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.

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.

Detectors for single-molecule fluorescence imaging and spectroscopy.

Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines.

Photon-Counting H33D Detector for Biological Fluorescence Imaging.

We have developed a photon-counting High-temporal and High-spatial resolution, High-throughput 3-Dimensional detector (H33D) for biological imaging of fluorescent samples. The design is based on a 25 mm diameter S20 photocathode followed by a 3-microchannel plate stack, and a cross delay line anode. We describe the bench performance of the H33D detector, as well as preliminary imaging results obtained with fluorescent beads, quantum dots and live cells and discuss applications of future generation detectors for single-molecule imaging and high-throughput study of biomolecular interactions.

A space- and time-resolved single photon counting detector for fluorescence microscopy and spectroscopy.

We have recently developed a wide-field photon-counting detector having high-temporal and high-spatial resolutions and capable of high-throughput (the H33D detector). Its design is based on a 25 mm diameter multi-alkali photocathode producing one photo electron per detected photon, which are then multiplied up to 10 times by a 3-microchannel plate stack. The resulting electron cloud is proximity focused on a cross delay line anode, which allows determining the incident photon position with high accuracy.

Fluorescence lifetime microscopy with a time- and space-resolved single-photon counting detector.

We have recently developed a wide-field photon-counting detector (the H33D detector) having high-temporal and high-spatial resolutions and capable of recording up to 500,000 photons per sec. Its temporal performance has been previously characterized using solutions of fluorescent materials with different lifetimes, and its spatial resolution using sub-diffraction objects (beads and quantum dots). Here we show its application to fluorescence lifetime imaging of live cells and compare its performance to a scanning confocal TCSPC approach.

ASIC-based event-driven 2D digital electron counter for TEM imaging.

A two-dimensional application specific integrated circuit (ASIC) based detector, designed for X-ray protein crystallography, has been tested to determine its suitability as a direct electron detector for TEM imaging in the voltage range of 20-400 keV. Several markedly different properties of this device distinguish it from the charge coupled device (CCD) detectors: (1) the ASIC detector can be used directly under electron bombardment in the voltage range stated above, therefore requiring no scintillator screen; (2) each active pixel of the device is an electron counter and generates digital output independently; (3) the readout of the device is frameless and event driven; (4) the device can be operated at the room temperature and is nearly noise free; and (5) the counting dynamic range of the device is virtually unlimited. It appears that an imaging system based on this type of device would be ideal for low-dose TEM imaging and online diffraction observation and recording, as well as more conventional imaging, providing the many advantages of direct digital readout for almost all applications.