Basics of Intravital Microscopy Webinar 1
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This is the first of a 4-part series introducing Scintica’s newly formed relationship with IVIM Technology and their IntraVital Microscopy platform (IVM). In this session, we introduced the fundamentals of fluorescence microscopy, review some example images, and focus on this technique's intravital imaging applications. This webinar focused on formulating a basic understanding of the imaging modality to further understand the IVM system's capabilities throughout the rest of the webinar series. First, the fundamental principles of fluorescence imaging were explained, along with their advantages and challenges with applied in an in vivo setting. Next, we highlighted intravital microscopy's advantages and its role in oncology research and other scientific areas. We also provided an overview of the most commonly used animal models for intravital imaging. Finally, we focused on the importance of acquiring quantitative imaging data and navigate around some pitfalls. Key examples from the research field were collected in this webinar. After attending this webinar, attendees will have: a basic understanding of the fundamentals of fluorescence microscopy, an overview of intravital imaging advantages and applications, an overview of the most commonly used intravital imaging animal models, an understanding of what to pay attention to in order to acquire quantitative imaging data.
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Basics of Intravital Microscopy Webinar 1
- 1. The Basics of Intravital Fluorescence Microscopy Sara Rapic MSc, PhD Product Manager, Imaging Division, Scintica
- 2. WWW.SCINTICA.COM 2 Topics of Discussion • Fundamentals of Fluorescence Imaging • Intravital Microscopy • Systems • Advantages • Models • Quantification • Key Research Applications
- 4. WWW.SCINTICA.COM Choosing an Imaging Modality Ghita et al. Cancers, 2019 4
- 5. WWW.SCINTICA.COM Optical Imaging Nguyen et al. EMJ Radiology, 2020 5
- 7. WWW.SCINTICA.COM In Vivo Optical Imaging Scatter & Absorption Calderhead and Tanaka. Photomedicine: Advances in Clinical Practice, 2017 7
- 8. WWW.SCINTICA.COM http://en.wikipedia.org/wiki/Optical_window_in_biological_tissue In Vivo Optical Imaging Tissue Absorption Absorption in blood Absorption in water 8
- 9. WWW.SCINTICA.COM In Vivo Optical Imaging Optimal Imaging Window Adapted from Ruggiero et al. Dalton Transactions, 2016 Optimal Imaging Window Tissue Penetration (mm) UV IR VIS + NIR H2O H2O Relative Absorbance 100 10 1 0.1 0.01 Hb HbO2 Hb 9
- 11. WWW.SCINTICA.COM Fluorescence Imaging Fluorophores • Genetically modified fluorescent proteins: • Organic dyes: FITC/TRITC, Alexa (conjugated antibodies) • Inorganic dyes: Quantum Dots • Endogenous species: elastin, collagen, NADH/FAD 11
- 13. WWW.SCINTICA.COM https://www.youtube.com/watch?v=BYwHLhgP1qI Fluorescence Microscope Components 13
- 14. WWW.SCINTICA.COM https://www.youtube.com/watch?v=BYwHLhgP1qI Fluorescence Microscope Components 14
- 15. WWW.SCINTICA.COM Confocal/ Two-Photon Microscope Mostany et al. Advanced Fluorescence Microscopy, 2014 Upright 100 um 500 um Upright Confocal Two-Photon 15
- 16. WWW.SCINTICA.COM Confocal/ Two-Photon Microscope Mostany et al. Advanced Fluorescence Microscopy, 2014 Single Photon Excitation Two-Photon Excitation 16
- 17. WWW.SCINTICA.COM 17 Conclusions Optical imaging has excellent resolution but poor penetration depth Propagation of light through biological tissue is affected by hemoglobin and water The optimal window for in vivo optical imaging lies between 600 and 1000 nm Fluorescence is the commonly used optical technique for in vivo imaging Up to 4 fluorophores can be imaged simultaneously when chosen carefully to avoid spectral overlap
