In-Vivo Imaging: IVIS Lumina XR. William R. Anderson IVIS Product Specialist

Transcription

1 In-Vivo Imaging: IVIS Lumina XR William R. Anderson IVIS Product Specialist 1

2 What will be covered? Introduction Principles of optical In Vivo Imaging Key IVIS Hardware components Overview of Living Image Software Fluorescence & X-ray options Training Hands on Training 2

3 Why Optical In Vivo Imaging? Powerful labeling technique - gene expression results in production of luciferase Amount of light is proportional to number of active live cells Typical applications range from oncology studies, infectious disease (tracer) to imaging transgenic animals (functional) Non-invasive does not require subject to be euthanized Relatively simple instrumentation. Science 3

4 The Value of X-Ray + Optical Imaging X-Ray Provides a Fixed Anatomical Reference The Question: Where is the source origin relative to the surface signal? The Problem: Tissue attenuation/ scattering makes 2D optical signals difficult to locate at a defined location. The Solution: A co-registered X-ray image provides a fixed anatomical reference, defining skeletal structure and soft tissue organs and enabling better localization of the optical signal. 4

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7 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 CCD views the diffuse image on the surface of the subject Bioluminescent Source Science 7

8 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 8

9 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 9

10 X-ray vs Visible Light X-ray photons carry more energy than visible light which can penetrate thicker tissue X-rays which penetrate tissue are typically absorbed or scattered Energy, density, thickness and material type effect X-ray penetration Light appears on the tissue surface be means of the shortest path, X-rays penetrate through Bioluminescent Source 95% X-rays Out 85% 90% 50% 40% 35% 35% 100% 100% X-rays In Surface Radiance Science 10

11 How an X-Ray Image is Acquired CCD Optics X-rays will be attenuated in tissue differently resulting in an image on the scintillator The CCD views the scintillator resulting in a planar X-ray image Scintillator Stage X-ray and Optical images have different path lengths. To correct this geometrical difference, the X-ray image is registered to the optical image X-ray Source Close Up of Scintillator in Position Science 11

12 IVIS Lumina XR Hardware Customized for in-vivo imaging High sensitivity from nm Large dynamic range Hardware 12

13 The IVIS Lumina XR Imaging Chamber CCD Camera Cooled -90C Interchangeable Emission Filter Wheel f/1 Lens & Aperture Cesium Iodide (CsI(Tl)) scintillator Heated Sample Stage Light-tight and shielded imaging chamber Automated aluminum filter X-Ray Source (40kVp,100mA) Tungsten Anode Hardware 13

14 Living Image Software Controls all settings in the IVIS system (fully computer controlled) Provides advanced cataloging and browsing tools Provides analysis tools for quantification Instrument settings are analogous to photography Images are acquired in a multi-step process Software 14

15 Images are Composed of multiple layers: Software Acquisition 15

16 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 Light collected is proportional to how long the shutter is open (exposure time) Aperture (f/stop) controls the amount of light collected Digital pixel binning possible with CCD - for further increase in sensitivity Field of View A CCD Shutter Lens Aperture (f/stop) Emission Filters B C D Software Acquisition 16

17 Field-of-View (typical) FOV D = 12.5 x 12.5 cm FOV C = 10 x 10 cm Click # XQA Series: Fri, Sep 10, :08:43 Experiment: Bin:M (2), FOV12, f16, 0.2s Label: Comment: Camera: IVIS 13197, DW434 Analysis Comment: Filter: Open FOV B = 7.5 x 7.5 cm Click # XQA Fri, Sep 10, :10:04 Bin:M (2), FOV10, f16, 0.2s Filter: Open Camera: IVIS 13197, DW434 Series: Experiment: Label: Comment: Analysis Comment: FOV A = 5 x 5 cm Click # XQA Fri, Sep 10, :11:40 Bin:M (2), FOV7, f16, 0.2s Filter: Open Camera: IVIS 13197, DW434 Series: Experiment: Label: Comment: Analysis Comment: Click # XQA Fri, Sep 10, :12:40 Bin:M (2), FOV4, f16, 0.2s Filter: Open Camera: IVIS 13197, DW434 Series: Experiment: Label: Comment: Analysis Comment: Software Acquisition 17

