Courtesy of Matteo Iannacone. http://iannaconelab.com/
The role of stem cells
Stem cells can differentiate into specialized cells. Depending on the type of stem cell and influences on them, they can differentiate in every tissue or into tissue types. In addition, stem cells can repair the body by replenishing tissue in a continuous regeneration process.
By using mouse models, stem cells and their role in the body repair can be investigated with 2-photon microscopy. The hair follicle can be used as a model organism because it is easily accessible and has regenerative abilities. In addition, the hair follicle niche, the environment of stem cells, is a usable system because stem cells and differentiated cell types occupy distinct locations in the niche. These locations are consistent with their role in the hair growth.
With the TriM Scope II, the behavior of stem cells and their role in organ regeneration are studied by recording z-stacks of images at time intervals.
The aim is a time-lapse movie that shows e.g. the growth of a hair, cell dynamics, and the role of stem cells. Repeating measurements in the same areas over days, allows displaying the complete full hair regeneration cycle. The Spectral Detector enables the detection of up to six different labeled specimens by using sufficient dichroic mirrors and emission filters.
Additionally to standard 2-photon imaging experiments, the TriM Scope II makes it possible to perform laser ablation. An infrared laser beam with a sufficient wavelength, e.g. 900nm, and high power is focused on a defined area in the sample. By first ablating defined cells, it is possible to observe the role of the ablated cells in the process of hair growth.
 Deschene, E.R., Myung, P., Rompolas, P., Zito, G., Sun, T.Y., Taketo, M.M., Saotome, I., Greco, V., β-catenin activation regulates tissue growth non-cell autonomously in the hair stem cell niche, Science (2014), 343(6177), 1353–1356
Virtual projection down through the b cell follicle of the popliteal lymph node showing the follicular dendritic cell (FDC) network within a suspected developing germinal center.
Courtesy of Ann Haberman
Cell migration and interactions in the immune system
In studies about the immune system, the focus is on interactions between B-cells, T-cells, neutrophils, and proteins or other cells. Their interactions and how they influence the movement of different cells and the reaction of the immune system to a disease can be studied by intravital 2-photon microscopy.
In different organs, lymphocytes (T-cells, B-cells, and natural killer cells) interact with antigen-presenting cells. In addition, cells move permanently from blood into lymph nodes or into extralymphoid sites. So, to understand the reaction of the immune system, it is necessary to investigate the diverse interactions and the movement of cells. Also, the surrounding tissue has to be taken into account because it can influence the migration and interactions. LaVision BioTec’s TriM Scope II delivers all necessary functions to investigate the response of the immune system to diverse diseases.
To investigate the movements, interactions, and the influence of the tissue, mouse models can be used. The structures and cells of interest are labeled with fluorescent dyes, fluorescent proteins, or quantum dots. Labeled cells are injected into the mice, which are then anesthetized and the organ of interest, for example a popliteal lymph node, is surgically exposed. The TriM Scope II 2-photon microscope then allows the observation of the immune response in the organ. By using characteristic anatomical landmarks, the region of interest can be chosen and the fluorescence of the labeled particles can be detected with LaVision BioTec’s Spectral Detector.
This device allows the detection of up to six different colors with photomultiplier tubes (PMTs). With the TriM Scope II, 3D-Timelapse experiments can be performed to follow cells over time in a 3D tissue. Z-stacks of images up to more than 1000µm are recorded and repeated with a sufficient frequency. By using sufficient analysis programs, the 4D volumes (x-y-z-t) can be reconstructed and the movement of different cells can be tracked over time. The reconstruction of B-cell tracks allows the determination of track distributions, track speeds, turning angles, and motility coefficients for these cells. The influence of proteins on the cell movement can be investigated simultaneously.
Beside lymphocytes, the role of neutrophils is also studied. Neutrophils are a type of white blood cells, formed from stem cells in the bone marrow. They are part of the immune defense but, so far, not much is known about their role in the lymph node. With a TriM Scope II 2-photon microscope the movement of neutrophils in the lymph node of a living mouse can be investigated. Moreover, the role of the neutrophils in the initiation of an immune response against different pathogens can be observed.
The type of experiment is comparable to the one for the observation of lymphocytes. The recording of 3D stacks over a certain time in vivo allows the detection of cell movement, interactions, and influences of other cells, proteins, or tissue. 2-photon microscopy helps to understand the complexity of the immune system and the immune response against pathogenic germs.
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