Silicon photon-counting avalanche diodes for single-molecule fluorescence spectroscopy.

Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with applications in cell biology, biochemistry and biophysics. The basic feature of this technique is to excite and collect light from a very small volume and work in a low concentration regime resulting in rare burst-like events corresponding to the transit of a single molecule. Detecting photon bursts is a challenging task: the small number of emitted photons in each burst calls for high detector sensitivity.

Toward single-molecule optical mapping of the epigenome.

The past decade has seen an explosive growth in the utilization of single-molecule techniques for the study of complex systems. The ability to resolve phenomena otherwise masked by ensemble averaging has made these approaches especially attractive for the study of biological systems, where stochastic events lead to inherent inhomogeneity at the population level. The complex composition of the genome has made it an ideal system to study at the single-molecule level, and methods aimed at resolving genetic information from long, individual, genomic DNA molecules have been in use for the last 30 years.

Development of new photon-counting detectors for single-molecule fluorescence microscopy.

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.

Phasor imaging with a widefield photon-counting detector.

Fluorescence lifetime can be used as a contrast mechanism to distinguish fluorophores for localization or tracking, for studying molecular interactions, binding, assembly, and aggregation, or for observing conformational changes via Förster resonance energy transfer (FRET) between donor and acceptor molecules. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful technique but its widespread use has been hampered by demanding hardware and software requirements. FLIM data is often analyzed in terms of multicomponent fluorescence lifetime decays, which requires large signals for a good signal-to-noise ratio.

Parallel multispot smFRET analysis using an 8-pixel SPAD array.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales.

Superresolution optical fluctuation imaging (SOFI).

Superresolution microscopy has shifted the limits for fluorescence microscopy in cell -biology. The possibility to image cellular structures and dynamics of fixed and even live cells and organisms at resolutions of several nanometers holds great promise for future biological discoveries. We recently introduced a novel superresolution technique, based on the statistical evaluation of stochastic fluctuations stemming from single emitters, dubbed "superresolution optical fluctuation -imaging" (SOFI).

New photon-counting detectors for single-molecule fluorescence spectroscopy and imaging.

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. Two typical geometries can be used for these experiments: point-like and widefield excitation and detection. In point-like geometries, the basic concept is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule.

Microchannel Plate Imaging Photon Counters for Ultraviolet through NIR Detection with High Time Resolution.

Cross strip and cross delay line readout microchannel plate detectors in 18 mm, 25 mm and 40 mm active area formats including open face (UV/particle) and sealed tube (optical) configurations have been constructed. These have been tested with a field programmable gate array based electronics for single event encoding. Using small pore MCPs (6 μm) operated in a pair, we achieve gains of >1 × 10 which is sufficient to provide spatial resolution of ~17 μm FHWM with the 18 mm and 40 mm cross strip readouts.

Ultra high-throughput single molecule spectroscopy with a 1024 pixel SPAD.

Single-molecule spectroscopy is a powerful approach to measuring molecular properties such as size, brightness, conformation, and binding constants. Due to the low concentrations in the single-molecule regime, measurements with good statistical accuracy require long acquisition times. Previously we showed a factor of 8 improvement in acquisition speed using a custom-CMOS 8x1 SPAD array.

High-throughput FCS using an LCOS spatial light modulator and an 8 × 1 SPAD array.

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.

Achieving increased resolution and more pixels with Superresolution Optical Fluctuation Imaging (SOFI).

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.

High-throughput single-molecule fluorescence spectroscopy using parallel detection.

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy.

High-throughput multispot single-molecule spectroscopy.

Solution-based single-molecule spectroscopy and fluorescence correlation spectroscopy (FCS) are powerful techniques to access a variety of molecular properties such as size, brightness, conformation, and binding constants. However, this is limited to low concentrations, which results in long acquisition times in order to achieve good statistical accuracy. Data can be acquired more quickly by using parallelization.

Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI).

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.

Single-quantum dot imaging with a photon counting camera.

The expanding spectrum of applications of single-molecule fluorescence imaging ranges from fundamental in vitro studies of biomolecular activity to tracking of receptors in live cells. The success of these assays has relied on progress in organic and non-organic fluorescent probe developments as well as improvements in the sensitivity of light detectors. We describe a new type of detector developed with the specific goal of ultra-sensitive single-molecule imaging.

Phasor-based single-molecule fluorescence lifetime imaging using a wide-field photon-counting detector.

Fluorescence lifetime imaging (FLIM) is a powerful approach to studying the immediate environment of molecules. For example, it is used in biology to study changes in the chemical environment, or to study binding processes, aggregation, and conformational changes by measuring Förster resonance energy transfer (FRET) between donor and acceptor fluorophores. FLIM can be acquired by time-domain measurements (time-correlated single-photon counting) or frequency-domain measurements (with PMT modulation or digital frequency domain acquisition) in a confocal setup, or with wide-field systems (using time-gated cameras).

A novel fluorescence lifetime imaging system that optimizes photon efficiency.

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.

Surgical treatment of the infected hip implant. Two-stage reimplantation with a one-month interval.

Successful treatment of the infected hip prosthesis demands careful planning for infection control and reimplantation. Inadequate surgical debridement dooms well-executed revision surgery to infection failure. Reimplantation is difficult after resection arthroplasty, and this difficulty is increased with longer intervals between resection and reimplantation.

Case report 689. Metastatic atrial myxoma to the pubis.

We have presented a 23-year follow-up of a patient with left atrial myxoma with continuing slowly growing, skeletal metastases. The relatively indolent nature of the metastatic disease and the lack of mitotic figures suggest that the metastatic lesions may be treated locally. The patient underwent therapeutic tumor embolization and local resection of the pelvic lesion, with good results as she continues to do well with close follow-up.

Magnetic resonance imaging of extremity masses.

Twenty-seven adults with extremity masses were examined by magnetic resonance imaging (MRI). In 26/27 cases, computed tomography (CT) scans were available for comparison. Imaging with multiple pulse sequences is necessary to optimize diagnosis by MRI.

Surgical stabilization of pathological neoplastic fractures.

The most important factor to consider in deciding between treatment options in the management of metastatic bone disease is the level of the patient's dysfunction and pain. Severe dysfunction or pain demands a treatment that predictably leads to a quick resumption of the painless activities of daily living. A treatment that predictably will restore function in months may seem reasonable in patients with a normal remaining life span, but is untenable if those months represent a high percentage of remaining life span, as they do in metastatic disease afflicted patients.

An aggressive treatment approach for adult osteomyelitis.

Osteomyelitis in the adult patient has been associated with failure of eradication, late recurrence, nonunion, and prolonged hospitalization. A staged aggressive approach has been used for the past seven years to treat 53 patients with adult osteomyelitis. This approach includes: evaluation of bone necrosis and identification of the etiologic organisms by deep bone culture; radical surgical debridement of devascularized tissue; intensive systemic antibiotics; and early bone and soft tissue reconstruction.