18 Field of View (X-ray) FOV C 10 x 10 cm FOV B 7.5 x 7.5 cm FOV A 5 x 5 cm Software Acquisition 18

19 Setting Sensitivity Luminescent Signal Level The IVIS CCD camera has a raw signal range of 0 to 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 19

20 Living Image Control Panel Controls Sensitivity Affects Sensitivity Software Acquisition 20

21 Signal level is directly proportional to exposure time Exposure Time Shorter exposure time improves throughput. {Recommended min exposure time > 0.5 secs} Longer exposure time increases signal {Recommended max exposure time < 5 mins} 2 sec f/1 small binning ~5000 counts peak 10 sec f/1 small binning ~25000 counts peak Software Acquisition 21

22 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/stop (Lens Aperture) f/1 f/16 Software Acquisition 22

23 Binning refers to the grouping of pixels into a larger super-pixel Pixel Binning (CCD Resolution) Large Binning (8) Higher Sensitivity/ Lower Resolution Medium Binning (4) Small Binning (2) Higher Resolution / Lower Sensitivity 10 seconds f/2 Large Binning 10 seconds f/2 Medium Binning 10 seconds f/2 Small Binning Software Acquisition 23

24 Auto-exposure feature available for bioluminescence / fluorescence Autoexposure User definable autoexposure settings Software Acquisition 24

25 dp/ded X-ray parameters X-ray Dose vs FOV (and exposure time) Binning controls resolution Two Energy Settings: Animal: Tuned for live animal imaging, filter in place to reduce dose Specimen: Tuned for thin tissue samples, filter out to increase contrast Dose (Gray): Amount if ionizing radiation absorbed into tissue Average Dose: 4.4 mgray/image 2.0x x x x X-ray Emission Spectrum Energy (kev) Unfiltered Al Filter Software Acquisition 25

26 Summary of Basic Camera Settings Controls Sensitivity Affects Sensitivity Software Acquisition 26

27 Acquisition Single Image Overlay will automatically take Photo + Luminescent Single Image Acquisition Software Acquisition 27

28 Acquisition Sequential mode Allows automatic acquisition of a series of images separated by fixed time points. Starts Sequential Image Acquisition User Friendly Sequence Editor Software Acquisition 28

29 Imaging Wizard Select for assistance in setting up bioluminescence or fluorescence sequences 29

30 Image Labeling Good labeling practices are necessary for effective data browsing Software Cataloging 30

31 Image Cataloging & Browsing Tools User defined information Preview window Select multiple images from several days and load as group for simultaneous analysis Software Cataloging 31

32 Quantification Tool palette for adjusting scale/opacity etc. Region of interest (ROI) tools to measure surface intensities Software - Analysis 32

33 Regions of Interest Tools ROI shapes available: Square Circle Contour Grid ROI s can be created: Manually Automatically Free Draw Important to be consistent with ROI selections Software Analysis 33

34 Measurement Table Measurement table displays information about each ROI Table is user configurable and can be exported to a spreadsheet Software - Analysis 34

35 Calibrated Physical Units Living Image automatically compensates for device settings: Exposure 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 exposure, f/stop 1, Small binning ~5000 counts peak 2.82 x 10 8 photons/sec 10 sec exposure, f/stop 1, Small binning ~25000 counts peak 2.82 x 10 8 photons/sec Software - Analysis 35

36 Calibrated Physical Units vs Raw Signal - Example Raw Signal (Counts) Exp time: 30 sec 30 sec 60 sec 60 sec 60 sec 60 sec Binning: small small small small medium medium Day: Peak Counts Software - Analysis 36

37 Calibrated Physical Units vs Raw Signal- Example Calibrated Signal (Photons per second) Exp time: 30 sec 30 sec 60 sec 60 sec 60 sec 60 sec Binning: small small small small medium medium Day: Radiance: Photons per second Software - Analysis 37

38 Counts X-Ray Units Relative Absorption Correction Living Image automatically compensates for X-ray settings: Integration Time, f/stop, Binning, and Field of View X-Ray Absorption correction improves contrast and density correlation Raw Image - I raw Absorption Image - I abs Average intensities of the same mouse bone tissue acquired at various FOV, binning, f-stop and exposure time. The raw images showed significant variation due to changes in instrument settings, but the converted absorption images reported nearly constant measurements. 2.0x x10 4 Raw Absorption 1.0x x Instrument Settings Software Analysis 38

