Chemistry Reference
In-Depth Information
Figure 3.1 Optical arrangements used for imaging single
molecules by total internal reflection fluorescence microscope
microscopy. A, objective-type TIRF microscope. B. prism-type
TIRF microscope. The clear regions between the glass slides and
the tapes are the flow chambers. The sample is located on the
bottom cover-slip in A and the top cover-slip in B.
arrow, the laser beam in the microscope objective moves laterally maintaining its
direction of propagation parallel to the optic axis. When the beam at the back focal
plane reaches a critical radius (explained further below), then the incident angle is
large enough to re
ect all of the energy at the interface, and the non-propagating
evanescent wave extends a few hundred nanometers into the aqueous sample
compartment. Commercial objective-type TIRF illuminators are also available that
use a movable
fiber optic source instead of L1 and M1.
In prism-type TIRFmicroscopy (Figure 3.1B), the evanescent wave is generated at
the opposite side of the sample compartment from the objective. The collimated
excitation laser beam is projected through a fused silica slide or prism to the aqueous
interface. Quartz or fused silica is used for this purpose because its intrinsic auto-
fluorescence is lower than that of glass. Polarization of the evanescent
eld is easier to
predict with this arrangement, but it adds complexity. The imaging objective in this
case is meant to operate at a longer working distance through the water of the sample
chamber, reducing the maximum NA
o
and brightness.
In either arrangement,
fluorescent material located at the re
ecting surface is
excited by the oscillating electromagnetic
field and emits
fluorescence. The emission
is collected by the objective lens and is projected onto the photo-detector, either a
camera or a photo-diode. Fluorescence frommaterial suspended in the medium but
within the 100
-
200-nm extent of the evanescent wave also contributes to the image.
This effect poses an upper limit of approximately 50 to 100 nM on the concentration
of
uorescent substrates or other
fluorescent ligands that bind to the protein
immobilized on the surface. An approach to circumvent this limitation is to use
