Search

Single-molecule fluorescence spectroscopy (SMFS), based on the detection of individual molecules freely diffusing through the excitation spot of a confocal microscope, has allowed unprecedented insights into biological processes at the molecular level, but suffers from limited throughput. We have recently introduced a multispot version of SMFS, which allows achieving high-throughput SMFS by virtue of parallelization, and relies on custom silicon single-photon avalanche diode (SPAD) detector arrays. Here, we examine the premise of this parallelization approach, which is that data acquired from different spots is uncorrelated. In particular, we measure the optical crosstalk characteristics of the two 48-pixel SPAD arrays used in our recent SMFS studies, and demonstrate that it is negligible (crosstalk probability ≤ 1.1 10-3) and undetectable in cross-correlation analysis of actual single-molecule fluorescence data.

Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Single-molecule fluorescence spectroscopy (SMFS), based on the detection of individual molecules freely diffusing through the excitation spot of a confocal microscope, has allowed unprecedented insights into biological processes at the molecular level, but suffers from limited throughput. We have recently introduced a multispot version of SMFS, which allows achieving high-throughput SMFS by virtue of parallelization, and relies on custom silicon single-photon avalanche diode (SPAD) detector arrays. Here, we examine the premise of this parallelization approach, which is that data acquired from different spots is uncorrelated. In particular, we measure the optical crosstalk characteristics of the two 48-SPAD arrays used in our recent SMFS studies, and demonstrate that it is negligible (crosstalk probability ≤ 1.1 10 −3 ) and is undetectable in cross-correlation analysis of actual single-molecule fluorescence data.

Single-molecule FRET (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser exci-tation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hin-ders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Single-molecule spectroscopy on freely-diffusing molecules allows detecting conformational changes of biomolecules without perturbation from surface immobilization. Resolving fluorescence lifetimes increases the sensitivity in detecting conformational changes and overcomes artifacts common in intensity-based measurements. Common to all freely-diffusing techniques, however, are the long acquisition times. We report a time-resolved multispot system employing a 16-channel SPAD array and TCSPC electronics, which overcomes the throughput issue. Excitation is obtained by shaping a 532 nm pulsed laser into a line, matching the linear SPAD array geometry. We show that the line-excitation is a robust and cost-effective approach to implement multispot systems based on linear detector arrays.

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

Single-molecule spectroscopy on freely-diffusing molecules allows detecting conformational changes of biomolecules without perturbation from surface immobilization. Resolving fluorescence lifetimes increases the sensitivity in detecting conformational changes and overcomes artifacts common in intensity-based measurements. Common to all freely-diffusing techniques, however, are the long acquisition times. We report a time-resolved multispot system employing a 16-channel SPAD array and TCSPC electronics, which overcomes the throughput issue. Excitation is obtained by shaping a 532 nm pulsed laser into a line, matching the linear SPAD array geometry. We show that the line-excitation is a robust and cost-effective approach to implement multispot systems based on linear detector arrays.

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. Bursts are very brief, requiring detectors with fast response time and capable of sustaining high count rates. Finally, many bursts need to be accumulated to achieve proper statistical accuracy, resulting in long measurement time unless parallelization strategies are implemented to speed up data acquisition. In this paper we will show that silicon single-photon avalanche diodes (SPADs) best meet the needs of single-molecule detection. We will review the key SPAD parameters and highlight the issues to be addressed in their design, fabrication and operation. After surveying the state-of-the-art SPAD technologies, we will describe our recent progress towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. The potential of this approach is illustrated with single-molecule Förster resonance energy transfer measurements.

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.

