Single-molecule detection by fluorescence microscopy is limited by the presence of an excess of out-of-focus fluorescent molecules. How does a TIRF microscope uniquely overcome this limitation?
A) By removal of the out-of-focus molecules by selective destruction.
B) By using a deconvolution algorithm that reverses the convolution of signals due to the out-of-focus molecules.
C) By a confocal set-up that eliminates out-of-focus signals.
D) By exciting only in-focus molecules via an evanescent field.
E) By using an objective lens with extremely high numerical aperture.
20-21 Atomic Force Microscopy
Atomic force microscopy (AFM) is used in an experiment to unfold a multidomain protein by applying mechanical force. The protein contains several copies of an immunoglobulin domain that are unfolded one by one as the two ends of the molecule (one attached to a cover slip, and the other to the AFM tip) are being pulled apart, resulting in the "sawtooth" force-extension curves shown below. The same experiment is done twice, once in the presence and once in the absence of a chaperone protein that stabilizes the immunoglobulin domains. Answer the following questions based on this graph.

Question

Quizplus · Accepted Answer

In a total internal reflection fluorescence (TIRF) microscope, an evanescent wave excites only those fluorescent molecules that lie within an extremely thin plane adjacent to the cover slip.

Single-Molecule Detection by Fluorescence Microscopy Is Limited by the Presence