Antonino Ingargiola | University of California, Los Angeles (original) (raw)

Papers by Antonino Ingargiola

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its perform... more 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 Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interact... more Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by software implementation. In particular, despite the widespread application of smFRET, no complete software package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and well-documented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to customize the analysis for their own needs. By bundling analysis description, code and results in a single document, FRETBursts allows to seamless share analysis workflows and results, encourages reproducibility and facilitates collaboration among researchers in the single-molecule community.

Biophysical Journal, 2016

Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interact... more Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool in studying cellular processes at the molecular scale [21]. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume it emits a burst of photons that can be detected by single-photon avalanche detectors (SPADs). The intensities of donor and acceptor fluorescence can then be related to the distance between the two dyes. The analysis of smFRET experiments involves identifying photon bursts from single-molecules in a continuous stream of photon, estimating the background and other correction factors, filtering and finally extracting the corrected FRET efficiencies for each sub-population in the sample. In this paper we introduce FRETBursts, a software for confo-cal smFRET data analysis which allows executing a complete burst analysis pipeline by using state-of-the-art algorithms. FRETBursts is an open source python package that we envision both as toolkit for research and new developments in burst analysis and as reference implementation of commonly employed algorithms. We follow the highest standard in software development to ensure that the source is easy to read, well documented and thoroughly tested. Moreover, in an effort to lower the barriers to computational reproducibil-ity, we embrace a modern workflow based on Jupyter notebooks that allows to capture of the whole process from raw data to figures within a single document.

We introduce Photon-HDF5, an open and efficient file format to simplify exchange and long-term ac... more We introduce Photon-HDF5, an open and efficient file format to simplify exchange and long-term accessibility of data from single-molecule fluorescence experiments based on photon-counting detectors such as single-photon avalanche diode, photomultiplier tube, or arrays of such detectors. The format is based on HDF5, a widely used platform- and language-independent hierarchical file format for which user-friendly viewers are available. Photon-HDF5 can store raw photon data (timestamp, channel number, etc.) from any acquisition hardware, but also setup and sample description, information on provenance, authorship and other metadata, and is flexible enough to include any kind of custom data. The format specifications are hosted on a public website, which is open to contributions by the biophysics community. As an initial resource, the website provides code examples to read Photon-HDF5 files in several programming languages and a reference Python library (phconvert), to create new Photon-HDF5 files and convert several existing file formats into Photon-HDF5. To encourage adoption by the academic and commercial communities, all software is released under the MIT open source license.

We introduce Photon-HDF5, an open and efficient file format to simplify exchange and long term ac... more We introduce Photon-HDF5, an open and efficient file format to simplify exchange and long term accessibility of data from single-molecule fluorescence experiments based on photon-counting detectors such as single-photon avalanche diode (SPAD), photomultiplier tube (PMT) or arrays of such detectors. The format is based on HDF5, a widely used platform- and language-independent hierarchical file format for which user-friendly viewers are available. Photon-HDF5 can store raw photon data (timestamp, channel number, etc...) from any acquisition hardware, but also setup and sample description, information on provenance, authorship and other metadata, and is flexible enough to include any kind of custom data.
The format specifications are hosted on a public website, which is open to contributions by the biophysics community. As an initial resource, the website provides code examples to read Photon-HDF5 files in several programming languages and a reference Python library (phconvert), to create new Photon-HDF5 files and convert several existing file formats into Photon-HDF5. To encourage adoption by the academic and commercial communities, all software is released under the MIT open source license.

One of the main drawbacks of single-photon avalanche diode arrays is optical crosstalk between ad... more One of the main drawbacks of single-photon
avalanche diode arrays is optical crosstalk between adjacent
detectors. In the past, this phenomenon was basically ascribed
to light propagating from one detector to another through a
direct optical path. Accordingly, deep trenches coated with metal
were introduced as optical isolation barriers between pixels. This
solution, however, was unable to completely prevent the crosstalk.
In this letter, we demonstrate that a strong contribution to optical
crosstalk comes from photons reflected at the bottom of the chip.
These photons can bypass trenches making them less effective.

