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. Therefore the
advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data
acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation
and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate
the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence
measurements obtained with different novel multipixel single-photon counting detectors.
We have recently developed a wide-field photon-counting detector (the H33D detector) having high-temporal and highspatial
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.
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<sup>7</sup> 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.
In this paper we give a brief report on the development of simple direct- and indirect-detection imagers for proton radiography experiments. We outline a conceptual design for a novel, multi-frame 5 mega frames per second (Mfs) hybrid imager. The high-density interconnect is identified as a critical enabling technology. We present a description of a 3D electronics packaging cube, which was completed in a recent feasibility study.
The need for higher throughput, higher dynamic range detectors for protein crystallography is stimulating work on new detector concepts. Current detectors such as photo- cathode converters optically coupled to Charge Coupled Devices (CCDs) use a two step detection process. The incoming x-ray is converted in the photo-cathode in low energy photons. These longer wavelength photons can be efficiency detected in the CCD where they create an electrical charge. The overall process is quite inefficient as few photons are generated in the primary converter and fewer are collected in the CCD. Due to the scarcity of photons per x-ray an event driven processing of the information is impossible. New approaches favors the direct detection of the incoming photon using either semiconductor detectors, the most commonly used being silicon, or gas detectors with internal amplification. The incoming photons directly create change in the detector at levels sufficient to be processed on a per event basis. All these approaches share common features: event level processing, large dynamic range, and fast readout leading to high throughput. While the primary application is in photon counting these detectors are also capable of providing multi-parameter data such as the measure of the x-ray energy and its time of occurrence. Several groups are working on the development of these detectors using different approaches to the capture of the information and its readout. The capabilities and limitations of these implementations are reviewed.
A 2D photon counting digital pixel array detector is being designed for static and time resolved protein crystallography. This room temperature detector will significantly enhance monochromatic and polychromatic protein crystallographic throughput data rates by more than two or three orders of magnitude when compared to present data collection systems. The detector has an unbounded photon counting dynamic range and exhibits superior spatial resolution when compared to present crystallographic phosphor imaging plates or phosphor coupled CCD detectors. The detector is a high resistivity N-type Si with a pixel pitch of (150 X 150) microns, and a thickness of 300 microns that is bump bonded to an application specific integrated circuit. The event driven readout of the detector is based on the column architecture and allows an independent pixel bit rate above 1 million photons/sec. The device provides energy discrimination and sparse data readout that yields minimal dead time. This type of architecture allows an almost continuous (frame-less) data acquisition, a feature not found in any current detector being used for protein crystallographic applications. For the targeted detector size of (1000 X 1000) pixels, average hit rates greater than 10<SUP>11</SUP> photons/sec for the complete detector appears achievable. This paper will present an overview of the hybridized detector performance which includes the analog amplifier response and the photon counting capabilities of the (16 X 16) array operating with both digital and analog circuitry. Also the operation of the serial interface will be described.
A high frame rate optically shuttered CCD camera for radiometric imaging of transient optical phenomena has been designed and several prototypes fabricated, which are now in evaluation phase. the camera design incorporates stripline geometry image intensifiers for ultra fast image shutters capable of 200ps exposures. The intensifiers are fiber optically coupled to a multiport CCD capable of 75 MHz pixel clocking to achieve 4KHz frame rate for 512 X 512 pixels from simultaneous readout of 16 individual segments of the CCD array. The intensifier, Philips XX1412MH/E03 is generically a Generation II proximity-focused micro channel plate intensifier (MCPII) redesigned for high speed gating by Los Alamos National Laboratory and manufactured by Philips Components. The CCD is a Reticon HSO512 split storage with bi-direcitonal vertical readout architecture. The camera main frame is designed utilizing a multilayer motherboard for transporting CCD video signals and clocks via imbedded stripline buses designed for 100MHz operation. The MCPII gate duration and gain variables are controlled and measured in real time and up-dated for data logging each frame, with 10-bit resolution, selectable either locally or by computer. The camera provides both analog and 10-bit digital video. The camera's architecture, salient design characteristics, and current test data depicting resolution, dynamic range, shutter sequences, and image reconstruction will be presented and discussed.
A 2D pixel array image sensor module has been designed for time resolved Protein Crystallography. This smart pixels detector significantly enhances time resolved Laue Protein crystallography by two or three orders of magnitude compared to existing sensors like films or phosphor screens coupled to CCDs. The resolution in time and dynamic range of this type of detector will allow to study the evolution of structural changes that occur within the protein as a function of time. This detector will also considerably accelerate data collection in static Laue or monochromatic crystallography and make better use of the intense beam delivered by synchrotron light sources. The event driven pixel array detectors, based on the column architecture, can provide multiparameter information (energy discrimination, time), with sparse and frameless readout without significant dead time. The prototype module consists of a 16 by 16 pixel diode array bump-bonded to the integrated circuit. Different detector materials (Silicon, CdZnTe) are evaluated. The detection area is 150 by 150 micrometers<SUP>2</SUP> connected to the readout electronics. The individual pixel processor consists of a low-noise amplifier shaper followed by a differential threshold comparator which provides the counting of individual photons with an energy above a programmable threshold. To accommodate the very high rates, above 5 by 10<SUP>8</SUP>/cm<SUP>2</SUP>/s, each pixel processor has a 3 bit pre-scaler which divides the event rate by 8. Overflow from the divider which defines a pseudo fourth bit will generate a readout sequence providing the pixel address. Addresses, generated locally as analog signals, are converted off-chip and used to increment a location in an histogramming memory to generate the computerized image of the Laue diagram.