controls the amount of light received by
  the CCD
• f/1 is wide open
• Max light collection, default for luminescent
• f/8 is smallest aperture
• best resolution, default for photo

f/1
f/8
Software Acquisition
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Pixel Binning (CCD Resolution)

• Binning refers to the grouping of pixels into a
  larger super-pixel
• Large Binning High Sensitivity/ Lower
  resolution
• Small Binning High Resolution/ Lower
  Sensitivity

10 seconds f/2
10 seconds f/2
10 seconds f/2
Large Binning
Medium Binning
Small Binning
Software Acquisition
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Summary of Basic Camera Settings
Controls Sensitivity
Affects Sensitivity
Software Acquisition
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Acquisition Single Image
Overlay will automatically take Photo
Luminescent
Single Image Acquisition
Software Acquisition
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Acquisition Sequential mode
Allows automatic acquisition of a seriesof
images separated by fixed time points.
Starts Sequential Image Acquisition
Software Acquisition
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Image Labeling

• Good labeling practices are necessary for
  effective data browsing

Series 3 weeks, 6 weeks Experiment M626 Label
M626-34, M626-61 Comment Dorsal View, Level
B Analysis Comment
Software Cataloging
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Standard Label Sets for Cataloging Images
Software Cataloging
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Image Cataloging Browsing Tools
Software Cataloging
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Regions of Interest Tools

• ROIs available are
• Contour
• Circle
• Square
• Grid

Software Analysis
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Calibrated Physical Units

• Living Image automatically compensates for
  device settings Integration Time, f/stop,
  Binning, and Field of View.
• Calibrated units are Photons per Second,
  representing the flux radiating
  omni-directionally from a user defined region.

2 sec f/2 Small Binning 5000 counts peak 2.82
x 108 photons/sec
10 sec f/2 Small Binning 25000 counts peak 2.82
x 108 photons/sec
Software - Analysis
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Measurement Table

• Measurement Table displays information about
  each
• Region of Interest in the image.
• Measurement Table is user configurable and can
  be
• exported to a spreadsheet

Software - Analysis
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Fluorescence
Fluorophore
Emission Wavelength
Excitation Wavelength
excited state
ground state
Fluorescence
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IVIS Fluorescence System

• Twenty-two position computer
• controlled Emission filter wheel
• Twelve position computer controlled Excitation
  filter wheel
• 150 Watt Tungsten/Halogen lamp
• with computer controlled intensity
• Low Auto Fluorescence optics and fibers

Fluorescence
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Fluorescence Acquisition
Lamp level High / Low
Select Fluorescent Imaging Mode
Select filters
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Locks Excitation and Emission filters together
Fluorescence
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Excitation and Emission Filters
normalized intensity
Fluorescence
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Instrument Background Correction
Uncorrected Image
Corrected Image
Instrument Measurement
Fluorescence
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Fluorescent Calibrated Units Efficiency
Excitation Light Pattern
GFP Well Plate Corrected Efficiency
GFP Well Plate Uncorrected Photons or Counts
Fluorescence
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Animal Diet and Autofluorescence in Control Mice
Regular Rodent Food
Alfalfa Free Rodent Food
Cy5.5
Cy5.5
Fluorescence
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Autofluorescence of Control Mice
Alfalfa Free Rodent Food
Fluorescence
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Autofluorescence Background Excitation Filters
Animal autofluorescence Image using background
excitation filter
Fluorescent Image
Corrected Image
Fluorescence
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Background Excitation Filters

normalized intensity
Fluorescence
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Advanced Topics Diffuse Luminescent Imaging
Tomography (DLITTM)
580 nm
600 nm
620 nm
640 nm
Fluorescence
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For an In Depth Study Software Manual
Software
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What weve covered . . .

• Science
• Light is scattered and absorbed by tissue
  dependant on wavelength of Calibrated physical
  units compensate for device settings
• Hardware
• Custom designed for in vivo bioluminescent
  imaging
• Settings are analogous to photography
• Software
• Images are acquired in a two step process
• Sensitivity is controlled by Integration Time,
  f/stop, and Binning
• Living Image controls IVIS and provides image
  analysis tools
• Fluorescence
• Tissue Autofluorescence and Instrument Background
  can be subtracted

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