- 19. WWW.SCINTICA.COM Intravital Microscopy | When? Applications Publications Developments 1930 1940 1950 1960 1970 1980 1990 2000 2010 Luminescent light IVM First IVM publication IVM in cancer research Fluorescence Microscopy with Fluorophores Anatomy of living organisms Static in vivo processes Longitudinal in vivo processes Immune response & capillaries Tumor metastasis & pathophysiology ~ 15 Multiphoton Microscopy Spinning Disk Microscopy ~ 40 ~ 50 ~ 100 ~ 150 ~ 150 ~ 400 1000+ 1000+ Confocal Microscopy 19
- 20. WWW.SCINTICA.COM Intravital Microscopy | Why? Macroscopic-scale Imaging & Diagnosis (Organ/Tissue-level : Structural Information) Microscopic-scale Imaging & Analysis (3D Cell-level : Molecular/Functional Information) Intravital microscopy (IVM) Islet cell (MIP-GFP) Vessel (CD31) PET / CT / MRI X-Ray Ultrasound Cancer cell (B16F10-GFP) Vessel (CD31) Sinusoid (LYVE-1) Blood circulation (FITC-Dextran) Neutrophil (Ly6G) disease stage, development, and treatment response In situ Spatial information Real-time Quantitative Longitudinal Reduces animals 20
- 21. WWW.SCINTICA.COM 13 • Transgenic animals (endogenous fluorescence) • Injection of fluorescent agent(s) (cells/antibodies/dyes) Intravital Microscopy | Animal Models McKenzie at al., 2018 Asokan et al., 2020 Stoletov et al., 2018 Bogoslowski et al., 2020 21
- 22. WWW.SCINTICA.COM Intravital Microscopy | Where? 22
- 23. WWW.SCINTICA.COM Intravital Microscopy | How? Coste et al. Cytometry, 2019 23
- 24. WWW.SCINTICA.COM Intravital Microscopy | Window Chambers Dorsal Skinfold Chamber - Cancer Xenograft imaging Cranial Imaging Window - Long-term repeated brain imaging Abdominal Imaging Window - Long-term abdominal organ imaging 24
- 25. WWW.SCINTICA.COM https://youtu.be/2ozH_FOtkBI Intravital Microscopy | Window Chambers Dorsal Skinfold Chamber - Cancer Xenograft imaging 25
- 26. WWW.SCINTICA.COM Disruption of Biology • Surgical manipulation • Antibiotics/anti-inflammatory • Animal stress • Prolonged laser exposure • Antibody binding effects Changed microenvironment Figley et al. PLOS One, 2013 Iba-1 Iba-1 protein expression 26
- 27. WWW.SCINTICA.COM Intravital Microscopy | What? For cancer and drug development, intravital microscopy enables a direct imaging analysis of the tumor development and drug delivery to target tissue as well as efficacy, and mode of action (MOA) of new therapeutic candidates at a microscopic, cellular level in various preclinical model of human disease. Cancer Metastasis - CTC Cancer cell dissemination to circulation Drug Delivery - Nanoparticle Anti-cancer nanoparticle delivery Circulating Tumor cell (CTC) Drug carrier Cancer cell Vessel Bone Marrow Transplanted BM cell, HSPC 27
- 28. WWW.SCINTICA.COM Skin Inflammatory response KARS Granulocyte Intravital Microscopy | What? Intravital microscopy enables dynamic 3D imaging of various cellular-level dynamics such as cell trafficking, cell-cell interaction, and cell-microenvironment interaction inside the living body in vivo, providing a new insight in the processes of human disease development. Cancer Xenograft - T cell Triple Negative Human Breast Cancer T cell Cancer cell Vessel Lung Microcirculation in Sepsis Model 28
- 29. WWW.SCINTICA.COM Intravital Microscopy | Image Parameters Contrast Sensitivity Speed Resolution Depth 29
- 30. WWW.SCINTICA.COM Intravital Microscopy | Motion Artifacts Uncorrected Motion Artifacts Corrected for Motion Artifacts 30
- 31. WWW.SCINTICA.COM Intravital Microscopy | Spectral Crosstalk McRae et al. PLOS One, 2019 31
- 32. WWW.SCINTICA.COM Intravital Microscopy | Autofluorescence http://zeiss-campus.magnet.fsu.edu/ 32
- 33. WWW.SCINTICA.COM Intravital Microscopy | Quantification Intra-/extravasation Cell motion tracking Cellular interactions Intracellular changes Cell area/count Vascular analysis Adapted from Evans et al. Experimental Neurology, 2019 33
- 34. WWW.SCINTICA.COM 34 Conclusions Successful intravital imaging depends on: • Before imaging System chosen Animal model optimization Window chamber quality • During imaging Animal positioning Animal maintenance Imaging parameters • After imaging Pre-processing corrections Post-processing quantification