39 Fluorescence Fluorophore Excitation Wavelength excited state Emission Wavelength ground state Fluorescence 39

40 IVIS Lumina s Fluorescence Components Fully computer controlled Emission filter wheel (user changeable) Twelve position Excitation filter wheel Low Auto-Fluorescence optics and fibers 150 Watt Tungsten/Halogen lamp with computer controlled intensity Fluorescence 40

41 Transmission % % Fluorescence Filters Excitation Filters Standard Emission Filter Choices Wavelength (nm) Filter nm Bandwidth Standard Filter Set 4 Standard Emission Filters Low (500 Series)/ Mid (600 Series) 7 Narrow Band Pass Emission Filters Spectral unmixing and planar spectral imaging Mid-High/ High (700 Series) 7 Narrow Band Pass Emission Filters Spectral unmixing Optimal for NIR imaging 41

42 Normalized Intensity Standard Filter Set For Imaging Multiple Reporters 4 Standard Emission Filters ICG nm QD800, AlexaFluor 750, DiR, IRDye800CW nm nm Cy nm AlexaFluor 680 and 700, Cy nm nm DsRed nm mcherry, mtomato, DsRed, QD605, TurboFP nm nm GFP nm GFP, EGFP, FITC nm nm normalized transmission of light through 1cm of tissue Wavelength (nm) 42

43 Filter FITC GFP YFP RFP/DsRed mtomato QD605 AF595 mcherry XenoLight 680 Osteosense 680 Cy5.5 XenoLight 750 MMPSense 750 Cy7 IRDye 800CW Xenolight DIR QD805 ICG High Efficiency Narrow Bandwidth Filter Wheel Packages RFP/DsRed mtomato QD605 AF595 mcherry mplum TurboFP635 QD705 mplum TurboFP635 XenoLight 680 Osteosense 680 XenoLight 750 MMPSense 750 Cy7 IRDye 800CW Xenolight DIR QD805 43

44 Fluorescence Acquisition Select Fluorescent Imaging Mode Lamp level High / Low Select filters Fluorescence 44

45 Fluorescence Acquisition Select Fluorescent Imaging Mode Lamp level High / Low Select filters Fluorescence 45

46 Fluorescent Calibrated Units: Radiant Efficiency Radiant Efficiency = Emission Light (photons/sec/cm 2 /str) Excitation Light (mw/cm 2 ) Fluorescence 46

47 Counts Radiant Efficiency Fluorescent Calibrated Units: Radiant Efficiency Excitation Light Pattern Units of Radiant Efficiency compensates for non-uniform excitation light pattern GFP Well Plate Uncorrected GFP Well Plate Corrected vs. 47

48 Auto-fluorescence of Control Mice l = 550nm l = 610nm l = 740nm l = 840nm Fluorescence 48

49 Animal Diet Auto-fluorescence in Control Mice Regular Rodent Food Alfalfa Free Rodent Food l = 740nm l = 740nm Fluorescence 49

50 Auto-fluorescence Subtraction Image math Filters set to target the background auto-fluorescence (lower l) Filters optimized to target the signal (+ background noise) Fluorescence 50

51 Spectral Un-mixing Raw Spectral Images - 605nm excitation filter Unmixed AutoFl Unmixed XF680 Unmixed XF750 Subcutaneous injection of molecules of XenoFluor 680 (scruff) + Subcutaneous injection of molecules of XenoFluor 750 (lower dorsal region Fluorescence 51

52 For an In Depth Study Living Image Software Manual IVIS University 52

53 IVIS Bioware and Reagents Bioware Bioware Ultra Bioware Ultra Red Suzen O Coin (508) suzen.ocoin@caliperls.com NIR Fluorescent Reagents 680, 750, 770nm Protein Labeling Kits DiR D-Luciferin Substrate RediJect D-Luciferin RediJect D-Luciferin Ultra 53

54 Summary Imaging principles Light is scattered and absorbed by tissue - dependant on wavelength and depth Calibrated physical units compensate for device settings Hardware Custom designed for in-vivo bioluminescent & fluorescent imaging Settings are analogous to photography Software Living Image used for acquisition and analysis Images are acquired in a two step process Sensitivity is controlled by Exposure time, f/stop and binning Fluorescence Tissue and Instrument Auto-fluorescence can be subtracted 54

55 Questions please 55