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NEW -> Brown University announced the winners of their 2007
Seed Fund Awards. Jay Tang and his co-PIs have been awarded $90,000 for the study of Integrin Mediated Adhesion and
Retraction during T Cell Migration.
We are just beginning a research project focusing on integrin mediated force generation in migrating T cells. Migration is
is an important cellular process that has been shown to be involved in a wide range of applications including tissue repar,
immune surveillance, vascular diseases, chronic inflammations and even things like cancer, multiple sclerosis and mental retardation.
In our research we focus on the migration of T cells, which through a cyclic process of protrustion, adhesion and retraction are able to
migrate over substrates. We will be using Total Internal Reflection Fluorescence (TIRF) to probe the role of integrin and other proteins in
the process of migration and adhesion. The TIRF system will focus on the thin adhesion layer that it is in direct connection with the substrate
allowing distinct visualization of only those integrin that are involved in the migration process. In addition we will take adavantage of the
laboratory's Atomic Force Microscope (AFM) to probe the elastic nature and response of the migrating cells.
Total Internal Reflection Fluorescence Microscopy (TIRFM)
Total internal reflection is a well known optical phenomenon which relies upon the angle of incidence and the indices of refraction of
the two materials forming the interface. By introducing the light at an extremely sharp angle the light will be totally internally reflected
within the glass coverslip and the light will propogate parallel to the surface. This angle is determined through Snell's law:
When i > io we immediately see that sin r > 1, which means that r is a purely imaginary cosine.
The resulting physical meaning of this complex angle is immediately obvious when the value is inserted into the propagation factor of
the refracted wave.
The wave propagates parallel to the surface and has an exponential attenuation beyond the interface. This exponential attenuation is taken
advantage of in imaging adhesion sites of the cells. Because the light is attenuated within ~ 100 nm, only those fluorophores right next
to the surface of the glass coverslip can be excited. Everything beyond this penetration depth is left unexcited. The resulting image
shows almost no background fluorescence and makes focal adhesion sites easily identifiable.
Our TIRFM system is set up on a Nikon TE-2000 equipped with the Nikon white light TIRF illuminator.
TIRFM AFM Combination
We've recently added the capability to image cells simultaneously using the TIRFM in addition to a tradiational Atomic Force Microscope
(AFM). A diagram depicting the setup is shown below:
This setup allows us to both measure local forces using the AFM, and to use the AFM tip as a probe to stimulate the cell on the
coverslip. If the tip is used as a probe the TIRF can provide real time monitoring of changes in the adhesion sites on the cell. It
will be possible to engage cells as they migrate across the coverslip surface.
 An example of a T cell labeled with Alexa 488 and Cy 3 and imaged using both the TIRFM and AFM.
With this new capability we hope to achieve the following goals:
- Measure and track the dynamic activity of activated integrin as the cell migrates
- Record how actiev integrins redistribute when the cell is subjected to applied local forces from the AFM tip
- Measure the retraction force of the cell as it migrates using the AFM
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