Single-molecule FRET experiments with a red-enhanced custom technology SPAD
Panzeri, F., Ingargiola, A., Lin, R. R., Sarkhosh, N., Gulinatti, A., Rech, I., Ghioni, M., Weiss, S., Cova, S., Michalet, X.
Proceedings of SPIE 8590: 85900D (2013)
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 (ěs-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.
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8-spot smFRET analysis using two 8-pixel SPAD arrays
Ingargiola, A., Panzeri, F., Sarkhosh, N., Gulinatti, A., Rech, I., Ghioni, M., Weiss, S., Michalet, X.
Proceedings of SPIE 8590: 85900E (2013)
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.
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Large Area and High Efficiency Photon Counting Imaging Detectors with High Time and Spatial Resolution for Night Time Sensing and Astronomy
Siegmund, O. H. W., Vallerga, J. V., Tremsin, A. S., McPhate, J. B., Michalet, X., Weiss, S., Frisch, H., Wagner, R., Mane, A., Elam, J., Varner, G.
Proceedings of AMOS 2012: 1-6 (2012)
Imaging sensors using GaAs photocathodes of 18 mm active area format with microchannel plates and cross strip readouts in sealed tubes have been developed. The GaAs photocathodes have peak quantum efficiency of ~30% from 550 nm to 850 nm and background rates of ~50 kHz at 8° C. The sensor system including our processing electronics achieves spatial resolution of ~40 µm FHWM (at gains of ~ 106), can process high event rates (5 MHz input rates), has event timing accuracy of ~360 ps and local area counting rates of up to 20 kHz (100 µm spot). We have also made significant progress on development of novel large area (20 cm) microchannel plates and their implementation into sealed tube devices. These microchannel plates have demonstrated better than 100 µm spatial resolution, low background rates (0.1 events cm-2 sec-1), and very good lifetime stability to 7 C cm-2 of charge extraction. Considerable progress in development of a 20 cm format sealed tube device has also been made including the demonstration of 20 cm bialkali photocathodes with stable peak (360 nm) quantum efficiency of > 20%.
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Parallel multispot smFRET analysis using an 8-pixel SPAD array
Ingargiola, A., Colyer, R.A., Kim, D., Panzeri, F., Lin, R., Gulinatti, A., Rech, I., Ghioni, M., Weiss, S., Michalet, X.
Proceedings of SPIE 8228: 82280B (2012)
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.
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Microchannel Plate Imaging Photon Counters for Ultraviolet through NIR Detection with High Time Resolution
Siegmund, O. H. W., Vallerga, J. V., Tremsin, A. S., Michalet, X., Colyer, R.A., Weiss, S.
Proceedings of SPIE 8033: 80330V (2011)
Cross strip and cross delay line readout microchannel plate detectors in 18 mm, 25 mm and 40 mm active area formats including open face (UV/particle) and sealed tube (optical) configurations have been constructed. These have been tested with a field programmable gate array based electronics for single event encoding. Using small pore MCPs (6 µm) operated in a pair, we achieve gains of >1 x 106 which is sufficient to provide spatial resolution of ~17 µm FHWM with the 18 mm and 40 mm cross strip readouts. New cross strip electronics can process high output event rates (> 4 MHz) with high spatial resolution, and self triggered event timing accuracy of ~1.5 ns for sealed tube XS optical sensors. A peak quantum efficiency of between 13% and 19% at 500 nm has been achieved with SuperGenII photocathodes with response from 400 nm to 900 nm for both cross strip and cross delay line sealed tubes. Local area counting rates of up to 40 kHz (100 µm spot) have been attained with XS sealed tubes, along with image linearity and stability to better than 50 µm. 25 mm cross delay line tubes achieve ~50 µm resolution and > 2 MHz output event rates.
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New photon-counting detectors for single-molecule fluorescence spectroscopy and imaging
Michalet, X., Colyer, R.A., Scalia G., Weiss, S., Siegmund, O. H. W., Tremsin, A. S., Vallerga, J. V., Villa, F., Guerrieri, F., Rech, I., Gulinatti, A., Tisa, S., Zappa, F., Ghioni, M., Cova, S.
Proceedings of SPIE 8033: 803316 (2011)
Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. Two typical geometries can be used for these experiments: point-like and widefield excitation and detection. In point-like geometries, the basic concept 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. In widefield geometries, the same issues of background reduction and single-molecule concentration apply, but the duration of the experiment is fixed by the time scale of the process studied and the survival time of the fluorescent probe. Temporal resolution on the other hand, is limited by signal-to-noise and/or detector resolution, which calls for new detector concepts. We will briefly present our recent results in this domain.
