Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/ intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.cumulants ͉ fluorescence ͉ quantum dots ͉ superresolution microscopy ͉ intermittency

Superresolution Optical Fluctuation Imaging (SOFI) as initially demonstrated allows for a resolution enhancement in imaging by a factor of square-root of two. Here, we demonstrate how to increase the resolution of SOFI images by re-weighting the Optical Transfer Function (OTF). Furthermore, we demonstrate how cross-cumulants can be exploited to obtain a fair approximation of the underlying Point-Spread Function. We show a two-fold increase of resolution (over the diffraction limit) of near-infrared quantum dot labeled tubulin-network of 3T3 fibroblasts

Fluorescence lifetime imaging (FLIM) is a powerful microscopy technique for providing contrast of biological and other systems by differences in molecular species or their environments. However, the cost of equipment and the complexity of data analysis have limited the application of FLIM. We present a mathematical model and physical implementation for a low cost digital frequency domain FLIM (DFD-FLIM) system, which can provide lifetime resolution with quality comparable to time-correlated single photon counting methods. Our implementation provides data natively in the form of phasors. On the basis of the mathematical model, we present an error analysis that shows the precise parameters for maximizing the quality of lifetime acquisition, as well as data to support this conclusion. The hardware and software of the proposed DFD-FLIM method simplifies the process of data acquisition for FLIM, presents a new interface for data display and interpretation, and optimizes the accuracy of lifetime determination. Microsc. Res. Tech. 71:201-213, 2008. V V C 2007 Wiley-Liss, Inc. INTRODUCTIONWe have developed a physical implementation and statistical model of a new method for fluorescence lifetime imaging (FLIM) data collection and analysis. Our approach falls in the category of the ''frequency domain'' approach to lifetime acquisition, yet uses a detector operating in the photon counting mode. This digital frequency domain (DFD) method overcomes the problems of duty cycle, modulation of the detector gain and expensive radio frequency synthesizers used in the classical analog frequency domain approach. In our approach we implemented all the operations performed in a frequency-domain lifetime instrument in a digital form using a single field programmable chip. Since all operations including the generation of the light modulation frequency, the generation of the cross-correlation sampling frequency and the assignment of the time of arrival of a photon to a bin are digital, there are no calibrations or adjustments to be performed. The mathematical model presented below fully accounts for all the elements of the DFD method. In addition, the mathematical model reproduces, as a limiting case, the principle of the time-correlated single photon counting (TCSPC) approach. Therefore, on a common statistical basis we can compare the two approaches and derive some general conclusions about the relative precision of the two methods. We found that with proper system design the two methods can be made to have comparable precision. More importantly, the mathematical model was used to maximize the precision of the DFD implementation and to determine which parameters are crucial to reach optimal performance. In terms of precision of the lifetime measurement, we were able to fully quantify the effect of the instrument response including the jitter of the detection system.Fluorescence lifetime is a fundamental spectroscopic quantity that allows quantitative analysis through several approaches, including the identification of molecu-

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level

We present a novel approach to high-throughput Fluorescence Correlation Spectroscopy (FCS) which enables us to obtain one order of magnitude improvement in acquisition time. Our approach utilizes a liquid crystal on silicon spatial light modulator to generate dynamically adjustable focal spots, and uses an eight-pixel monolithic single-photon avalanche photodiode array. We demonstrate the capabilities of this system by showing FCS of Rhodamine 6G under various viscosities, and by showing that, with proper calibration of each detection channel, one order of magnitude improvement in acquisition speed is obtained. More generally, our approach will allow higher throughput single-molecule studies to be performed.