In vivo multiphoton imaging facility
Intravital multiphoton imaging of immune cells
The most relevant experimental setting to address the mechanisms that navigate immune cells into, within and out of lymphoid and non-lymphoid tissues remains the live animal, regardless of the value of alternative experimental systems to analyze immunological and molecular functions in vitro. The major aim of this central bio-imaging platform (infrastructure core facility) is to provide access to state-of-the-art two- and multiphoton in vivo imaging microscopy (MP-IVM) to the members of the Collaborative Research Centre 914 (CRC 914).
We provide animal models allowing MP-IVM of immune cells in several mouse tissues and organs, including bone marrow, lymph node, arterial wall, spleen, lungs, gastric mucosa, ear, placenta and cremaster muscle. We use transgenic report mice expressing fluorescent proteins in distinct cell lineages, including hematopoietic progenitor cells, monocytes, neutrophils, dendritic cells, or T cells. Imaging is performed under both non-infectious and infectious S2 safety conditions.
We provide professional advice and assistance in planning and setting up microscopy experiments, sample preparation and imaging as well as in post-imaging reconstruction and analysis using Volocity™, ImageJ™, ImSpector™ and Imaris software.
Booking and usage
Before starting experiments, all researchers must contact the facility manager Dr. Hellen Ishikawa-Ankerhold to provide basic details on the experiments planned (e.g. type of experiment, animal project license, safety conditions, anesthesia required, etc.).
Trained researchers can use the MP-IVM after booking a timeslot in the calendar of the imaging facility (via email to the manager). Untrained researchers can use the equipment under our supervision.
We offer two MP-IVM systems that are located in air-conditioned darkrooms (S1 and S2 laboratory). The preparatory room features an anesthesia device, a dissecting microscope, a laminar air flow cabinet and basic equipment for preparation of antibodies and chemicals.
Short description of the multiphoton microscopy (MP-IVM):
- Multiphoton microscope TrimScope I is equipped with a Ti:Sa (titanium:sapphire) laser Mai-Tai 1020 HP (Spectra-Physics) tunable in the range of 690 to 1020 nm (laser class 4). The system has four direct PMT detectors for single beam point scanning, and for multi-beam imaging a highly sensitive Hamamatsu EM CCD camera. It has a manual sample stage.
- Multiphoton microscope TrimScope II is equipped with a Ti:Sa laser Chameleon Ultra II (Coherent) tunable in the range of 680 to 1080 nm (laser class 4) and additionally an OPO compact to support the range of 1000 to 1600 nm. The system has four high sensitive GaAsP (Gallium arsenide phosphide) detectors for single beam scanning, and one additional PMT detector for Dodt contrast. It has a Hamamatsu EM CCD camera Orca Flash 2.8 and it is equipped with a motorized sample stage.
- Both microscopes have HBO fluorescence light sources, four fluorescent filter cubes and bright field trans illumination light source.
- Both microscopes feature a diverse range of filters and objectives that can be used according to the experimental model and imaging needs.
- We also offer animal triggered in vivo-imaging, where ECG and respiration signals are used to limit the impact of motional perturbation.
General information on fluorescence, confocal and multiphoton imaging microscopy: http://www.mdpi.com/1420-3049/17/4/4047In