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Optical Imaging: Fluorescent and Bioluminescent Techniques

Explore the scientific basis of optical imaging, including basic fluorescent and bioluminescent imaging techniques, penetration depths, and clinical applications. Discover the benefits and advantages of using fluorescent imaging in medical research and surgery.

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Optical Imaging: Fluorescent and Bioluminescent Techniques

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  1. Add for 2016 • methylene blue • More radiology related and less cardiology related examples (GI??) • http://medicalphysicsweb.org/cws/article/research/63518

  2. Optical Imaging FRCR Part 1Scientific Basis of Imaging Mr John Saunderson, consultant medical physicist john.saunderson@hey.nhs.uk, CHH ext (76)1329 8/12/2015

  3. FRCR Part 1 syllabus Scientific Basis of Imaging • 4.22 Optical Imaging • Basic fluorescent imaging • Basic bioluminescent imaging • Difference between different optical imaging techniques • Penetration depths • Clinical applications New subject for 2016 exam!

  4. Basic fluorescent imaging

  5. Fluorescent Imaging • Administer dye • Into vessel to view flow, or • Into tissue for labelling (e.g. tumour) • Illuminate with excitation light • different dyes require different wavelengths • Source may be filtered white light or monochromatic laser • Observe fluorescence produced • may be visible to human eye • may require infrared camera to detect

  6. Fluorescent Imaging

  7. Light Sources

  8. Imagers • Usually CCD camera • May be direct view for surface or intraoperative procedures, or • Via endoscope

  9. Benefits of Fluorescent Imaging • high contrast, • high sensitivity – can see extremely small concentrations • Gives molecular information: makes some (bio) chemistry spatially and temporally visible; • great tools for research: several possible imaging modes, most of which are unique; • cheap: the optical instrumentation and computing needed are quite simple; • easy to use: resembles classical staining. • Advantages over traditional stain include • Can see below surface • Can be useful to take image with and without excitation light. A Review of Indocyanine Green Fluorescent Imaging in Surgery, JT Alander, et al, International Journal of Biomedical Imaging, Volume 2012, Article ID 940585, 26 pages, doi:10.1155/2012/940585

  10. Assessment of perfusion (intraoperative) Endoscopic view https://www.youtube.com/watch?v=SogiHaFg-_E&list=PLO8wpJwEq4tUDg3yXyoQ5BLNr-_PYkaux

  11. Examples of fluorescent dyes • Sodium fluorescein • Indocyanine green (ICG)

  12. Sodium fluorescein • Blue light (490 nm) causes yellow-green light (525 nm) to be emitted • Main use ophthamology - angiography of retina and choroid • Dye injected intravenously • White light from a flash is passed through a blue excitation filter. • Unbound fluorescein fluoresce, emitting yellow-green light • A barrier filter blocks any reflected light so that the images capture only light emitted from the fluorescein. • Images are acquired for ten minutes depending on the pathology being imaged. The images are recorded digitally.

  13. Fluoroscein Accidental Arterial Injection

  14. Indocyanine green (ICG) • Sometimes used in ophthalmology • Growing use in surgery (ICGA = ICG angiography) • Excite with infrared (IR) light of 600-800 nm • Causes IR light of 750-900 nm to be emitted, so must be viewed using an IR camera • Infrared penetrates deeper than blue, so shows deeper lying blood vessels than fluoroscein A Review of Indocyanine Green Fluorescent Imaging in Surgery, JT Alander, et al, International Journal of Biomedical Imaging, Volume 2012, Article ID 940585, 26 pages, doi:10.1155/2012/940585

  15. Indocyanine green angiography - rat

  16. Assessment of perfusion (intraoperative) Endoscopic view https://www.youtube.com/watch?v=SogiHaFg-_E&list=PLO8wpJwEq4tUDg3yXyoQ5BLNr-_PYkaux

  17. Intraoperative fluorescence angiographyfor the evaluation of coronary artery bypass graft patency • allows confirmation of the location of the coronary arteries and assessment of bypass graft function during coronary artery bypass procedures. • System - a video camera and a laser diode • camera guided by a range-detector diode, is positioned a safe distance above the heart. • A small amount of ICG dye is then administered as a central venous injection. • This dye fluoresces when illuminated using laser energy and the images are recorded digitally.

  18. Intraoperative Fluorescence Imaging System for On-Site Assessment of Off-Pump Coronary Artery Bypass Graft, K Waseda et al, JACC: Cardiovascular Imaging, vol 2 , no. 5, 2009

  19. Molecular guided surgery Zhu B, Sevick-Muraca EM. A review of performance of near-infrared fluorescence imaging devices used in clinical studies. Br J Radiol 2015;88: 20140547.

  20. Autofluorescence • No dye used • Normal and tumour tissue which look the same under white light, may fluoresce differently under monochromatic light • e.g. blue light causes endobronchial lung cancer to glow green (tumour cells have less flavins, which absorb green light)

  21. Figure 1. A, Autofluorescence imaging–negative (green) hyperplastic polyp. B, Autofluorescence imaging–positive (dark purple) adenoma. ASGE Technology Committee, Report on Emerging Technology - Autofluorescence imaging, Gastrointestinal Endoscopy Volume 73, No. 4 : 2011

  22. Basic bioluminescent imaging

  23. Basic bioluminescent imaging • A non-invasive imaging modality widely used in the field of pre-clinical oncology research. • Main chemical used is firefly luciferase • Chemical reaction - excitation light not needed • luciferin + ATP → luciferyl adenylate + PPi • luciferyl adenylate + O2 → oxyluciferin + AMP + light • Low amounts of light emitted • Too low for direct viewing • Sensitive camera needed • The luciferase gene can be cloned and spliced into target DNA of specific cells.

  24. Penetration depths

  25. > 1400nm Infrared B 740-1400 IRA 740-620 Red 620-585 Orange 585-575 Yellow 575-500 - Green 500-400 Blue/indigo/violet 400-315 Ultraviolet A 314-280 UVB 180-280 UVC i.e. infrared penetrates deeper, etc.

  26. Optical Radiation Safety(Not part of FRCR syllabus) Control of Artificial Optical Radiation at Work Regulations 2010 • Where there is a reasonably foreseeable risk to eyes or skin from optical radiation • Risk Assessment • Assess whether Exposure Limit Values (ELV) exceeded

  27. Laser safety classes • Class 1 – safety • Class 2 – blink reflex protects • Class 3R - blink reflex probably protects • Class 3B – direct beam or via mirror surface can damage eyes • Class 4 – even reflected beam from a matt surface could damage eyes. Might damage skin. Might cause fire. MUST HAVE THE CORRECT EYEWEAR FOR PARTICULAR LASER DEVICE BEING USED MHRA Guidance: Lasers, intense light source systems and LEDs, 2015 https://www.gov.uk/government/publications/guidance-on-the-safe-use-of-lasers-intense-light-source-systems-and-leds

  28. Possible references • An introduction to molecular imaging in radiation oncology: A report by the AAPM Working Group on Molecular Imaging in Radiation Oncology (WGMIR) http://www.aapm.org/pubs/reports/RPT_255.pdf • Optical Biopsy: A New Frontier in Endoscopic Detection and Diagnosis http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2169359/ • (Note, both above printed out and put in my filing cabinet under FRCR

  29. End

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