Multiple application

Transcription

1 Multiple application

2 Protein-protein Interaction Rapamycin-Induced Association of FRB and FKBP PNAS Piwnica-Worms group, 2004

3 Imaging GFP virus Infection on Plant

4 Image Min = e+07 Max = e+08 p/sec/cm^2/sr x Color Bar Min = e+07 Max = e x10 9 Click # XIC Fri, Sep 29, :19:49 Bin:HR (2), FOV5, f1, 30s Filter: Open Camera: IVIS 11221, DW434 Series: WellPlate Checkout Experiment: final checkout Label: Comment: Analysis Comment: bkg sub flat-fielded cosmic x Color Bar Min = e+08 Max = e Click # XIC_N _001 Series: WellPlate Checkout 2006 ~9 ë28 é 00:33:14 Experiment: final checkout Level=High, XIC _001 Em=GFP, Ex=GFP - XIC _002 Label: Bin:HR (2), FOV10, f2, 15s Comment: Camera: IVIS 11221, DW434 Analysis Comment: Color Bar Min = e+07 Max = e+08

5 Evaluating Therapeutics in a Zebrafish Tumor Xenograft Model Bioluminescence Fluorescence Zhao et al, 2009

6

7 Lui et al 2010 Common Radionuclides for Molecular Imaging

8 IVIS Imaging of Radionuclide Probes Positron emitters (eg 18 F) and Beta emitters (eg 131 I) routinely used in clinic Positron and electron emitting radionuclides can produce this light detectable with IVIS system 18 F-FDG tumor imaging Čerenkov Radiation Liu et al (2010) PLoS One 5 e9470

9 XenoLight D-Luciferin Your customers need Luciferin to monitor bioluminescence- so why shouldn t they buy XenoLight Luciferin from Caliper XenoLight Luciferin is: The ONLY Luciferin optimized for in vivo imaging with IVIS systems Validated on our Bioware and Bioware Ultra Cell Lines Used by Caliper scientists to calibrate IVIS Imaging Systems Supplied under stringent lot control to ensure experimental reproducibility Knowledgeable technical support for in depth biology Image on the left shows metastases by PC - 3M cells in an intracardiac model. Image on the top shows single cell imaging in vivo with our ultra bright 4T1 - luc2 cells. All data were generated using our D-Luciferin Buy the Luciferin that allowed us to image a single cell in vivo #6187 許先生

10

11

12

13

14

15 2-DeoxyGlucosone (DG)-750 is a fluorescent probe for in vivo targeting of tumors that typically exhibit elevated glucose uptake rate in comparison to surrounding tissues.

16

17 Light Producing Transgenic Animal LPTA Luciferase Animal Model Animal Model Application Ho-1 Oxidative Stress Cyp450 Drug Metabolism inos Epx Sepsis Asthma IL-2 Innate immunity Kap VEGF Androgen Angiogenesis Ins2 Kdr Diabetes Wound healing

18 Stem Cell/Adoptive Transfer Tracking stem cells Engraftment Differentiation Transplant biology Donor cells from LPTAs β-actin-luc GAPDH-luc Reporter specific (e.g., Rip-luc) Tracking immune response Lymphocytes Eosinophils Neutrophils

19 Fluorescent Imaging Agents and Tags

20 Targeting hydroxyapatite, a biomarker for osteoblast activity, is an essential component of normal bone

21 Integrin 750 Probe binds to avb3 integrin on the cell surface which are abundant in tumor masses

22 Where can I find datasheets on the website

23 Where can I find all datasheets on the website

24 IVIS Spectrum Imaging System - Hardware Customized for in vivo imaging Highly sensitive camera with a large dynamic range

25 Standard Images are Composed of Two Images Photographic + Luminescent = Overlay

26 Alignment Light Projector Allows rapid and reproducible positioning of subjects. Size changes with Field of View setting

27 Field-of-View (typical) FOV D: 21.5 cm FOV C: 12.5 cm 1.5x 2.5x FOV B: 6.5 cm FOV A: 4.0 cm 5x 8.7x

28 Living Image Control Panel Controls Sensitivity

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

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

31 Image Cataloging & Browsing Tools User defined information

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

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

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

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

36 Fluorescence Fluorophore Excitation Wavelength excited state Emission Wavelength ground state

37 IVIS Fluorescence Imaging 18 Emission filters (computer controlled) 10 Excitation filters (computer controlled) 150 Watt Tungsten/Halogen lamp (computer controlled intensity) Low Auto Fluorescence optics and fibers Emission filter wheel Excitation filter wheel Optical switch Trans-illumination Light source

38 Transmission % Excitation and Emission Filters Excitation filters 35 nm bandwidth nm nm nm nm Wavelength (nm) nm nm nm nm 18 Emission filters 20 nm bandwidth