- 35. WWW.SCINTICA.COM 35 Key Research Applications
- 36. Oncology
- 37. WWW.SCINTICA.COM Longitudinal Cancer Xenograft Imaging Intravital Imaging of dorsal skinfold chamber for cancer xenograft imaging Objective Lens Heating Pad Rectal Probe Chamber holder Dorsal Skinfold Chamber Dorsal Skinfold Chamber - Cancer Xenograft Imaging (H460, Human non-small cell lung carcinoma) Day 0 Day 4 SC. Injection of H460-GFP (Human Lung Cancer; NSCLC) H460-GFP (0.1 mil / 20 μL) Intravenous Injection of Tetramethyl-rhodamine dextran TAMRA dextran H460-GFP Vessel (TMR-dextran) 37
- 38. WWW.SCINTICA.COM Longitudinal Cancer Xenograft Imaging Intravital Imaging of dorsal skinfold chamber for cancer xenograft imaging No Treatment Anti-Angiogenic Treatment Day7 Day10 Day13 Day10 Day13 Day7 LLC-GFP Vessel (CD31) 50 μm Objective Lens Heating Pad Rectal Probe Chamber holder Dorsal Skinfold Chamber Dorsal Skinfold Chamber - Monitoring of anti-angiogenic treatment effect in vessel morphology and dilation 38
- 39. WWW.SCINTICA.COM Longitudinal Cancer Xenograft Imaging Intravital Imaging of dorsal skinfold chamber for cancer xenograft imaging Dorsal Skinfold Chamber - Monitoring of nanoparticle delivery to triple-negative breast cancer, MDA-MB-231 50 μm 2 hour 6 hour 24 hour MDA-MB-231-GFP Nanoparticle Vessel (CD31) Objective Lens Heating Pad Rectal Probe Chamber holder Dorsal Skinfold Chamber 39
- 40. WWW.SCINTICA.COM Longitudinal Cancer Xenograft Imaging Intravital Imaging of dorsal skinfold chamber for cancer xenograft imaging Dorsal Skinfold Chamber - Monitoring of nanoparticle delivery to triple-negative breast cancer, MDA-MB-231 50 μm 2 hour 6 hour 24 hour MDA-MB-231-GFP Nanoparticle Vessel (CD31) Objective Lens Heating Pad Rectal Probe Chamber holder Dorsal Skinfold Chamber 40
- 42. WWW.SCINTICA.COM Real-time Abdominal Imaging Abdominal Window Chamber Monitoring of cellular behavior in kidney, pancreas, and spleen Cover glass Kidney Spleen Abdominal Imaging chamber Pancreas Islet β cell (MIP-GFP) Vessel (Evans Blue) MIP-GFP mouse - endogenously expresses GFP in pancreatic beta-cells under the control of mouse insulin 1 promotor Monocyte/DC (CX3CR1-GFP) Vessel (TAMRA-dextran) CX3CR1-GFP mouse - endogenously expresses GFP in monocytes, macrophage, brain microglia and DCs under control of endogenous Cx3cr1 locus Pancreas Spleen 100 μm Kidney Islet β cell (MIP-GFP) Vessel (Evans Blue) 42
- 43. WWW.SCINTICA.COM Real-time Gastric Imaging Intravital Imaging of colon cancer implanted in caecum 250 µm Cancer (CT26:H2B-GFP) Vessel (CD31) Cancer (CT26:H2B-GFP) Vessel (CD31) Vessel (CD31) 43
- 44. WWW.SCINTICA.COM Intravital Imaging of Liver Disease Biomed. Opt. Express 11(8):4835 (2020) Hepatic Lipid Droplet (SF44) Liver Sinusoid (CD31) Portal Vein Periportal Sinusoid 44
- 45. WWW.SCINTICA.COM Intravital Imaging of Liver Disease Normal diet MCD, 2 days MCD, 7 days MCD, 14 days Hepatic Lipid Droplet (SF44) Liver Sinusoid (CD31) Intravital Imaging of Non-alcoholic Fatty Liver Disease (NAFLD) Biomed. Opt. Express 11(8):4835 (2020) 45
- 46. Immunology
- 47. WWW.SCINTICA.COM Heating pad Objective Lens Motorized XYZ translational stage Heating pad sensor Rectal probe: body temp. monitoring Cover glass holder Dynamic Immune Cell Imaging In Vivo Blood Vessel Imaging - Endothelial cell labeled in vivo by intravenous injection of anti- CD31 antibody conjugated with far-red fluorophore LysM-GFP mouse - endogenously expresses green fluorescence protein (GFP) in the neutrophil and macrophage by genetically knocking eGFP gene into the lysozyme M (LysM) locus Intravital Imaging of ear skin Neutrophil/Macrophage (LysM-GFP) Vessel (CD31) J. Cell Biology, 216(7):2201 (2017) 48