Single-molecule fluorescence spectroscopy of freely diffusing molecules in solution is a powerful... more Single-molecule fluorescence spectroscopy of freely diffusing molecules in solution is a powerful tool used to investigate the properties of individual molecules. Single-Photon Avalanche Diodes (SPADs) are the detectors of choice for these applications. Recently a new type of SPAD detector was introduced, dubbed red-enhanced SPAD (RE-SPAD), with good sensitivity throughout the visible spectrum and with excellent timing performance. We report a characterization of this new detector for single-molecule fluorescence resonant energy transfer (smFRET) studies on freely diffusing molecules in a confocal geometry and alternating laser excitation (ALEX) scheme. We use a series of doubly-labeled DNA molecules with donor-to-acceptor distances covering the whole range of useful FRET values. Both intensity-based (μs-ALEX) and lifetime-based (ns-ALEX) measurements are presented and compared to identical measurements performed with standard thick SPADs. Our results demonstrate the great potential of this new detector for smFRET measurements and beyond.

One of the main drawbacks of Single Photon Avalanche Diode arrays is the optical crosstalk betwee... more One of the main drawbacks of Single Photon Avalanche Diode arrays is the optical crosstalk between adjacent detectors. This phenomenon represents a fundamental limit to the density of arrays, since the crosstalk increases with reducing the distance between adjacent devices. In the past, crosstalk was mainly ascribed to the light propagating from one detector to another through a direct optical path. Accordingly, deep trenches coated with metal were introduced as optical isolation barriers between pixels. This solution, however, was unable to completely prevent the crosstalk. In this paper we present experimental evidence that a significant contribution to crosstalk comes from photons reflected internally at the bottom of the chip. These photons can bypass trenches making them ineffective. We also propose an optical model suitable to predict the dependence of crosstalk on the position within the array. Downloaded From: http://proceedings.spiedigitallibrary.org/ on 11/10/2015 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx Proc. of SPIE Vol. 6771 677111-2 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 11/10/2015 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx Proc. of SPIE Vol. 6771 677111-7 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 11/10/2015 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx

In recent years a growing number of applications demands always better timing resolution for Sing... more In recent years a growing number of applications demands always better timing resolution for Single Photon Avalanche Diodes. The challenge is pursuing the improved timing resolution without impairing the other device characteristics such as quantum efficiency and dark counts. This task requires a clear understanding of the physical mechanisms necessary to drive the device engineering process. Past studies state that in Si-SPADs the avalanche injection position statistics is the main contribution to the photon-timing jitter. However, in recent re-engineered devices, this assumption is questioned. For the purpose of assessing for good this contribution we developed an experimental setup in order to characterize the photontiming jitter as a function of the injection position by means of TCSPC measurements with a laser focused on the device active area. Results confirmed not only that the injection position is not the main contribution to the photon-timing jitter but also evidenced a radial dependence never observed before. Furthermore we found a relation between the photon-timing jitter and the specific resistance of the devices. To characterize the resistances we studied the avalanche current density distribution in the device active area by imaging the photo-luminescence due to hot-carrier emission.

One of the main issues of Single Photon Avalanche Diode arrays is optical crosstalk. Since its in... more One of the main issues of Single Photon Avalanche Diode arrays is optical crosstalk. Since its intensity increases with reducing the distance between devices, this phenomenon limits the density of integration within arrays. In the past optical crosstalk was ascribed essentially to the light propagating from one detector to another through direct optical paths. Accordingly, reflecting trenches between devices were proposed to prevent it, but they proved to be not completely effective. In this paper we will present experimental evidence that a significant contribution to optical crosstalk comes from light reflected internally off the bottom of the chip, thus being impossible to eliminate it completely by means of trenches. We will also propose an optical model to predict the dependence of crosstalk on the distance between devices.

Single Molecule Spectroscopy and Superresolution Imaging VI, 2013

Single-molecule Förster resonance energy transfer (smFRET) techniques are now widely used to addr... more Single-molecule Förster resonance energy transfer (smFRET) techniques are now widely used to address outstanding problems in biology and biophysics. In order to study freely diffusing molecules, current approaches consist in exciting a low concentration (<100 pM) sample with a single confocal spot using one or more lasers and detecting the induced single-molecule fluorescence in one or more spectrally- and/or polarization-distinct channels using single-pixel Single-Photon Avalanche Diodes (SPADs). A large enough number of single-molecule bursts must be accumulated in order to compute FRET efficiencies with sufficient statistics. As a result, the minimum timescale of observable phenomena is set by the minimum acquisition time needed for accurate measurements, typically a few minutes or more, limiting this approach mostly to equilibrium studies. Increasing smFRET analysis throughput would allow studying dynamics with shorter timescales. We recently demonstrated a new multi-spot excitation approach, employing a novel multi-pixel SPAD array, using a simplified dual-view setup in which a single 8-pixel SPAD array was used to collect FRET data from 4 independent spots. In this work we extend our results to 8 spots and use two 8-SPAD arrays to collect donor and acceptor photons and demonstrate the capabilities of this system by studying a series of doubly labeled dsDNA samples with different donor-acceptor distances ranging from low to high FRET efficiencies. Our results show that it is possible to enhance the throughput of smFRET measurements in solution by almost one order of magnitude, opening the way for studies of single-molecule dynamics with fast timescale once larger SPAD arrays become available.

