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. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy.

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. With the expected improvements in photocathode sensitivity and increase in detector throughput, this technology appears as a promising alternative to the current lifetime imaging solutions.

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 107 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. The imaging and fluorescence lifetime measurement performances of the H33D detector installed on a standard epifluorescence microscope will be presented. We compare them to those of standard single-molecule detectors such as single-photon avalanche photodiode (SPAD) or electron-multiplying camera using model samples (fluorescent beads, quantum dots and live cells). Finally, we discuss the design and applications of future generation of H33D detectors for single-molecule imaging and high-throughput study of biomolecular interactions.

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.

Electronic and optical moi? Interference with microchannel plates: artifacts and benefits.

The spatial resolution of position-sensitive detectors that use stacks of microchannel plates (MCP's) with high-resolution anodes can be better than 20-microm FWHM [Proc. SPIE 3114, 283-294 (1997)]. At this level of accuracy, channel misalignments of the MCP's in the stack can cause observable moiré interference patterns. We show that the flat-field detector response can have moiré beat pattern modulations of as great as approximately 27% with periods from as small as a few channel diameters to as great as the size of a MCP multifiber. These modulations, however, may be essentially eliminated by rotation of the MCP's or by a mismatch of the channel sizes. We also discuss how the modulation phenomena can be a useful tool for mapping the metric nonlinearities of MCP detector readout systems. Employing the optical moiré effect, we demonstrate a simple, but effective, technique for evaluation of geometrical deformations simultaneously over a large MCP area. For a typical MCP, with a 60-channel-wide multifiber, we can obtain accuracies of 1.2 mrad for multifiber rotations and twists and 35/(L/p) mrad for channel-long axis distortions (where L/p is MCP thickness to interchannel distance ratio). This technique may be used for the development of MCP x-ray optics, which impose tight limitations on geometrical distortions, which in turn are not otherwise easily measurable with high accuracy.

Low density of neutral hydrogen and helium in the local interstellar medium: Extreme Ultraviolet Explorer photometry of the Lyman continuum of the hot white dwarfs MCT 0501-2858, MCT 0455-2812, HZ 43, and GD 153.

The first comprehensive sky survey of the extreme ultraviolet (EUV) spectral range performed by the Extreme Ultraviolet Explorer (EUVE) has uncovered a handful of very bright sources at wavelengths longer than the He I 504 angstroms photoionization edge. Among these objects are four white dwarfs with exceptionally low interstellar medium (ISM) column densities along the line of sight. Analysis of EUV photometry of the He-rich DO white dwarf MCT 0501-2858 and the H-rich DA white dwarf MCT 0455-2812 along one line of sight and of the DA white dwarfs HZ 43 and GD 153 near the north Galactic pole indicates that the overall minimum column density of the neutral material centered on the Sun is N(H I)= 0.5-1.0 x 10(18) cm-2. In the case of MCT 0501-2858, EUV photometric measurements provide a clear constraint to the effective temperature (60,000-70,000 K). Given these neutral hydrogen columns, the actual contribution to the density of neutral species from the immediate solar environment (the "local fluff") would only cover a distance of approximately 2-3 pc (assuming an average density n(H I) = 0.1 cm-3) leaving these lines of sight almost entirely within the hot phase of the ISM. A preliminary examination of the complete EUVE long-wavelength survey indicates that these lines of sight are exceptional and set a minimum column density in the solar environment.

Small blaze angle gratings with various surface treatments for use in the extreme ultraviolet.

Three replica gratings were made from a single master; two were epoxy overcoated with different thicknesses of gold, and one was replicated directly in gold. Their performance in EUV light at grazing incidence angles was measured and compared.

Design of the Extreme Ultraviolet Explorer long-wavelength grazing incidence telescope optics.

Designing optics for photometry in the long-wavelength portion of the EUV spectrum (400-900 A) poses different problems from those arising for optics, operating shortward of 400 A. The available filter materials which transmit radiation longward of 400 A are also highly transparent at wavelengths shortward of 100 A. Conventional EUV optics, with grazing angles of less, similar10 degrees , have very high throughput in the EUV, which persists to wavelengths shortward of 100 A. Use of such optics with the longer-wavelength EUV filters thus results in an unacceptably large soft x-ray leak. We have overcome this problem by developing a mirror design with larger graze angles >/=20 degrees , which has high throughput at wavelengths longer than 400 A but at the same time very little throughput shortward of 100 A.

Composite thin-foil bandpass filter for EUV astronomy: titanium-antimony-titanium.

Thin metallic foils of antimony and titanium have been investigated in an attempt to develop an EUV filter with a bandpass from 350 to 550 A. A composite filter has been developed composed of antimony sandwiched between two titanium foils. The transmissions of sample composite foils and of pure titanium foils from 130 to 1216 A are presented. The absorption coefficients of antimony and titanium and the effect of titanium oxide on the transmission are derived. The composite filter has been found to be quite stable and mechanically rugged. Among other uses, the filter shows substantial promise for EUV astronomy.