Development of ultrafast camera-based single fluorescent-molecule imaging for cell biology.

The spatial resolution of fluorescence microscopy has recently been greatly enhanced. However, improvements in temporal resolution have been limited, despite their importance for examining living cells. Here, we developed an ultrafast camera system that enables the highest time resolutions in single fluorescent-molecule imaging to date, which were photon-limited by fluorophore photophysics: 33 and 100 µs with single-molecule localization precisions of 34 and 20 nm, respectively, for Cy3, the optimal fluorophore we identified. Using theoretical frameworks developed for the analysis of single-molecule trajectories in the plasma membrane (PM), this camera successfully detected fast hop diffusion of membrane molecules in the PM, previously detectable only in the apical PM using less preferable 40-nm gold probes, thus helping to elucidate the principles governing the PM organization and molecular dynamics. Furthermore, as described in the companion paper, this camera allows simultaneous data acquisitions for PALM/dSTORM at as fast as 1 kHz, with 29/19 nm localization precisions in the 640 × 640 pixel view-field.

Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics.

Using our newly developed ultrafast camera described in the companion paper, we reduced the data acquisition periods required for photoactivation/photoconversion localization microscopy (PALM, using mEos3.2) and direct stochastic reconstruction microscopy (dSTORM, using HMSiR) by a factor of ≈30 compared with standard methods, for much greater view-fields, with localization precisions of 29 and 19 nm, respectively, thus opening up previously inaccessible spatiotemporal scales to cell biology research. Simultaneous two-color PALM-dSTORM and PALM-ultrafast (10 kHz) single fluorescent-molecule imaging-tracking has been realized. They revealed the dynamic nanoorganization of the focal adhesion (FA), leading to the compartmentalized archipelago FA model, consisting of FA-protein islands with broad diversities in size (13-100 nm; mean island diameter ≈30 nm), protein copy numbers, compositions, and stoichiometries, which dot the partitioned fluid membrane (74-nm compartments in the FA vs. 109-nm compartments outside the FA). Integrins are recruited to these islands by hop diffusion. The FA-protein islands form loose ≈320 nm clusters and function as units for recruiting FA proteins.

Internal dynamics of supercoiled DNA molecules.

The intramolecular diffusive motion within supercoiled DNA molecules is of central importance for a wide array of gene regulation processes. It has recently been shown, using fluorescence correlation spectroscopy, that plasmid DNA exhibits unexpected acceleration of its internal diffusive motion upon supercoiling to intermediate density. Here, we present an independent study that shows a similar acceleration for fully supercoiled plasmid DNA. We have developed a method that allows fluorescent labeling of a 200-bp region, as well as efficient supercoiling by Escherichia coli gyrase. Compared to plain circular or linear DNA, the submicrosecond motion within the supercoiled molecules appears faster by up to an order of magnitude. The mean-square displacement as a function of time reveals an additional intermediate regime with a lowered scaling exponent compared to that of circular DNA. Although this unexpected behavior is not fully understood, it could be explained by conformational constraints of the DNA strand within the supercoiled topology in combination with an increased apparent persistence length.