- 48. WWW.SCINTICA.COM Dynamic Immune Cell Imaging KARS protein - KARS protein labelled with far-red fluorophore Alexa647 was intradermally injected by using microinjector 3 hours after KARS injection 6 hours after KARS injection 0 hours after KARS injection J. Cell Biology, 216(7):2201 (2017) LysM-GFP mouse - endogenously expresses green fluorescence protein (GFP) in the neutrophil and macrophage by genetically knocking eGFP gene into the lysozyme M (LysM) locus 49
- 49. WWW.SCINTICA.COM Real-time Lymphatics Imaging Heating pad Popliteal lymph node Cover glass Warm water circulator Rectal probe: body temp. monitoring Temperature Sensor Objective Lens Cover glass holder Motorized XYZ translational stage Tail vein catheter Intravital Imaging of popliteal lymph node Parenchyma HEV Lumen Intravital imaging of extravasation of T cells & B cells in the high endothelial venule (HEV) of Lymph Node • T cell & B cell obtained from actin-DsRed & actin-GFP mice then adoptively transferred to wildtype C57BL/6 mouse • FRC labeled by anti-ER-TR7 antibody conjugated with Alexa Fluor 647 • HEV Lumen labeled by IV injection of 2MD FITC-Dextran 50
- 51. WWW.SCINTICA.COM Longitudinal Transplanted Cell Imaging Day 1 Day 3 Day 4 1 mm Heating pad Cover glass Cranium Stereotaxic Instrument: Mouth Adapter, Ear Bar Objective Lens Motorized XYZ translational stage Coronal suture Central vein Sagittal suture Cranium Transplanted cell Vessel (CD31) Longitudinal repetitive wide-area intravital imaging of cranial bone marrow after bone marrow transplantation of c-kit+ BM cell (DsRed) Intravital Imaging of cranial bone marrow PLoS ONE, 12(11):e0187660 (2017) 52
- 52. WWW.SCINTICA.COM Longitudinal Transplanted Cell Imaging H2B-GFP / β-actin-DsRed mouse - expresses green fluorescence protein (GFP) in the nucleus and DsRed in cytoplasm Longitudinal repetitive wide-area intravital imaging of cranial bone marrow after bone marrow transplantation of c-kit+ BM cell (DsRed) Day 1 Intravital Imaging of cranial bone marrow Day 3 Day 4 1 mm Transplanted cell Vessel (CD31) PLoS ONE, 12(11):e0187660 (2017) 53
- 53. Infection
- 54. WWW.SCINTICA.COM Real-time Thoracic Imaging Intravital Imaging of lung Stabilized lung imaging - Suction-assisted lung stabilization imaging window chamber Illuminating fiber Ventilator Lung Imaging Window Chamber Heating Pad Motorized XYZ translational stage Pulse Oximetry Objective Lens Homeothermic Controller Temperature Sensor Chamber Titling Mount Protected Ag Mirror Cover glass Mouse Intubation Imaging chamber holder Tilting mount Lung Lung Imaging Window chamber Suction tube Suction hole 55
- 55. WWW.SCINTICA.COM Real-time Thoracic Imaging Intravital Imaging of lung, intravascular neutrophil Real-time imaging of RBC flowing in pulmonary microvasculature - Monitoring of pulmonary microcirculation Blood flow (FITC-Dextran) Neutrophil (Ly6G) Track Speed Mean (μm/sec) 0 1500 50μm 50 μm Vessel (Tie2-GFP) RBC (DiD) European Respiratory Journal 2019, 53:1800736 56
- 56. Inflammation
- 57. WWW.SCINTICA.COM Longitudinal Brain Imaging Intraperitonealinjection 3-NP(20mg/kg) Intravenousinjection TRITC-Dextran(Angiography) Cranial window (30 days recovery) LysM-GFP mouse Longitudinal Intravital imaging (30 days) OBJ Day 1 Day 4 Day 7 Day 10 Sham Control 3-NP LysM-GFP Vessel (TRITC-Dextran) Biomed. Opt. Express 11(8):4835 (2020) Intravital Imaging of brain Motorized XYZ translational stage Objective Stereotaxic stage with heating function 58
- 58. WWW.SCINTICA.COM Longitudinal Brain Imaging Intravital Imaging of brain Motorized XYZ translational stage Objective Stereotaxic stage with heating function Biomed. Opt. Express 11(8):4835 (2020) Day 1 Day 4 Day 7 Day 10 59
- 59. WWW.SCINTICA.COM Longitudinal Brain Imaging LysM-GFP Vessel / BBB Leakage Vascular Leakage Vascular Leakage Intravital Imaging of brain Motorized XYZ translational stage Objective Stereotaxic stage with heating function Biomed. Opt. Express 11(8):4835 (2020) 60
- 60. WWW.SCINTICA.COM WL I R CFM svOCT 2 mm 1 mm Ex Vivo Correlation + Validation In Vivo 61 Multimodal Imaging H&E H&E CD31 0.25 mm g-H2AX
- 63. Audience Poll