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Ultrahigh-throughput single molecule spectroscopy with a 1024 pixel SPAD
Colyer, R.A., Scalia, G., Villa, F. A., Guerrieri, F., Tisa, S., Zappa, F., Cova, S., Weiss, S., Michalet, X.
Proceedings of SPIE 7905: 790503 (2011)
Single-molecule spectroscopy is a powerful approach to measuring molecular properties such as size, brightness, conformation, and binding constants. Due to the low concentrations in the single-molecule regime, measurements with good statistical accuracy require long acquisition times. Previously we showed a factor of 8 improvement in acquisition speed using a custom-CMOS 8x1 SPAD array. Here we present preliminary results with a 64X improvement in throughput obtained using a liquid crystal on silicon spatial light modulator (LCOS-SLM) and a novel standard CMOS 1024 pixel SPAD array, opening the way to truly high-throughput single-molecule spectroscopy.
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Single-molecule fluorescence spectroscopy and imaging
Michalet, X., Colyer, R.A., Weiss, S.
SPIE Newsroom (October 2010)
New photon-counting cameras and multipixel photon-counting detector arrays are transforming single-molecule observational approaches.
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High-throughput multispot single-molecule spectroscopy
Colyer, R.A., Scalia, G., Kim, T., Rech, I., Resnati, D., Marangoni, S., Gulinatti, A., Ghioni, Cova, S., Weiss, S., Michalet, X.
Proceedings of SPIE 7571: 75710G (2010)
Solution-based single-molecule spectroscopy and fluorescence correlation spectroscopy (FCS) are powerful techniques to access a variety of molecular properties such as size, brightness, conformation, and binding constants. However, this is limited to low concentrations, which results in long acquisition times in order to achieve good statistical accuracy. Data can be acquired more quickly by using parallelization. We present a new approach using a multispot excitation and detection geometry made possible by the combination of three powerful new technologies: (i) a liquid crystal spatial light modulator to produce multiple diffraction-limited excitation spots; (ii) a multipixel detector array matching the excitation pattern and (iii) a low-cost reconfigurable multichannel counting board. We demonstrate the capabilities of this technique by reporting FCS measurements of various calibrated samples as well as single-molecule burst measurements.
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High-throughput single-molecule fluorescence spectroscopy using parallel detection
Michalet, X., Colyer, R.A., Scalia, G., Kim, T., Levi, M., Aharoni, D., Cheng, A., Guerrieri, F., Arisaka, A. Millaud, J., Rech, I., Resnati, D., Marangoni, S., Gulinatti, A., Ghioni, M., Tisa, S., Zappa, F., Cova, S., Weiss, S.
Proceedings of SPIE 7608: 76082D (2010)
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.
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High Speed Multichannel Charge Sensitive Data Acquisition System With Self-Triggered Event Timing
Tremsin, A. S., Siegmund, O.H.W., Vallerga, J.V., Raffanti, R., Weiss, S., Michalet, X.
IEEE Transactions on Nuclear Science 56 (3): 1148-1152 (2009)
A number of modern experiments require simultaneous measurement of charges on multiple channels at MHz event rates with an accuracy of 100–1000 e- rms. One widely used data processing scheme relies on application of specific integrated circuits enabling multichannel analog peak detection asserted by an external trigger followed by a serial/sparsified readout. Although this configuration minimizes the back end electronics, its counting rate capability is limited by the speed of the serial readout. Recent advances in analog to digital converters and FPGA devices enable fully parallel high speed multichannel data processing with digital peak detection enhanced by finite impulse response filtering. Not only can accurate charge values be obtained at high event rates, but the timing of the event on each channel can also be determined with high accuracy. We present the concept and first experimental tests of fully parallel 128-channel charge sensitive data processing electronics capable of measuring charges with an accuracy of 1000 e- rms. Our system does not require an external trigger and, in addition to charge values, it provides the event timing with an accuracy of 1 ns FWHM. One of the possible applications of this system is high resolution position sensitive event counting detectors with microchannel plates combined with cross strip readout. Implementation of fast data acquisition electronics increases the counting rates of those detectors to multi-MHz level, preserving their unique capability of virtually noiseless detection of both position (with an accuracy of ~ 10 um FWHM) and timing ( 1 ns FWHM) of individual particles, including photons, electrons, ions, neutrals, and neutrons.
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Phasor-based single-molecule fluorescence lifetime imaging using a wide-field photon-counting detector
Colyer, R.A., Siegmund, O.H.W., Tremsin, A., Vallerga, J.V., Weiss, S., Michalet, X.
