Xenogen IVIS Lumina Introductory In-Vivo Imaging Training Will Hauser Field Service Engineer
What Will Be Covered Introduction Science of in vivo Imaging Xenogen IVIS Lumina Series Hardware Overview Living Image Software Overview Fluorescence System Training Hands on Training
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 themselves lab instrument, not imaging center Science
Tissue is not Transparent - Light Absorbance Depends on Wavelength Luciferase Emission Spectra and Tissue Transmission Relative Transmission 1.0 0.8 0.6 0.4 0.2 0 400 500 600 700 800 λ nm Science Renilla Bacteria Firefly Transmission of light through 1 cm of tissue
Tissue is not Transparent Light Absorbance Depends on Wavelength Tissue window λ nm
Photons Diffuse Through Tissue and the IVIS Views this Signal on the Surface of the Subject CCD Optics 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 Bioluminescent Source Science
Photons Diffuse Through Tissue and the IVIS Views this Signal on the Surface of the Subject CCD Optics 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 Bioluminescent Source Science
Photons Diffuse Through Tissue and the IVIS Views this Signal on the Surface of the Subject CCD Optics 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 Bioluminescent Source Science
IVIS Imaging System Lumina Series Hardware Customized for in vivo imaging High sensitivity from 300-900 nm Large dynamic range Living Image software Hardware
The IVIS Imaging Chamber Camera Cover Optical Filter Wheel CCD Array f/1 Lens & Aperture Shutter LED Illuminators Heated Sample Stage Light-tight Enclosure Electronics Tray Hardware
Living Image Software Controls all settings in the IVIS Imaging 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
Standard Images are Composed of Two Images Photographic + Luminescent = Overlay Software Acquisition
Camera and Lens Settings are Analogous to Those Used in Standard Photography Field of View (FOV) is dependent on the distance from the lens to the sample CCD Shutter Lens Aperture (f/stop) Emission Filters Light collected is proportional to the time the shutter is open (exposure time) Aperture (f/stop) controls amount of light collected and depth-of-field CCD Binning is the resolution setting A B C Field of View D Software Acquisition
Field of View FOV D = 12.5 x 12.5 cm FOV C = 10 x 10 cm FOV B = 7.5 x 7.5 cm FOV A = 5 x 5 cm Click # XQA20040910141240 Software Acquisition Series:
Setting Sensitivity Luminescent Signal Level The IVIS CCD camera has a raw signal range of 0 to 65535 Analog to Digital Counts (2 16 ). 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) Instrument is calibrated to automatically compensate for changes in sensitivity settings Software
System Control Panel Controls Sensitivity Affects Sensitivity Software Acquisition
Exposure Time Luminescent 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
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/16 is smallest aperture Best resolution, default for photo f/1 f/16 Software Acquisition
Binning refers to the grouping of pixels into a larger super-pixel Large Binning = 16 High Sensitivity/ Lower Resolution Medium Binning = 8 Small Binning = 4 High Resolution / Lower Sensitivity 10 seconds f/2 Large Binning Pixel Binning (CCD Resolution) 10 seconds f/2 10 seconds f/2 10 seconds f/2 Medium Binning Small Binning Software Acquisition
Summary of Basic Camera Settings Controls Sensitivity Affects Sensitivity Software Acquisition
Acquisition Single Image Overlay will automatically take Photo + Luminescent Single Image Acquisition Software Acquisition
Acquisition Sequential mode Allows automatic acquisition of a series of images separated by fixed time points. Starts Sequential Image Acquisition Easy to use Sequence Editor Software Acquisition
