Tips and Tricks for Preclinical Optical Imaging
•Download as PPTX, PDF•
0 likes•69 views
Learning Objectives: How to add optical imaging capabilities to your lab How to detect very few cells How to differentiate multiple signals within the same animal How to properly acquire and analyze data How to do ex vivo imaging…and much more In this webinar, Katie will answer the most frequently asked questions in the preclinical optical imaging field. Whether you are just exploring the idea of adding optical capabilities to your lab or you’ve been imaging for years, you won’t want to miss the opportunity to add some of these tricks to your arsenal. Everything from substrates to image analysis will be discussed!
Recommended
More Related Content
Similar to Tips and Tricks for Preclinical Optical Imaging
Similar to Tips and Tricks for Preclinical Optical Imaging (20)
More from Scintica Instrumentation
More from Scintica Instrumentation (20)
Recently uploaded
Recently uploaded (20)
Tips and Tricks for Preclinical Optical Imaging
- 1. Tips & Tricks of Pre-clinical Optical Imaging Katie Parkins, PhD Applications Scientist kparkins@scintica.com
- 2. WWW.SCINTICA.COM Topics of discussion 2 Review of preclinical optical imaging and applications How to add optical imaging to your lab Considerations when designing your experiments Getting the most out of your imaging data
- 3. Review of optical imaging
- 4. WWW.SCINTICA.COM How does optical imaging work? 4 Fluorescence Imaging Bioluminescence Imaging James ML, Gambhir SS. Physiological reviews. 2012
- 5. WWW.SCINTICA.COM Applications of optical imaging 5 • Oncology • Neurology • Biodistribution studies • Treatment studies (BLI) • Cell tracking • Ex vivo imaging
- 6. How to add optical imaging to your lab
- 7. WWW.SCINTICA.COM Do I need to be an imaging expert to incorporate optical imaging into my lab? 7 What do I need? • Lab bench allowing animal work • Imaging substrate • Cells expressing relevant reporter OR optical dyes
- 8. WWW.SCINTICA.COM Getting started 8 • X-Ray/CT adds infrastructure/safety considerations • Start with fluorescence or Fluc BLI • Start with premade substrate (i.e. RediJect) to limit variability • Order cell lines that are already stably expressing your reporter gene • X-Ray/CT complementary? • Many different reporter genes to choose from • Many options to buy premade or buy in powder form; new substrates made for enhanced sensitivity • Can engineer new cell lines in house with gene of interest or source out to companies New to optical Comfortable with optical
- 9. WWW.SCINTICA.COM Do I need to have the expertise and infrastructure to engineer cells? 9 • ATCC • Biogene • Gentarget Inc • Biocytogen • Promega • Many more…
- 10. Considerations when designing your experiments
- 11. WWW.SCINTICA.COM How long do I need to image for? 11 • Always run a pilot study if possible to determine kinetics • Timeline will differ between disease models, strain of mice, subjects, time points • Without running a pilot, account for approx. half hour per mouse (if using Fluc) • Set up a sequence acquisition if possible and copy and paste ROI for each individual image • Image as frequently as needed until “peak signal” is clearly passed Keyaerts, M. et al. Eur J Nucl Med Mol Imaging 35, 999–1007 (2008).
- 12. WWW.SCINTICA.COM How often should I image? 12 • Again, pilot study important! • Consider how aggressive your disease model is (metastatic cancer cell line vs. PDX model) • Image as frequently as you can (but only if gaining new information) • If part of treatment study, choose timepoints accordingly i.e. image directly before starting drug to get true pre-treatment measurement • If you need to image more than once a week, always start with a pre- injection image to ensure substrate from your previous session has been cleared (this can change as the disease progresses) www.covance.com
- 13. WWW.SCINTICA.COM Will animal fur interfere with my signal? 13 Luciferase expressing metastases mCherry expressing cancer cells • Use nude mice when possible • White fur is better than dark fur • Shave/nair area of interest if possible • Avoid animal models that have skin colour variability (i.e. melanoma models) • Most important thing is to be consistent
- 14. WWW.SCINTICA.COM What else can interfere with my signal? 14 Scabbing/dry blood, stitches, organs.. Parkins KM et al. Theranostics, 2020.