Proceedings of SPIE 7185: 71850T (2009)
Fluorescence lifetime imaging (FLIM) is a powerful approach to studying the immediate environment of molecules. For example, it is used in biology to study changes in the chemical environment, or to study binding processes, aggregation, and conformational changes by measuring Förster resonance energy transfer (FRET) between donor and acceptor fluorophores. FLIM can be acquired by time-domain measurements (time-correlated single-photon counting) or frequency-domain measurements (with PMT modulation or digital frequency domain acquisition) in a confocal setup, or with wide-field systems (using time-gated cameras). In the best cases, the resulting data is analyzed in terms of multicomponent fluorescence lifetime decays with demanding requirements in terms of signal level (and therefore limited frame rate). Recently, the phasor approach has been proposed as a powerful alternative for fluorescence lifetime analysis of FLIM, ensemble, and single-molecule experiments. Here we discuss the advantages of combining phasor analysis with a new type of FLIM acquisition hardware presented previously, consisting of a high temporal and spatial resolution wide-field single-photon counting device (the H33D detector). Experimental data with live cells and quantum dots will be presented as an illustration of this new approach.
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Chapter 3: Peptide-Functionalized Quantum Dots for Live Diagnostic Imaging and Therapeutic Applications
Bentolila, L. A., Doose, S., Ebenstein, Y., Iyer, G., Li, J.J., Michalet, X., Tsay, J., Weiss, S.
Inorganic Nanoprobes for Biological Sensing and Imaging
Mattoussi, H., Cheon, J., Eds
Artech House, Boston, 2009
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Chapter 8: Molecular Imaging: Physics and Bioapplications of Quantum Dots
Michalet, X., Bentolila, L. A., Weiss, S.
Advances in Medical Physics
Wolbarst, A. B., Hendee, W. R., Eds
Medical Physics Publishing, Madison, 2008
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Hybrid photodetector for single-molecule spectroscopy and microscopy
Michalet, X., Cheng, A., Antelman, J., Suyama, M., Arisaka, K., Weiss, S.
Proceedings of SPIE 6862: 68620F (2008)
We report benchmark tests of a new single-photon counting detector based on a GaAsP photocathode and an electron-bombarded avalanche photodiode developed by Hamamatsu Photonics. We compare its performance with those of standard Geiger-mode avalanche photodiodes. We show its advantages for FCS due to the absence of after-pulsing and for fluorescence lifetime measurements due to its excellent time resolution. Its large sensitive area also greatly simplifies setup alignment. Its spectral sensitivity being similar to that of recently introduced CMOS SPADs, this new detector could become a valuable tool for single-molecule fluorescence measurements, as well as for many other applications.
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Chapter 3: Quantum optics: Colloidal fluorescent semiconductor nanocrystals (quantum dots) in single-molecule detection and imaging
Bentolila, L. A., Michalet, X., Weiss, S.
Single Molecules and Nanotechnology
Rigler, R., Vogel, H., Eds
Springer-Verlag, Berlin Heidelberg, 2008
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Detectors for Microscopy: Next-generation 3-D detector improves single-molecule imaging
Michalet, X., Siegmund, O.H.W., Vallerga, J.V., Jelinsky, P., Millaud, J.E., Arisaka, K., Weiss, S.
Laser Focus World 43: 97-101 (2007)
A high-throughput three-dimensional detector combines the advantages of wide-field detectors and high-temporal-resolution point detectors, proving instrumental for single-molecule imaging and the study of biomolecular interactions.
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Peptide Coated Quantum Dots for Biological Applications
Iyer, G., Pinaud, F., Tsay, J.,Li, J. J., Bentolila, L. A., Michalet, X.,Weiss, S.
IEEE Transactions on Nanobioscience, 5: 231-238 (2006)
Quantum dots (QDOTs) have been widely recognized by the scientific community and the biotechnology industry, as witnessed by the exponential growth of this field in the past several years. We describe the synthesis and characterization of visible and near infrared QDots—a critical step for engineering organic molecules like proteins and peptides for building nanocomposite materials with multifunctional properties suitable for biological applications.
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Fluorescence lifetime microscopy with a time- and space-resolved single-photon counting detector
Michalet, X., Siegmund, O.H.W., Vallerga, J.V., Jelinsky, P., Pinaud, F. F., Millaud, J.E., Weiss, S.
Proceedings of SPIE 6372: 63720E (2006)
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.
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Chapter 7: Molecular Imaging
Michalet, X., Bentolila, L., Weiss, S.
Advances in Medical Physics
Wolbarst, A. B., Zamenhof, R. G., Hendee, W. R., Eds
Medical Physics Publishing, Madison, 2006
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Using photon statistics to boost microscopy resolution
Michalet, X., Weiss, S.
