multidimensional single molecule spectroscopy

Multidimensional single-molecule fluorescence microscopy is a method to image the fluorescence from a population of single fluorophores discriminating in colour and polarisation. [See also Fluorescence Lifetime Imaging Microscopy (FLIM)).

The method has previously been used to generate two colour images of the fluorescence of individual tetramethylrhodamine-labelled biotin (FRET-donor) and Cy5-labelled strepavidin (FRET-acceptor), at two orthogonal polarisations, providing simultaneous single pair FRET (spFRET) and single molecule fluorescence polarisation (smFP) images (Cognet et al., 2000, App. Phys. Let. 77: 4052). SpFRET can measure the distance between two sites in a biomolecule (or between two interacting molecules) in the range between 1-10 nm, a length-scale encompassing the typical diameter of membrane proteins. The rate of energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor fluorophores, so time-dependent spFRET can report on very small fluctuations in the distance distribution of donor-acceptor pairs by ratioing the anti-correlated two-colour time-traces for donor and acceptor. In turn, smFP can completely determine the mean orientation of a single fluorophore relative to any convenient system of coordinates. Also, with a time resolution of 1 ms (typical time for a membrane protein to travel its diameter), smFP can characterise molecular rotations in a time scale of 10-100 ms.

There are several important gains to be made in studying the conformational dynamics at the single-molecule level. Individual molecules are intrinsically pure, homogeneous, and synchronised samples. At low densities, common under physiological conditions in the cell, the position of fluorescence-labelled proteins in the cell membrane can be imaged on a CCD detector with a 40 nm positional accuracy by non-linear least-squares fitting of the point spread function of the microscope. The location of these proteins can subsequently be tracked with a time resolution of a few ms at the same time as spFRET and smFP are measured. Unlike ensemble-averaged measurements, spFRET data from a single donor-acceptor pair can reveal transient conformation intermediates, even those with steady state concentrations too low to measure using conventional methods. Determining heterogeneity and degree of fluctuations can be crucial to understand EGFR function because seemingly identical copies of this receptor do not necessarily have the same function in the cell. Also, it is only at the single-molecule level that the four polar angles required to characterise a molecular orientation change, i.e. the two azimuthal (phi) and the two axial (theta) angles of the absorption and emission dipoles, can be simultaneously measured by smFP. This is because in single molecules only the fluorescence intensity is dependent on the direction of the excitation polarisation. The emission polarisation ratio does not depend on the latter, so absorption and emission polarisation ratios can be measured simultaneously. Simultaneous measurements of both polarisation ratios are not possible with ensemble-averaged techniques because the excitation polarisation biases the polarisation of the emitting light via photoselection. Also, the azimuthal angles relative to the excitation polarisation are lost in the ensemble average. SmFP polarisation ratios are determined from the intensity values of the anti-correlated parallel and perpendicular emission traces as a function of excitation polarisation. Molecular rotations will result in time-dependent changes in these polarisation ratios between successive time frames, and these rotations can then be characterised.

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