39

40 Transillumination Background removal,deep signal detection,flit

41 Normalized Intensity Use transillumination to remove background Left kidney implanted with 1x10 15 XenoFluor 680 Pillow EPI TRANS 100 Excitation Emission Ex=620 Em= Wavelength (nm) S/N=1.10 S/N=110

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

43 What is Spectral Unmixing? Calculates concentrations of different fluorescent components Requires images acquired at multiple wavelengths to perform the spectral analysis Emission or excitation scan Quantitative and qualitative results

44 In vitro Spectral Unmixing Example: Dyes in a Dish XF 680 XF 750 Top and bottom: XF680 and XF750 mixture: 1:1 Left middle: XF680 only Right middle: XF750 only 1 x10 14 molecules per spot Ex640, Em nm Composite

45 Image Overlay Capabilities Bioluminescent Tumor DHE Image Overlay

46 3D tomography for bioluminescence Spectral measurements provide information on depth of source In vitro ROI data, 20 nm bandpass filters Subcutaneous Chest Cavity

47 3D Tomography (Luminescence) Spectral scans with Emission filters allows for absolute quantification and source localization

48 3D tomography for fluorescence Select trans-illumination points Highlight multiple locations of interest and add to the image sequence Spatial scans with transillumination light source allows for absolute quantification and source localization 106

49 3D Tomography (Fluorescence) Spatial scans with trans-illumination light source allows for absolute quantification and source localization

50 In vivo dual modality tomography Fluorescence Imaging Tomography - FLIT Bioluminescence Imaging Tomography - DLIT Mouse Left Tumor Depth [mm] Mouse Right Tumor Depth [mm] DLIT FLIT

51 Utilize Well Plate Quantification to Determine Cell Number or pmol of Reporter Dilute your cells or dye and image Select Well Plate Quantification from Tools menu Choose library when reconstructing Enter cell number or concentration per well Save as a library

52 Utilize Well Plate Quantification to Determine Cell Number or pmol of Reporter 2D 5.44e5 cells vs e8 photons/sec 3D vs. 2.93e4 cells 1.79e7 photons/sec

53 Automatic Mouse Atlas Registration in LI4.0 Unregistered Coregistered

54 IVIS Spectrum CT Modalities uct With Co-Registration 2 mice simultaneously Low Scan time: s Low Dose: ~ mgy FOV: Up to 12x12x3 cm Voxel size: µm GPU Based Image Reconstruction Automatic Registration

55 IVIS Spectrum CT Configuration Turntable approach: Compact CT system minimally interferes with optical imaging 3072 x 864 pixels 75 m pixel pitch 50kV, 1 ma

56 Range of Resolutions on Spectrum CT Coarse Standard Medium Fine

57 Spectrum CT Operating Modes Mode Name Voxel Size (um) Est. Resolution (um) FOV LxWxH (cm) Filter # Views Binning Est. Dose (mgy) Total Time (s)* Fast x12x3 440 Al Standard, 1 Mouse x12x3 440 Al Standard, 2 Mouse x12x3 120 Cu Medium Res x6x3 440 Al High Res x2.4x2 120 Cu *Total Time: Times are approximate and include overhead, acquisition and reconstruction time. + High Res: One mode is available for the bottom 2cm of the FOV and another mode is available for the top of the FOV.

58 Reconstruction of Osteolytic Lesions MB-231-luc breast cancer cell line Intracardiac injection 1 x 10 6 cells

59 Reconstruction of Lung Cancer HD460-luc lung cancer cell line Intrathoracic injection of 1 x 10 6 cells suspended in Matrigel

60 Bacterial Targeting of Tumors Brevibacterium Delivered Orally to Mice with HTC116 Tumors HTC116 Cancer Signal Brevibacterium Bacterial Signal Dual Cancer/Bacterial Signal Mark Tangney and Cormac Gahan, University of Cork

61 BRAIN TARGETING NANODRUG 3D Fluorescent Imaging (FLIT) Brain Probe 15min Post Injection Note: skeleton added from mouse atlas Dr. Chen Jiang, Fudan University School of Pharmacy. Unpublished data

62 3D Reconstruction of Bioluminescent Glioma

63 Fat Imaging Tally-ho obese mice

64 Contrast Imaging Visipaque

65 Flux (Photons/second) 4D LONGITUDINAL IMAGING Day17 Day21 Day24 Day31 3D BLI Quantification of Metastases in Leg Joints 1E+09 1E+08 1E+07 1E+06 Right Left Days Post Injecion of Cells Experimental Details MB231 cells Intracardiac Injection DLIT - Luciferase CT Image Settings 236 m voxel 26 mgy Courtesy of Heng Xu & Ning Zhang

66 Co-Registration of BLI and MRI in Prion Infected GFAP-luc Mouse

67 Thanks for your attention