Intravital microscopy
This article needs additional citations for verification. (June 2013) |
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.[1]
In mammals, in some experimental settings a surgical implantation of an imaging window is performed prior to intravital microscopy. This allows repeated observations over several days or weeks. For example, if researchers want to visualize liver cells of a live mouse they will implant an imaging window into mouse's abdomen.[2] Mice are the most common choice of animals for intravital microscopy but in special cases other
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. This is useful for many areas of research including immunology[3] and stem cell research.[1]
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 an important role in intravital microscopy development.[1]
The possibility of generating appropriate transgenic mice is crucial for 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.[4]
Imaging
Intravital microscopy can be performed using several light microscopy techniques including widefield fluorescence, confocal, multiphoton, spinning disc microscopy and others. The main consideration for the choice of a particular technique is the penetration depth needed to image the area and the amount of cell-cell interaction details required.
If the area of interest is located more than 50–100 µm below the surface or there is a need to capture small-scale interactions between cells, multiphoton microscopy is required. Multiphoton microscopy provides considerably greater depth of penetration than single-photon confocal microscopy.[5] Multiphoton microscopy also allows visualizing cells located underneath bone tissues such as cells of the bone marrow.[6] The maximum depth for the imaging with multiphoton microscopy depends on the optical properties of the tissue and experimental equipment. The more homogenous the tissue is the better it is suited for intravital microscopy. More vascularized tissues are generally more difficult to image because red blood cells cause absorption and scattering of the microscope light beam.[1]
Fluorescence labeling of different cell lineages with differently coloured proteins allows visualizing cellular dynamics in a context of their
It might be useful to pair up multiphoton and confocal microscopy as this 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.[7] This latter is an example of the Three Rs principle implementation.
Imaging subcellular structures
In the past, intravital microscopy could only be used to image biological processes at tissue or single-cell levels. However, due to development of subcellular labeling techniques and advances in minimizing motion artifacts (errors generated by heartbeat, breath and
Limitations of intravital microscopy
One of the main advantages of intravital microscopy is the opportunity to observe how cells interact with their
References
External links
- Media related to Intravital microscopy at Wikimedia Commons