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Super-resolution imaging is achieved by localizing diffraction-limited spots with high accuracy. Here we combined two powerful approaches to image the chromosomal DNA inside cells. In one method, the accumulated, stochastic binding of fluorescent labels to an imaging target are localized, while in the other the fluorophore transitions between dark and bright states (compatible with binding, photobleaching, photo-activation, blinking, etc.), even when fluorophore images overlap, are localized. Combining the two techniques results in a robust microscopy that is faster than what is possible with either technique alone, requires less optimization, and corrects for cell autofluorescence. In addition, background noise due to fluorescent labels in solution can be virtually eliminated by using labels that fluoresce only when bound to the target.Many DNA-specific dyes show dramatic fluorescence enhancement upon binding to DNA, including SYTO, LOLO, and YOYO dyes. We used nanomolar concentrations of SYTO and LOLO to image lambda DNA attached to poly-L-lysine coated glass and chromosomal DNA in fixed HEK 293 and HeLa cells. We found average single-fluorophore localization errors of 36 nm and 24 nm on glass and in cells, respectively. These imaging techniques may prove useful in future studies of chromosomal DNA in cells, including chromatin structure and defects.This approach was further applied to imaging microtubules in vitro. We used commercial Oregon Green 488 paclitaxel to achieve a 10 nm average fluorophore localization error, and streptavidin S45A to transiently label biotinylated microtubules with Atto647N, resulting in an average of 18 nm fluorophore localization error. Future work will involve simultaneous imaging of DNA and proteins to answer important biological questions.

Quantum dots (q-dots) are bright and long-lasting fluorescence probes that are widely used in in vitro and in vivo experiments. Here, we report 2-photon (2P) widefield excitation of single q-dots in aqueous solutions which allows fast real time imaging. We show this on a Myosin V, labeled on one head with a 655 nm qdot. With 50 msec integration time, 2 μM ATP, 840 nm excitation, we achieve 1.0 nm spatial accuracy and can watch the motor move over 20 steps. This compares well with 1-photon excitation at 532 nm. We find that adding DTT or BME to the imaging buffer essentially eliminates blinking. We also demonstrate 2P microscopy of individual q-dots by scanning with a diffraction-limited spot, allowing z-resolution depth discrimination. In this optical arrangement, we can use an EMCCD, instead of the usual PMT, as the detector, which considerable simplifies matters. In this arrangement, we show 3D imaging of q-dot-labeled E.coli LamB receptors. Our technique opens great possibilities for fast live cell and tissue imaging.

Viral infection begins with the binding of a virus to a specific target on the surface of the host cell, followed by viral genome delivery into the host and a continuation of the infection process. Before binding occurs, the virus must first find its receptor by a process whose details are largely unknown. We applied high-resolution fluorescence microscopy and single-particle tracking to elucidate the target-finding process in bacteriophage λ as it infects an Escherichia coli cell. By monitoring the motion of individual viruses through the early stages of infection, we identified a unique spatial focusing process that allows a virus to arrive from its initial random landing site to its destination at the cell pole. The search process is governed by the interaction between the virus and the LamB receptors, and by the spatial organization of the receptor network on the cell surface. Our findings allowed us to develop a theoretical model for the target-finding process that reproduces the key features observed in experiment. We discuss the possible implications of our findings for the process of viral receptor-finding in higher systems.