So this past week we all did presentations on what we believed our projects were about. I had received three papers from a grad student working with my mentor and had read through them before arriving, but this presentation gave me a chance to do some outside research on the mechanics of TIRF microscopy and some of the various applications which were really interesting. An elaborated version of my presentation follow,
I will be working with Dr. Cédric Bouzigues this summer at Le labortoire d’Optiques et Biologies. We will be imaging interactions between a single protein and a strand of dna using TIRF microscopy.
With many forms of microscopy a specimen can be overwhelmed by background fluorescence and not be imaged clearly. TIRF eliminated this issue and allowed for the observations of thin (100 nm) layers of a specimen or even the imaging of single molecules. This allows scientists to study the position and dynamics of molecules in living culture cells.
Figure 1: A TIRF Microscope
TIRF uses an evanescent electromagnetic field whose intensity decays exponentially with distance. Because of this property, these evanescent waves illuminate only the top layer of the specimen. The portion of the specimen in the evanescent range is excited and emits fluorescent light which is what makes it visible. As seen in these two images the difference is extraordinary. The top image was created with EPI-fluorescence while the bottom one was created with TIRF.
Evanescent waves are generated from total internal reflection between the microscope and water interface. Total internal reflection occurs when a wave hits a surface at an angle greater than the critical angle of that surface. As a result the wave is fully reflected. Evanescent waves produced under normal conditions have zero net energy. In this diagram it propagtes in the x direction and would decrease exponentially in the z direction.
Figure 2: An EPI-fluorescent image (top) is contrasted with the higher resolution TIRF image (bottom).
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