Preclinical Imaging with Fluorescence In Vivo Endomicroscopy
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Laser confocal endomicroscopy (LCE) is the technology that bridges the gap and delivers the best of both worlds. Its confocal principle offers cellular resolution while probe-based endoscopic imaging facilitates real-time in vivo imaging of tissues with minimal invasiveness. The nickname of “virtual histology” is fitting, as it captures real-time microscale images comparable to histology. LCE is a cutting-edge imaging modality with endless possibilities, and numerous research groups are exploring this tool for their preclinical imaging needs. In this live webinar hosted by Scintica Instrumentation, Dr. Mohammedayaz Rangrez provides scientists with theoretical and practical knowledge of the FIVE2 (ViewnVivo) and its preclinical research applications. This webinar features a hands-on demonstration, imaging in different tissue types, Z sectioning and other key capabilities of the system. Participants will also learn about FIVE2 (ViewnVivo) software features, its integration with ImageJ, and 3-D video capture of tissue architecture. Topics discussed in this webinar include: - The advantages of laser confocal endomicroscopy over benchtop confocal microscopy and PET/MRI - Hardware functionality and software operation of the FIVE2 (ViewnVivo) – fluorescence in vivo endomicroscope - How to use the FIVE2 (ViewnVivo) for imaging tissue architecture with cellular resolution and for optical sectioning - How to apply the FIVE2 (ViewnVivo) in preclinical imaging - Troubleshooting and safety tips of the FIVE2 (ViewnVivo)
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Preclinical Imaging with Fluorescence In Vivo Endomicroscopy
- 1. Mohammedayaz Rangrez MSc, PhD Product Manager Scintica Instrumentation Phone: +1 (519) 914 5495 mrangrez@scintica.com PRECLINICAL IMAGING WITH FLUORESCENCE IN VIVO ENDOMICROSCOPY
- 2. A PhD degree with Animal surgeries and microscopy 4 years of teaching experience 16 YouTube e-Learning shows 4+ years of experience with microscopy
- 3. Fluorescent
- 4. Compound Microscope Fluorescence Microscope Confocal Microscope
- 5. CONTENT Background Introduction to the Technology Practical Parts of the instrument and their function Software operation Imaging X Y imaging Optical sectioning Applications Safety and trouble shooting Summary
- 6. BACKGROUND
- 7. Bright field Dark field
- 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 vivo imaging, 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 – FIVE2 is 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 confocal endomicroscope 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 IMAGING VS. T.S. HISTOLOGY
- 22. Image does not show 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 facilitates ease of operation
- 25. Wavelength (nm) 300 400 600 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
- 27. VIEWNVIVO DETECTION FILTERS Filte r 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
- 28. RODENT IMAGING
- 32. 1. General Non-specific Or 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 (10ml of 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 IN VIVO ENDOMICROSCOPY Cancer Vasculature Gastrointestinal Skeletal Skin
- 36. Courtesy of Dr Philip 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 courtesy of Researchers at the University of Melbourne, Faculty of Veterinary and Agricultural Sciences
- 39. Mitotic cell nucleus Ejecting epithelial cell Brush border & mucus layer Images courtesy of Cameron Nowell and researchers at Monash University, Melbourne, Australia
- 41. TUMOUR ANGIOGENESIS IN VIVO 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 DEFECT MODEL 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 RECTAL MUCOSAL 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 OVER BUNDLE FIBER TECHNOLOGY
- 46. Scanning mechanism offers far 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 PLANE TRANSFER 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 PLANE TRANSFER ALLOWS SMALL ANIMAL FLEXIBLE ENDOSCOPY No cellular details! Subcellular resolution!
- 49. Bundle Fiber “HD” Image VIEWNVIVO FULL SCREEN TRUE HD
- 50. LIMITATIONS OF FLUORESCENCE IN VIVO ENDOMICROSCOPY
- 52. TROUBLE SHOOTING, CLEANING AND SAFETY
- 55. SUMMARY
- 57. Bundle Fiber No cellular details! FIVE2 Subcellular resolution! APPLICATIONS OF FLUORESCENCE IN VIVO ENDOMICROSCOPY Cancer Vasculature Gastrointestinal Skeletal Skin
- 58. If you have any 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!