Maximum-Likelihood Position Sensing of Single Molecules in a Confocal Microscope

Abstract

In wide-field microscopy, the location of a single molecule can be measured to within tens of nanometers by imaging the point spread function over a number of camera pixels and finding the center of the image. However, for many single-molecule applications, confocal microscopy is preferable to wide-field imaging, as it provides improved signal-to-noise ratio due to the very small detection volume; it is necessary for two-photon excitation, which offers potential advantages for intracellular studies; and it facilitates monitoring of sub-millisecond dynamics and fluorescence lifetimes by use of a single-photon avalanche diode (SPAD) detector for time-resolved single-photon counting. Here, we report studies of the capabilities for sub-diffraction, single-nanoparticle position determination in a confocal two-photon microscope. To measure the position, the beam from a femtosecond laser is split and recombined at beam splitters to produce four beams that are focused to slightly offset spatial positions centered at the vertices of a tetrahedron, and with pulses that are temporally offset so as to yield pulse-interleaved excitation at the four overlapping focal volumes. Two-photon-excited fluorescence is collected from the entire four-beam excitation volume onto just one SPAD detector. Time-gated photon detection provides information on the volume from which each photon was most likely emitted and hence, the most likely position of the single particle. As this form of position sensing requires only a single SPAD, it is scalable to multiple detectors for multi-color observations, and can thus be used to find the separations of differently colored molecules over a distance range that is complementary to that achievable by FRET.