Webinar #4 Live Virtual Demonstration of the IVM System
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The document describes the IVM-MS intravital microscopy system from Scintica. The system provides an all-in-one solution for intravital imaging of various organs in live animals. It integrates hardware and software optimized for high-quality intravital imaging. The system includes components for animal maintenance during imaging and can perform functions like z-stack, mosaic, and time-lapse imaging. Examples shown include brain, tumor, lung, and heart imaging applications in window chamber models using confocal and two-photon microscopy.
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Webinar #4 Live Virtual Demonstration of the IVM System
- 1. IVM-MS Intravital Microscopy System Product Demonstration 26 August 2021
- 2. WWW.SCINTICA.COM IVIM’s All-in-One System Conventional Approach IVIM Technology
- 3. IVM- Intravital Microscopy System 3 • All-in-one single box package for easy installation, operation and maintenance • Co-optimized H/W and S/W for superb intravital imaging performance • Integrated devices for live animal maintenance • No limitation in imaging various internal organs • Ultrafast imaging speed • Live tissue motion compensation
- 4. WWW.SCINTICA.COM IVIM Technology | Product Lines • IVM-C (Confocal) • IVM-M (Two-Photon) • IVM-CM (Confocal& Two-Photon) • IVM-MS (Two-Photon Smart Ver.)
- 5. WWW.SCINTICA.COM Confocal Microscopy (IVM-C, -CM) Wide-area Intravital Microscopy
- 6. WWW.SCINTICA.COM Two-Photon Microscopy (IVM-M, -CM, -MS) Sarcomere (SHG) Vessel (CD31) Neuromuscular Junction (Thy1-YFP) Skeletal Muscle
- 7. Components of the system 7 • 1. Microscope Body • 2. Two-photon laser • 3. System Power Supply • 4. Monitor • 5. Heating pad controller • 6. Stage controller (Jog Dial) • 7. PC
- 8. System Accessories 8 • Body temperature control system • Inhalation anesthesia set • Imaging window chambers and holder • Stereotactic mount for brain imaging
- 9. WWW.SCINTICA.COM Platform for Live Animal Maintenance Body Temperature Monitoring & Feedback Heater Control: Rectal Probe & Body Plate Heater Imaging Tissue Temperature Monitoring & Heater Control: 2 Indicators & Cover Glass Heater
- 10. WWW.SCINTICA.COM Total Darkness with Black-Out Curtain 10 Sliding black-out curtain protects system from potentially polluting ambient light during imaging
- 11. Window Chamber Hardware 11 Dorsal Skinfold Chamber Cranial Imaging Window Abdominal Imaging Window Goldie et al. 2014 doi:10.1038/nprot.2014.165 Palmer et al. 2011 doi:10.1038/nprot.2011.349 Ritsma et al. 2012 DOI: 10.1126/scitranslmed.3004394
- 12. WWW.SCINTICA.COM Experimental Procedure • Ear skin Imaging • Pancreas and Spleen • Lung Imaging • Cranial Window Chamber Model • Dorsal Skinfold Window Chamber Models • Thoracic Window Chamber Organ Models Longitudinal Models
- 14. WWW.SCINTICA.COM GUI Overview • Capture • Control: 2p laser and detector operation • Z-Stack: Setting up automated images along Z axis • Mosaic: Setting up automated images on the XY plane for wide- field imaging • Time-lapse: Imaging over time at pre-set time intervals • Multi-position: • IVM Studio
- 15. WWW.SCINTICA.COM Adjusting laser power and detector sensitivity
- 16. WWW.SCINTICA.COM Getting Ready for Image Acquisition
- 18. WWW.SCINTICA.COM Cranial Window Chamber- Brain Imaging
- 19. WWW.SCINTICA.COM Cranial Window Chamber-Brain Imaging
- 20. WWW.SCINTICA.COM Cranial Window Chamber-Brain Imaging
- 21. WWW.SCINTICA.COM Dorsal Skinfold Window Chamber-Tumor Imaging
- 22. WWW.SCINTICA.COM Dorsal Skinfold Window Chamber-Tumor Imaging 50 μm 2 hour 6 hour 24 hour MDA-MB-231-GFP Nanoparticle Vessel (CD31) Monitoring of nanoparticle delivery to triple-negative breast cancer, MDA-MB-231
- 23. WWW.SCINTICA.COM Thoracic Window Chamber-Lung IntravitalMicroscopyof Lung BloodVessel, Neutrophil,Circulation
- 24. WWW.SCINTICA.COM Thoracic Window Chamber- Cardiac Imaging IntravitalImagingof heart with motioncompensation* Wide-area intravital imaging of heart 200 μm Lymphatics (Prox-1 GFP) Vessel (CD31) *IVIM Technology’sintravitalmicroscopy system is equippedwith fully-integratedmotion compensationfunction,providingautomatichassle-freehigh-precisionmotion compensated intravitalimagingresults
- 25. WWW.SCINTICA.COM Adipose Tissue Skeletal Muscle Lung Ear Skin Lymph Node Retina Small Intestine Kidney
- 26. WWW.SCINTICA.COM Q&A Session Please enter your questions in the Q&A section.
