Total Internal Reflection Fluorescence Microscopy (TIRFM)
We are using this technique to visualise single fluorophores (Cy-3 or Cy-5
labelled ATP).
Is it really possible to visualise single fluorophores in solution?
There are a number of difficult problems to overcome, but yes, it is possible
and has been done. The three main problems are: finding a sensitive enough
detector; eliminating background excitation light and eliminating out of
focus fluorescence from the bulk solution. These problems are solved by
using TIRFM.
How does TIRFM work?
Total internal reflection is an optical phenomenon which occurs when light
propagating in a dense medium (such as glass) meets an interface with a
less dense medium, such as water. If the light meets the interface at a
small angle, some of the light passes through the interface (is refracted)
and some is reflected back into the dense medium. At a certain angle, all
of the light is reflected. This angle is known as the critical angle, and
its value depends on the refractive indices of the media (n1,
n3):
However, some of the energy of the beam propagates a short distance (a
few hundred nanometres) into the water, generating an evanescent
wave. If this energy is not absorbed, it passes back into the glass.
However, if a fluorophore molecule is within the evanescent wave it can
absorb photons and be excited. In this way, it is possible to get fluorescence
with a very low background of excitation light.
How can the fluorescence be detected?
The levels of fluorescence from a single fluorophore are extremely low
(hundreds to thousands of photons per second). We plan to observe this
fluorescence in two ways. The first is to use an intensified CCD camera
which will produce an image, in which bound fluorophores will appear as
bright spots. This will enable us to identify sites on the surface where
myosin molecules may be bound. For detection during experiments, we plan
to image the fluorophore through a pinhole onto a photomultiplier tube
(PMT), with which we can count the number of photons detected. This should
give us a very high signal to noise ratio, and allow us clearly to determine
when the fluorophore binds to the myosin, and when it dissociates.