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Preclinical imaging with fluorescence in vivo endomicroscopy
The document provides a detailed overview of the Five2 fluorescence in vivo endomicroscopy technology, highlighting its applications in imaging cancer, gastrointestinal, skeletal, and skin tissues with significant advantages over traditional imaging methods. It discusses the optical sectioning capabilities, user-friendly design, and various types of contrast agents used for enhanced imaging results. Key features also include practical demonstrations, limitations, and troubleshooting for effective use in preclinical research.
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Preclinical imaging with fluorescence in vivo endomicroscopy
- 1. Mohammedayaz Rangrez MSc, PhD ProductManager Scintica Instrumentation Phone: +1 (519) 914 5495 mrangrez@scintica.com PRECLINICAL IMAGING WITH FLUORESCENCE IN VIVO ENDOMICROSCOPY
- 2. A PhD degreewith Animal surgeries and microscopy 4 years of teaching experience 16 YouTube e-Learning shows 4+ years of experience with microscopy
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- 4. Compound Microscope FluorescenceMicroscope Confocal Microscope
- 5. CONTENT Background Introduction to the Technology Practical Partsof the instrument and their function Software operation Imaging X Y imaging Optical sectioning Applications Safety and trouble shooting Summary
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- 7. Bright field Darkfield
- 10. Benchtop Microscopy: high resolution,trouble with in vivo imaging, possibilities of artifacts!
- 11. Intravital/Multiphoton Microscopy: high resolution,low flexibility, complicated protocols!
- 12. PET/MRI: in vivoimaging, but no cellular details!
- 13. INVASIVE - -- - MINIMALLY INVASIVE - - - NON INVASIVE nm---µm----RESOLUTION----mm---- In vivo Ex vivo MRI CLSM
- 14. INVASIVE - -- - MINIMALLY INVASIVE - - - NON INVASIVE nm---µm----RESOLUTION----mm---- In vivo Ex vivo MRI CLSM
- 15. RAT BRAIN GLIOBLASTOMA • Invasive! Fluorescence/Confocal Microscope? •Insufficient Resolution! MRI PET? • FIVE - ViewnVivo Laser Confocal Endomicroscopy!
- 16. LCE – FIVE2is a simple user-friendly tool!
- 17. Laser Objective lens Tissue Detector Optical fiber Imaging plane Scan mechanism Ex= 488nm Em > 515 nm FIBER OPTIC CONFOCAL SYSTEM Transformation and Miniaturization
- 18. • Standard Diameter:3.8mm • (1) Short Probe: 46mm Rigid Length • (2) Standard Probe: 66mm Rigid Length • (3) Laparoscopic Probe: 300mm Rigid Length 1 2 3 4mm diameter probe placed in gentle contact with tissue being imaged. MINIATURISED SCANNER CONFIGURATION Contact site is counted as Z = 0
- 19. 19 • The confocalendomicroscope is an “optical sectioning” device (Out of focus material turns black, instead of blurry) • It isolates A thin plane of cells, viewed en face (en face = parallel to tissue surface, unlike typical cut sections) • One of the most important user operations is the control of the imaging plane depth • Imaging depth up to 400um • Tissue dependent THE ‘Z’ DIMENSION Capturing 3D Volumes and Enabling 3D Visualization X Axis Y Axis Z Axis
- 20. 20 EN FACE IMAGINGVS. T.S. HISTOLOGY
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- 22. Image does notshow included 3-way footswitch, or optional items Confocal Processor PC, Monitor, Keyboard and Mouse Probe Animal Stage and Probe Holder Imager Application + Fiji (ImageJ) FIVE2 Components
- 23. Foot pedal facilitatesease of operation
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- 25. Wavelength (nm) 300 400600 700500 #Fluorophores(logscale) 800 600 400 200 0.0 ImagingDepth(μm) 1000 100 10 1 0.0 488 WHY BLUE (488NM) EXCITATION? Optimal Performance and Flexibility
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- 27. VIEWNVIVO DETECTION FILTERS Filter Wh e e l Con ce pt Illu stration *EMPTY Filter Positions Can Be Used For ‘Custom Filters’ Which May Be Specified And Purchased, At An Additional Cost, At The Time Of Purchasing The Viewnvivo System Filter colours and location on the filter wheel are used for illustration purposes only and do not represent the actual filter colour or the detection properties
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- 32. 1. General Non-specificOr Semi-specific Fluorophores • Mainly intercellular compartment 2. Acridines • Nuclear/Acidic Organelle Stains • Acriflavine, Proflavine, Acridine Orange… 3. Cycline Antibiotics • Tetracycline, Doxycycline, more.. 4. Membrane Dyes • Good for nerve tracing • Dii, DiO, 4-Di-2-Asp TYPES OF CONTRAST AGENTS 5. Specific Dyes • Conjugated Peptides • Conjugated Antibodies 6. Functional Dyes • Calcium Markers (G-CAMP, Fluo-3, Fluo-4) • Metabolic Markers • pH Markers • Live/Dead Markers 7. Transgenic Fluorophores • Produced by the living cells when a certain gene is expressed • GFP, Egfp, YFP can be imaged
- 33. Fluorescein Sodium (10mlof 10%): Intravenously InjectedTopically Applied Acriflavine 0.05% Images courtesy Prof Adrian Polglase, Cabrini Monash University Academic Surgical Unit, Melbourne Australia CONTRAST AGENT COMPARISON (COLONIC MUCOSA)
- 34. APPLICATIONS OF FLUORESCENCE INVIVO ENDOMICROSCOPY Cancer Vasculature Gastrointestinal Skeletal Skin
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- 36. Courtesy of DrPhilip Currie Cancer Fibrous Tissue Normal Tissue FIVE2 Images: Instant, easy, live and in vivo! Classical Histology: Tedious prep, slow, possible artifacts and ex vivo!