Image Labeling Good labeling practices are necessary for effective data browsing Software Cataloging
Image Cataloging & Browsing Tools Software Cataloging
Regions of Interest Tools ROI shapes available: Contour Circle Square Grid ROI s can be created: Manually Automatically Free Draw
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 10 8 photons/sec 10 sec f/2 Small Binning ~25000 counts peak 2.82 x 10 8 photons/sec Software Analysis
Calibrated Physical Units vs. Raw Signal - Example Raw Signal in Counts Time: 30 sec 30 sec 60 sec 60 sec 60 sec 60 sec Binning: small small small small medium medium Day Number: 1 2 3 4 5 6 Peak Counts 1600 1200 800 400 Software - Analysis
Calibrated Physical Units vs. Raw Signal - Example Calibrated Signal in Photons per second Time: 30 sec 30 sec 60 sec 60 sec 60 sec 60 sec Binning: small small small small medium medium Day Number: 1 2 3 4 5 6 Photons per second Software - Analysis
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
Fluorescence Process Fluorophore Excitation Light excited state Up Shifted Emission Light ground state Fluorescence
IVIS Lumina s Fluorescence Components Fully computer controlled Eight position Emission filter wheel Twelve position Excitation filter wheel Low Auto Fluorescence optics and fiber optics 150 Watt Tungsten/Halogen lamp with computer controlled intensity Fluorescence
Fluorescence Acquisition Select Fluorescent Imaging Mode Lamp level High / Low Select filters 7
Emission Spectra of Common Fluorophores Excitation Emission Tissue window
Excitation and Emission Filters 1 ICG 0.1 710-760 nm 810-875 nm normalized intensity 0.11 normalized intensity 0.1 0.11 0.1 1.0 Cy 5.5 DsRed 500-550 nm 575-650 nm 615-665 nm 695-770 nm 0.1 GFP 445-490 nm 515-575 nm normalized transmission of light through 1cm of tissue 400 500 600 700 800 wavelength / nm Fluorescence
Excitation and Emission Filters 100 10 excitation filters 30nm bandwidth Transmission % 80 60 40 20 710-760 nm 810-875 nm 615 0-665 nm 695-770 nm 400 440 480 520 560 600 640 680 720 760 Wavelength (nm) 500-550 nm 575-650 nm 445-490 nm 515-575 nm 7 Red emission filters 20 nm bandwidth 720nm 840 nm
Fluorescent Calibrated Units: Efficiency Efficiency (%) = Emission Light (photons/sec) Excitation Light (photons/sec) Fluorescence
Fluorescent Calibrated Units: Efficiency Excitation Light Pattern Units of Efficiency compensates for non-uniform excitation light pattern GFP Well Plate Corrected: Efficiency GFP Well Plate Uncorrected: Photons or Counts Fluorescence
Autofluorescence of Control Mice Alfalfa Free Rodent Food GFP DsRed Cy5.5 ICG Fluorescence
Animal Diet and Autofluorescence in Control Mice Regular Rodent Food Alfalfa Free Rodent Food Cy5.5 Cy5.5 Fluorescence
Adaptive Fluorescence Background Subtraction (Filters/Corrections Palette) Mask setup include just the animal No Subtraction Adaptive Subtraction
Autofluorescence Background Excitation Filters Fluorescent Image Animal autofluorescence image using background excitation filter Corrected Image Fluorescence
Background Excitation Filters normalized intensity normalized intensity 1 0.1 1 0.1 1 0.1 ICG BkG, ICG Cy 5.5 BkG, Cy 5.5 DsRed Bkg, DsRed 460-490 500-550 nm 580-610 575-650 nm 615-665 nm 665-695 710-760 nm 695-770 nm 810-875 nm 1 GFPBkg, GFP 0.1 410-440 445-490 nm 515-575 nm normalized transmission of light through 1cm of tissue 400 500 600 700 800 900 wavelength / nm Fluorescence
For an In Depth Study Software Manual Software
What we ve covered... Science Light is scattered and absorbed by tissue dependent on wavelength of light 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 tool Fluorescence Tissue Autofluorescence and Instrument Background can be subtracted www.caliperls.com
Technical Support (508)435-9761 Tech.Support@CaliperLS.com Will Hauser (508)259-7931 Will.Hauser@CaliperLS.com