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May 03, 2023 414 likes •514 views

What is Intravital Microscopy? Four Requirement for Intravital Microscopy Intravital Microscopy Imaging Ann Seynhaeve Surgical Oncology, Erasmus MC - Daniel den Hoed Cancer Center, the Netherlands Laboratory of Experimental Surgical Oncology

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Ann Seynhaeve Surgical Oncology, Erasmus MC - Daniel den Hoed Cancer Center, the Netherlands Laboratory of Experimental Surgical Oncology

Intravital Microscopy Imaging

“Biomedical Research Techniques” What is Intravital Microscopy? Research tool to visualize and directly observe the microcirculation of

rgans in anesthetized or conscious animals in vivo,

and to analyze complex biological interactions and the potential mechanisms of disease. Four Requirement for Intravital Microscopy MOUSE I: Animal Model: mouse/rat Shaving of back of animal. Formation of skin flap and removal of skin and muscular layer. Construction of window chamber around skin flap. Insertion of tumor fragment or injection of tumor cells. Closer of window chamber with cover glass. RAT MOUSE I: Animal Model: Dorsal Skin-fold Chamber

Dorsal
Breast
Cranial
Abdominal

I: Animal Model: Other Chambers

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Bones
Embryonic skin
Lung
Heart

I: Animal Model: Other Implantations day 5 day 8 day 12 B16BL6 mouse melanoma II: Probes: Light Microscopy Angiogenesis (LLC) Co-option (B16) Lewis Lung Carcinoma B16BL6 II: Probes: Light microscopy Tumor blood-flow in soft tissue sarcoma II: Probes: Fluorescent Animals: Expression of a fluorescent protein

eNOS-GFP mouse: Green in endothelial cells
Cspg4-DsRed mouse: Red in perivascular cells
PDGFRb-Cre x mTmG: Red in all cells, green in perivascular cells

eNOS GFP Fluorescent injectables

Drugs: Doxil, Metoxantrone, Idarubicin,…
Bloodmarkers: Albumins, Dextrans, liposomal carriers,…
Small molecules: Hoechst, DAPI,…

II: Probes: Fluorescent eNOS GFP Cspg4 DsRed II: Probes: Fluorescent All mTomato Bloodflow marker Hoechst

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III: Microscope and equipment Microscope + camera

Multiphoton
Confocal
Fluorescent microscope
High performance digital camera

For image acquisition

and analysis Other

Heated stage to fix the chamber
Anesthesia unit

Short term Fixation: pre-heated metal plate Sedation: injection Most simple version: Pre-heated fixation plate for short term imaging III: Microscope and equipment III: Microscope The complete package: Multiphoton, anesthesia unit and heated stage for long-term evaluation Long term Fixation: plate is temperature controlled. Sedation: inhalation anesthetics.

Advantages of Intravital Imaging Real-time Imaging

Kinetics of response to therapy unknown: Histology over 5 day period. Problem: which time-point do I choose, or how many animals.

below: n= 2, 1 treatment plus control, 5 time points (Before, 4h, 24h, 48h, 72h), n=20.

Solution: intravital microscopy does not require predefined time-point.

below: n= 2, 1 treatment plus control, continuous kinetics

T C T C ENOUGH? Real Time Imaging: do not miss the point

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Study the Vascular Effects by Photodynamic Therapy laser Objective lens microscope Rat with window and sarcoma Injection of photosensitive drug before PDT t = 30 min (PDT) end of PDT (47 min) t = 80 min t = 4 h t = 24 h t = 48 h t = 72 h Real-time Monitoring of Tumor Vascular Effects 5 min 24hr 48hr Real-time Monitoring of multiple events eNOS GFP Drug

Imaging dynamic processes

Day 7 8 10 13 Day 14 15 16 Kinetics of vessel growth Tissue: B16BL6 tumor Green: Endothelial cells Red: Blood marker Kinetics of blood flow eNOS GFP Bloodmarker

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Kinetics of vessel invasion eNOS GFP Cspg4 DsRed Kinetics of pericyte invasion Kinetics of association and morphology eNOS GFP Cspg4 DsRed 3D reconstruction of association and morphology

Imaging fast processes

Endothelial tip cell movement T=4hrs

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Endothelial tip cell movement T=60min Filopodia movement

Imaging Rare Events

TCR Transduced T-cell Accumulate in Tumor eNOS GFP Bloodmarker

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Association of pericytes at the tip cell Pdgfrb GFP All mTomato 3D reconstruction of pericytes at the tip cell

Imaging Drug Delivery

Monitoring Intratumoral Drug Distribution Chemotherapeutics are injected systemically Question: what is the effect of certain (co)treatments on intratumoral drug distribution Injection of drug (fluorescent) Intratumoral Drug Distribution Tissue: Tumor Green: Blood marker Red: Drug carrier Tissue: Tumor Red: Drug Green: Drug carrier Intracellular Drug Localization

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Treatment effect on endothelial and perivascular cells Tissue: Tumor Green: Endothelial cells Red: perivascular cells Purple: Drug carrier eNOS GFP Cspg4 DsRed Pre-treatment effects Tissue: Tumor Green: Endothelial cells Purple: Drug carrier Blue: Hoechst eNOS GFP 2h 24h 72h Treatment effect on endothelial and perivascular cells Tissue: Tumor Green: Endothelial cells Red: perivascular cells Purple: Drug carrier eNOS GFP Cspg4 DsRed Hyperthermia Triggered Drug Release Tissue: Tumor Green: Content Red: Drug carrier Tissue: Tumor Green: Endothelial cells Red: Drug Hyperthermia Triggered Drug Release eNOS GFP

Imaging of Immunological Processes

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37°C T-cell activation by dendritic cells in the lymph node: lessons from the movies. Philippe Bousso. Nature Reviews Immunology 8, 675-684 (September 2008) In vivo imaging of leukocyte trafficking in blood vessels and tissues. Thorsten R Mempel et al. Current Opinion in Immunology. Volume 16, Issue 4, August 2004, Pages 406–417 37°C Innovation: Functional immunoimaging: the revolution continues. Nature Reviews Immunology 12, 858-864, 2012. Philippe Bousso & Hélène D. Moreau Intravital Imaging of Immune Processes Limitations of Intravital Microscopy

Invasive
Limited penetration depth
Movement of animal
Limited by light properties

Conclusions

allows real-time evaluation of processes in living

animals.

intracellular processes can be studied.
correct interpretation of treatment effects.
able to study process kinetics in detail.

Choose the right imaging technology for your research question. Medical University of South Carolina

D. Haemmerich

Surgical Oncology Erasmus MC T.L.M. ten Hagen

T. Lu
Dept. Cell Biology

Erasmus MC

R. de Crom
R. van Haperen

Helmholtz Zentrum München L.H. Lindner Max Planck Institute, Munster R.H. Adams

S. Adams

H.M. Eilken People involved a.seynhaeve@erasmusmc.nl