Inductor current sensing is becoming widely used in current programmed controllers for microprocessor applications. This method exploits a low-pass filter in parallel with the inductor to provide lossless current sense. A major drawback of inductor current sensing is that accurate sense the dc and ac components of the current signal requires precise matching between the low-pass filter time constant and the inductor time constant (L/[R.sub.L]). However, matching accuracy depends on the tolerance of the components and on the operating conditions; therefore it can hardly be guaranteed. To overcome this problem, a novel digital autotuning system is proposed that automatically compensates any time constant mismatch. This autotuning system has been developed for voltage regulator module (VRM) current programmed controllers. It makes it possible to meet the adaptive voltage positioning requirements using conventional and low cost components, and to solve problems such as aging effects, temperature variations, and process tolerances as well. A prototype of the autotuning system based on a field-programmable gate array and a commercial dc/dc controller has been designed and tested. The experimental results fully confirmed the effectiveness of the proposed method, showing an improvement of the current sense precision from about 30 % up to 4%. This innovative solution is suitable to fulfill the challenging accuracy specifications required by the future VRM applications. Index Terms--Current mode control, inductor current sensing techniques, VRM.

This paper investigates a mixed-signal fixed frequency digital voltage-mode controller for dc-dc converters. Switch turn-on is determined by system clock, while switch turn-off is determined asynchronously by comparing a signal proportional to the derivative of the output voltage and the voltage ramp driven by the digital-to-analog converter (DAC). One of the most important features is that the derivative action of the proportional-integral-derivative (PID) voltage-mode controller is inherently obtained by a combination of the analog front-end and the hard-wired digital logic, without requiring the numerical computation of the derivative action nor analog reactive elements (capacitors), which are usually needed for the derivative action in the analog domain and which are critical for the IC controller integration. Moreover, as compared to conventional digital approaches based on analog-to-digital converters (ADCs) and digital pulsewidth modulators (DPWMs), the phase-lag due to the uniformly sampled DPWM and due to the output voltage sampling is avoided, thus improving phased margin at the crossover frequency where the derivative contribution is usually prevailing. This property potentially enables faster controllers with improved dynamic response, breaking bandwidth limitation of conventional digital control architectures. The proposed control architecture is also effective from the IC point of view, since it is based on a DAC, a simple analog front-end, including a sample and hold, a comparator and an operational amplifier, and low digital signal-processing requirement (~5000 gates). Simulation and experimental results on a 1.2 V-20 A synchronous buck converter confirm the properties of the proposed solution. Index Terms--DC-DC converters, mixed-signal control, point-of-load converters, voltage-mode control.

This paper investigates a mixed synchronous/asynchronous digital voltage-mode controller for DC-DC converters. In the proposed control architecture, the turn-on switching event is determined asynchronously by comparing the converter output voltage and a synchronously generated voltage ramp driven by the digital control using a low-resolution digital-to-analog converter. Switch turn-off is determined synchronously by the system clock. In the proposed approach, the derivative action of the proportional-integral-derivative voltage-mode controller is inherently obtained by the frequency modulation, without requiring the digital computation of the derivative action. A simplified small-signal model is also derived in order to analyze the performance achievable by the proposed solution. This control architecture features good dynamic performance, and frequency modulation during transients. Simulation and experimental results on a synchronous buck converter, where the digital control has been implemented in field programmable gate array, confirm the effectiveness of the proposed solution. Index Terms--Digital control, DC-DC converters, mixed-signal control, voltage-mode control.

This paper proposes a simple autotuning technique for digitally controlled dc-dc converters. The proposed approach is based on the relay feedback method and introduces perturbations on the output voltage during converter soft-start. By using an iterative procedure, the tuning of proportional-integral-derivative parameters is obtained directly by including the controller in the relay feedback and by adjusting the controller parameters based on the specified phase margin and control loop bandwidth. A nice property of the proposed solution is that output voltage perturbations are introduced while maintaining the relay feedback control on the output voltage. The proposed algorithm is simple, requires small tuning times, and it is compliant with the cost/complexity constraints of integrated digital integrated circuits. Simulation and experimental results of a synchronous buck converter and of a dc-dc boost converter confirm the effectiveness of the proposed solution. Index Terms--Controller autotuning, dc-dc converters, digital control.