- 37. Images courtesy of:Martin Goetz and Ralf Kiesslich, Mainz University Hospital, Germany MOUSE BRAIN MICROVASCULAR IMAGING & CELL TRACKING
- 38. DOG STOMACH Image courtesyof Researchers at the University of Melbourne, Faculty of Veterinary and Agricultural Sciences
- 39. Mitotic cell nucleus Ejecting epithelialcell Brush border & mucus layer Images courtesy of Cameron Nowell and researchers at Monash University, Melbourne, Australia
- 41. TUMOUR ANGIOGENESIS INVIVO Images courtesy of: Dr Liem Vo & Peter Anikijenko, Monash University, Melbourne, Australia. Normal Murine Dermal Microvasculature Melanoma-Affected Dermal Microvasculature
- 42. TISSUE ENGINEERING -CARTILAGE Chondrocytes Contrast Agent: Fluorescein Sodium 0.5% in PBS; pH 7.4 fov=500µm fov=~100µm Images courtesy of Curtain University, Perth Western Australia Wu JP, Kirk TB, Zheng MH. Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique. J Orpthop Surg 2008; 3: 29.
- 43. SHEEP CARTILAGE DEFECTMODEL Condyle Contrast Agent: Acriflavine 0.05% Defect area Normal condyle Defect area post ACI Normal condyle Wu JP, Kirk TB, Zheng MH. Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique. J Orpthop Surg 2008; 3: 29. fov=500µm fov=500µm
- 44. IN VIVO RECTALMUCOSAL BARRIER FUNCTION Gracie Vargas, Kathleen Listiak Vincent, Yong Zhu, David Szafron, Tyra Caitlin Brown, Paula Patricia Villarreal, Nigel Bourne, Gregg N. Milligan, Massoud Motamedi. In Vivo Rectal Mucosal Barrier Function Imaging in a Large-Animal Model by Using Confocal Endomicroscopy: Implications for Injury Assessment and Use in HIV Prevention Studies. ASM 2016; 60
- 45. 45 ADVANTAGES OF FIVE2 OVERBUNDLE FIBER TECHNOLOGY
- 46. Scanning mechanism offersfar better resolution compared to Bundle fibre technology! 0.5µ 5µ PSF dimension – FIVE2 Specimen Lens Galvanometer Y scanner Galvanometer X scanner Laser beam
- 47. BUNDLE IMAGING PLANETRANSFER ALLOWS SMALL ANIMAL FLEXIBLE ENDOSCOPY Mouse Colon, Accessed Endoscopically, Probe Diameter = 1mm, Signal Level = 0.08 Mouse Colon, Surgically Exposed, Probe Diameter = 6.3mm, Signal Level = 1.0
- 48. BUNDLE IMAGING PLANETRANSFER ALLOWS SMALL ANIMAL FLEXIBLE ENDOSCOPY No cellular details! Subcellular resolution!
- 49. Bundle Fiber “HD” Image VIEWNVIVOFULL SCREEN TRUE HD
- 50. LIMITATIONS OF FLUORESCENCEIN VIVO ENDOMICROSCOPY
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- 57. Bundle Fiber No cellulardetails! FIVE2 Subcellular resolution! APPLICATIONS OF FLUORESCENCE IN VIVO ENDOMICROSCOPY Cancer Vasculature Gastrointestinal Skeletal Skin
- 58. If you haveany questions related to FIVE2 technology, please reach out to our product manager. Mohammedayaz Rangrez MSc, PhD Product Manager Scintica Instrumentation Phone: +1 (519) 914 5495 mrangrez@scintica.com THANK YOU!