- 15. WWW.SCINTICA.COM I’m looking to detect very few cells… 15 • Optimized high-sensitivity reporters/substrates • Injection site (as shallow as possible) • Use nude mice if possible • Position the area of interest as close to the camera as possible i.e. if camera is over the animal and looking for lung signal, position animal belly up • Increase binning (i.e. 16 or “large binning”) • Increase exposure time • Start with single source of signal to simplify results • Adjust FOV MFP Brain Lungs/abdomen
- 16. Getting the most out of your imaging data
- 17. WWW.SCINTICA.COM What units do I report? 17 • Total flux vs. average radiance • Consider the disease model (single tumor or whole-body measurement?) • Be consistent • If comparing signal over multiple timepoints- copy and paste the same ROI J. Tavares et al. / Methods 127 (2017) 37–44
- 18. WWW.SCINTICA.COM How do I know where my signal is coming from? 18 • Use optical to guide other complementary imaging techniques i.e. MRI, CT, US
- 19. WWW.SCINTICA.COM T1 Weighted • Bioluminescence helps to identify where the tumors may be located, however differentiating tumors from one another, and measuring tumor volume is better done using an anatomical imaging modality such as MRI or Ultrasound Kidney Splenic Vein Tumour Intestine IP Injection of SKOV3 Ovarian Tumour Cells Complementary imaging modalities?
- 20. WWW.SCINTICA.COM T2 Weighted • Bioluminescence helps to confirm viability of the tumor cells, as they express luciferase, approximate volumes may be possible from the BLI signal; anatomical images help to confirm tumor volume - ultrasound (263mm3) or MRI (273mm3) Orthotopic Mammary Fat Pad Tumour (MDA-MB-231) Complementary imaging modalities?
- 21. WWW.SCINTICA.COM T2 Weighted • A significant positive correlation was observed between BLI and MRI measurements Complementary imaging modalities? Parkins KM et al. Scientific Reports, 2016.
- 22. WWW.SCINTICA.COM How do I know where my signal is coming from? 22 • Use optical to guide other complementary imaging techniques i.e. MRI, CT, US • Take multiple images of the same signal, rotating the animal to better predict origin of signal Brannen A. et al. Scientific Reports, 2018.
- 23. WWW.SCINTICA.COM How do I know where my signal is coming from? 23 • Use optical to guide other complementary imaging techniques i.e. MRI, CT, US • Take multiple images of the same signal, rotating the animal to better predict origin of signal • Perform ex vivo imaging and take images at different levels of dissection until signal is found
- 24. WWW.SCINTICA.COM How do I know where my signal is coming from? 24 • Use optical to guide other complementary imaging techniques i.e. MRI, CT, US • Take multiple images of the same signal, rotating the animal to better predict origin of signal • Perform ex vivo imaging and take images at different levels of dissection until signal is found • Use 3D tomography to co-register signal with organ/bone library
- 25. WWW.SCINTICA.COM 3D Tomography 25 Co-registration with digital Organ & Bones Atlas • Choose to add or hide individual organs and bones • Axial, Sagittal and Coronal Views • Quantification of the signal in units of volume
- 26. WWW.SCINTICA.COM 3D Tomography 26 Co-registration with digital Organ & Bones Atlas • Choose to add or hide individual organs and bones • Axial, Sagittal and Coronal Views • Quantification of the signal in units of volume
- 27. WWW.SCINTICA.COM How do I manage multiple signals within the same animal/sample? 27 Sasportas LS, et al. PLoS ONE 9(9): e105079.
- 28. WWW.SCINTICA.COM Multiplex Imaging 28 Green 520nm Green 580nm Red 640nm IR 740nm Blue 480nm NIR 680nm IR 780nm Blue 420nm • Simultaneous (or near simultaneous) acquisition of multiple signals • Transmitted through a shared medium (one or multiple channels) and subsequently de-multiplexed Modalities with multiplexing capabilities • Bioluminescence imaging • Fluorescence imaging • PET imaging
- 29. WWW.SCINTICA.COM Multiplex Imaging 29 mCherry IRDye800 • Simultaneous monitoring of red and green fluorescent signals by dual-color in vivo imaging • Signals can be overlaid so that several reporters can be visualized simultaneously Useful applications: • Cell tracking (i.e. cancer cells and therapeutic cells) • Immunology (i.e. T-cell populations) • Neurology
- 30. WWW.SCINTICA.COM Help! My data looks weird! 30 • Take advantage of whole-body imaging modality • Understand what your signal means (does your substrate require oxygen and ATP etc.? • Molecular changes occur before anatomical changes (the size of your tumor may not be representative of viable cells) • Perform in vitro experiments to characterize each cell line • Luminometer device can be used for calibration purposes Vandergaast, R., et al. Cancer Gene Ther 27, 179–188 (2020)
- 31. WWW.SCINTICA.COM Summary 31 • Be consistent! (positioning, reporting, ROIs, substrates etc) • Be prepared for variability!! • Embrace signals you weren’t looking for • Always use the peak signal for quantification
- 32. Scintica Instrumentation Phone: +1 (519) 857-6199 kparkins@scintica.com Q&A SESSION: To ask a question, click the Q&A Button, type your question and click send. Any questions that are not addressed during the live webinar will be answered following the event. Thank you for participating! Katie Parkins, PhD Applications Scientist