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Intravital microscopy is a form of microscopy that allows observing biological processes in live animals (in vivo) at a high resolution that makes distinguishing between individual cells of a tissue possible. Before an animal can be used for intravital microscopy imaging it has to undergo a surgery involving implantation of an imaging window. For example, if researchers want to visualize liver cells of a live mouse they will implant an imaging window into mouse’s abdomen. Mice are the most common choice of animals for intravital microscopy but in special cases other rodents such as rats might be more suitable. Animals are always anesthetized throughout surgeries and imaging sessions. Intravital microscopy is used in several areas of research including neurology, immunology, stem cell and others. This technique is particularly useful to assess a progression of a disease or an effect of a drug.

Basic Concept
Intravital microscopy involves imaging cells of a live animal through an imaging window that is implanted into the animal tissue during a special surgery. The main advantage of intravital microscopy is that it allows imaging living cells while they are in the true environment of a complex multicellular organism. Thus, intravital microscopy allows researchers to study the behavior of cells in their natural environment or in vivo rather than in a cell culture. Another advantage of intravital microscopy is that the experiment can be set up in a way to allow observing changes in a living tissue of an organism over a period of time that is useful for many areas of research including cancer and stem cell research. High quality of modern microscopes and imaging software also permits subcellular imaging in live animals that in turn allows studying cell biology at molecular level in vivo. Advancements in fluorescent protein technology and genetic tools that enable controlled expression of a given gene at a specific time in a tissue of interest also played important role in intravital microscopy development.

The possibility of generating appropriate transgenic mice is crucial for an intravital microscopy studies. For example, in order to study the behavior of microglial cells in Alzheimer’s disease researchers will need to crossbreed a transgenic mouse that is a mouse model of Alzheimer’s disease with another transgenic mouse that is a mouse model for visualization of microglial cells. Cells need to produce a fluorescent protein to be visualized and this can be achieved by introducing a transgene.

Imaging
If there is a need to capture close interactions between cells multiphoton microscopy is preferable as it provides greater depth of image than single-photon confocal microscopy. Multiphoton microscopy also allows visualization of cells located underneath bone tissues such as cells of the bone marrow. It can be useful to pair up multiphoton and confocal microscopy that allows getting more information from every imaging session. This includes visualization of more different cell types and structures to obtain more informative images and using a single animal to obtain images of all the different cell types and structures that are of interest for a given experiment. This is an example of The Three Rs principle implementation.

Limitations of Intravital Microscopy
One of the main advantages of intravital microscopy is the opportunity to observe how cells interact with their microenvironment. However, visualization of all types of cells in the microenvironment is limited by the number of distinguishable fluorescent labels available. It is also widely accepted that some tissues such as brain can be visualized easier than others such as skeletal muscle. In addition, generating transgenic mice with a phenotype of interest and fluorescent proteins in appropriate cell types is often challenging and time consuming.