Philosophical Transactions of the Royal Society B: Biological Sciences, Feb 5, 2013

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-li... more 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. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting dist... more 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. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting dist... more 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. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

IEEE Journal of Quantum Electronics, 2011

In recent years, a growing number of applications demand better timing resolution from single-pho... more In recent years, a growing number of applications demand better timing resolution from single-photon avalanche diodes (SPADs). The challenge is pursuing improved timing resolution without impairing other device characteristics such as quantum efficiency and dark count rate. This task requires a clear understanding of the statistical phenomena involved in the avalanche current growth in order to drive the device engineering process. Past studies state that in Si SPADs the avalanche injection position statistics is the main contribution to the photon-timing jitter. However, in recent re-engineered devices, this assumption has been questioned. To address this issue, we developed an experimental setup capable of characterizing the photon-timing jitter as a function of the injection position by means of a laser focused on the device active area. The results not only confirmed that the injection position statistics is not the main contribution to photon-timing jitter, but also evidenced interesting dependences of the timing performances on the injection position. Furthermore, we found a relationship between the photon-timing jitter and the specific resistance of the devices, which has been investigated by means of photoluminescence measurements.

IEEE J. Select. Topics Quantum Electron.

Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with app... more 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 toward 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.

A growing number of applications require arrays of single-photon avalanche diode (SPAD) detectors... more A growing number of applications require arrays of single-photon avalanche diode (SPAD) detectors with low timing jitter. In order to improve jitter without compromising other performance parameters, a clear understanding of avalanche dynamics and statistics is necessary. In this work, a noninvasive electro-luminescence technique has been employed to investigate the current growing in a SPAD device. The obtained results let us assess, for the first time experimentally, the avalanche spreading speeds and also confirmed our assumption that the current growth pace and its statistics critically depend on the space charge effects.

Software by Antonino Ingargiola

PyBroMo is an open-source simulator for Brownian-motion diffusion and photon emission of fluoresc... more PyBroMo is an open-source simulator for Brownian-motion diffusion and photon emission of fluorescent particles excited by a diffraction limited laser spot. PyBroMo allows to simulate timestamps of photons emitted during smFRET experiments, including sample background and detectors dark counts.

FRETBursts is a software toolkit for burst analysis of confocal single-molecule FRET (smFRET) mea... more FRETBursts is a software toolkit for burst analysis of confocal single-molecule FRET (smFRET) measurements.

FRETBursts is an effort to bring reproducible computing to the field of single-molecule confocal microscopy. It provides a standard implementation of state-of-the-art algorithms for confocal smFRET analysis. FRETBursts is opensource and contributions are welcome.

Biophysical Journal, 2016

Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interact... more Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool in studying cellular processes at the molecular scale [21]. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume it emits a burst of photons that can be detected by single-photon avalanche detectors (SPADs). The intensities of donor and acceptor fluorescence can then be related to the distance between the two dyes. The analysis of smFRET experiments involves identifying photon bursts from single-molecules in a continuous stream of photon, estimating the background and other correction factors, filtering and finally extracting the corrected FRET efficiencies for each sub-population in the sample. In this paper we introduce FRETBursts, a software for confo-cal smFRET data analysis which allows executing a complete burst analysis pipeline by using state-of-the-art algorithms. FRETBursts is an open source python package that we envision both as toolkit for research and new developments in burst analysis and as reference implementation of commonly employed algorithms. We follow the highest standard in software development to ensure that the source is easy to read, well documented and thoroughly tested. Moreover, in an effort to lower the barriers to computational reproducibil-ity, we embrace a modern workflow based on Jupyter notebooks that allows to capture of the whole process from raw data to figures within a single document.

Single Molecule Spectroscopy and Superresolution Imaging VI, 2013

Philosophical Transactions of the Royal Society B: Biological Sciences, Feb 5, 2013

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting dist... more 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. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

IEEE Journal of Quantum Electronics, 2011

IEEE J. Select. Topics Quantum Electron.