- 28. System Accessories 28 • Body temperature control system
- 30. WWW.SCINTICA.COM Thoracic Window Chamber 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
Hinweis der Redaktion
- Thanks for the introduction. hello everyone! thank you for attending our virtual demo of the IVM system today. My name is Niloufar Khosravi, I am going to get you started by a short introduction to the Intravital microscopy system and its components. To refresh your memory on the system and give a little background to those who missed our previous webinars. Then my co-host Dr. Selina will show you the procedure for animal preparation and imaging of various external and internal organs of the body
- IVIM is an All-in-One intravital confocal/two-photon microscopy system which is designed and optimized for imaging of live animals. Those who are familiar with the world of intravital microscopy would know how using convectional confocal microscopes for animal studies can be technically challenging. As users have to prepare every single component and functions by yourself. The main challenges and Limitations are: The depth of imaging Motion artifacts due to lack of proper stabilization And taking care of the animal welfare and temperature control
- IVIM Technologies has come up with a solution to make intravital microscopy easier and reproducible for non experts and experts. The All-in-one single box package enables easy installation, operation and maintenance. hardware and software are co-optimized for ultrafast imaging and no limitation in imaging various internal organs. The system is equipped with live tissue motion compensation functions, can get the high-quality images of moving organs. Specific hardware has been integrated in the system to maintain animals’ normal physiological conditions.
- There are 4 setup options for the system: a confocal only system, a 2-photon system, confocal and 2 photon combined, and IVM-MS a system with a laser fixed at 920 nm wavelength for deep tissue imaging
- Very quickly remind you about Confocal and two photon: Confocal microscopy, is an optical imaging technique that uses a pinhole to block out-of-focus light coming from the background tissue. Therefore, increases optical resolution and contrast of images acquired
- 2p microscopy is a technique that utilizes 2 photons of longer wavelength but lower energy to excite fluorophores. As you might know components of tissue, such as hemoglobin, absorb and scatter incident excitation light of most visible wavelengths. This limits light penetration, resulting in low emission signals that are difficult to detect consistently. 2P technique uses near-infrared (NIR) excitation light. Since tissue is effectively transparent at these wavelengths, the background signal is strongly suppressed. Combination of these effects increase penetration depth and reduce photo bleaching. For deep tissue microscopy 2p is used instead of confocal. 2 photon laser also allows for label free imaging such as second harmonic generation often used to look at fibrous tissues.
- Here are the components of our system.
- The animal maintenance platform comes with a plate heater that can maintain body temperature. It is connected to a rectal probe which sends feedback to the system and the temperature is automatically adjusted. If you need to move an organ slightly out of the body to image, there is a tissue temperature heater with integrated glass covers slip that comes in contact to the tissue, and it maintains the temperature.
- The system is designed like a closed box with a sliding black-out door so protects the experimental area from ambient light. Unlike other microscopes that need to be placed in a dark rooms with black out drapes, you can place this system in any room you desire.
- If you are planning to perform longitudinal studies, you are required to place window chambers in your animals to optically expose the organ or tissue of interest, to preserve the area from also to protect it against infections. This allows you to image one animal over time, so you will reduce variation in your data and the number of animals used in your cohort.
- We have collected a series of videos to show you examples of imaging procedure with or without window chambers, Due to ethical concerns, we were unable to do a live demonstration. IN these videos we will walk you through the procedure and show you examples of the results Today we have picked a few models to demonstrate: Ear skin, Internal organ: Spleen, and Lung Also 2 window chamber models: cranial and dorsal skinfold
- Switching on the Machine First the laser and after 5 minutes he machine
- Open control to set the laser power Normally 20% for tumor tissues. we use high power cause tumor is dense so to see the core we crank up the laser For deep tissue imaging we use higher power The detector is the gain, if you increase the gain, you will receive more signal but you also increase the background to you have to find a right balance between the power and the gain for every channel.
- Set folder and the directory for the file> Select objective lens it goes from 4X to 60X. Some of these lasers are water immersion and oil. Water increases the resolution. We set the pixel 512 by 512 or 1024x1024.
- We select the channel GFP then we assign a pseudo color to the channel GFP green. Finding the focal plane. At this point system will be ready to acquire images or record videos, I’m passing it over to Selina to show you the procedure for animal preparation and image acquisition
- So you observed the basic steps through the imaging process and looked at acute models of various organs, I am going to quickly show you examples of placing window chambers for longitudinal imaging Let’s start with the cranial window chamber model
- This ia an example for brain imaging in blood brain barrier model. And perform 3D analysis of vascular network
- Go up to 1 mm
- Next, DS model. We have these customs designed hardware that we implant in dorsal skin and suture fix. We make an incision on one side of the skin, and inject tumor cells on other side of tissue, covered with coverglass and fix it. And image.
- We can perform imaging over months to monitor tumor progression and study the effect of therapeutics. Here you see an example of drug delivery with nano particles shown in red to triple negative breast cancer, images at 2 6 and 24 hr post delivery
- We want to end today's demo with imaging through the thoracic window chamber: Similar animal preparation procedure to other models. Liver is one of those moving organs in order to get images we need to stabilize it. In this custom design window model there is whole in the chamber which will be connected to a vacuum pump. This we make a small portion of the lung stable for imaging. And the motion compensation function we can get this clear and high resolution image of lung
- Imaging cardic will also be achieved with the same window model as it is also moving so fast. With the integrated motion compensation function you can get clear images, here you see lymphatic vessels and blood vessels in hert.
- Lymph node: Lymphatic vessels and lymphocytes Small Intestine- Mucosal Tissue and tubular glands Kidney-Ducts and tubules
- We want to end today's demo with imaging through the thoracic window chamber: In vivo Liver imaging can be applied to investigating drug delivery and uptake & clearance efficacy, and liver fibrosis mouse models. Thanks for attending IVIM Seminar and If there’s any question or inquiries, please feel free to ask us now or later through the contacts in display.