Proceedings of the National Academy of Sciences 103 (13): 4797-4798 (2006)
Commentary to an article by Sripad Ram, E. Sally Ward, and Raimund J. Ober in PNAS 103 (12): 4457-4462 (2006): Beyond Rayleigh's criterion: A resolution measure with application to single-molecule microscopy
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Near-infrared peptide-coated quantum dots for small animal imaging
Iyer, G., Li, J. J., Pinaud, F., Tsay, J. M., Bentolila, L. A., Michalet, X., Weiss, S.
Proceedings of SPIE 6096: 60960B (2006)
We have synthesized high quality type-II CdTe/CdSe near infrared quantum dots using successive ion layer adsorption and reaction chemistry. Transmission electron microscopy reveals that CdTe/CdSe can be synthesized layer by layer yielding quantum dots of narrow size distribution. Excitation and photoluminescence spectra reveal discrete type-II transitions, which correspond to energy lower that type-I bandgap. We have used a peptide coating technique on type-II and commercial near infrared quantum dots for delivery in live animals and cultured cells.
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Development of an Ultra-fast Single-Photon Counting Imager for Single-Molecule Imaging
Ohnukia, T., Michalet, X., Tripathia, A., Weiss, S., Arisaka, K.
Proceedings of SPIE 6092: 60920P (2006)
We have begun developing an innovative ultra-fast single-photon counting imager which comprises a mega-pixel CMOS array and a newly-designed Image Intensifier. It is expected to have single photon sensitivity with 100 psec time resolution, operational at a total counting rate exceeding 1MHz. The readout is based on dead-time-free flash ADC, running at 1-2GS/s, followed by a FPGA for real-time parallel data processing. Such a device has not been realized before and is expected to revolutionize time-resolved fluorescence imaging and spectroscopy from a single-molecule to whole animal level. To evaluate the design principle, an Image Intensifier with a GaAsP photocathode (>40% quantum efficiency at 400-600 nm) followed by double MCP was evaluated together with an existing CMOS camera. In our future design, the image from CMOS Camera will be combined with the MCP output, followed by a set of FPGA and CPU for real time data processing. This stream line method will allow ultra fast single-photon counting with 100 psec time resolution and 20 µm position resolution (1M pixel imaging). In this paper, we present the design principle and preliminary results on its performance. Our future plan and the design goals are also described.
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A space- and time-resolved single-photon counting detector for fluorescence microscopy and spectroscopy
Michalet, X., Siegmund, O.H.W., Vallerga, J.V., Jelinsky, P., Millaud, J.E., Weiss, S.
Proceedings of SPIE 6092: 60920M (2006)
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.
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A Cross Delay Line Detectors for High Time Resolution Astronomical Polarimetry and Biological Fluorescence Imaging
Siegmund, O.H.W., Michalet, X., Vallerga, J.V., Jelinsky, P., Millaud, J.E., Weiss, S.
IEEE Nuclear Science Symposium Conference Record N14-55: 448-452 (2005)
Ground based high time resolution astronomical polarimetry, imaging, and biological time-resolved molecular fluorescence lifetime imaging require specialized detectors. Photon counting detectors that combine high spatial resolution imaging with fast event timing capability for these uses have been a significant technical challenge. We have developed a hightemporal and spatial resolution, high-throughput sealed tube microchannel plate detector with electronic readout as tool for these applications. The design is based on a 25 mm diameter S20 photocathode followed by a microchannel plate stack, read out by a cross delay line anode with timing and imaging electronics. The detector supports 500 kHz global count rate, 10 kHz local count rate, 100 ps timing resolution and 40 µm spatial resolution. We describe the performance of the detector, as well as imaging results obtained with quantum dots and live cells.
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New Light on Quantum Dot Cytotoxicity
Tsay, J. M., Michalet, X.
Chemistry & Biology 12 1159-1161 (2005)
As quantum dots are beginning to be used for in vivo imaging, the question of their long-term effect on cell viability is becoming critical. In this issue of Chemistry & Biology, Lovric and colleagues examine the likely role of reactive oxygen species in quantum dot cytotoxicity.
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Thoughtful peer review is worth the time it takes
Michalet, X.
Nature 435 1160 (2005)
I wrote this letter in less than an hour as an amused and, I hoped, humorous reaction to somebody's comments about the problems associated with deciphering articles submitted online. However, space constraints forced the editors to perform significant cuts and rewriting. The end result reads like a pundit's lesson of ethics, which I never pretended nor have the interest to give. For the sake of completeness, and because it might be instructive for some readers or candidate writers, I provide here the original version
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Peptide-coated semiconductor nanocrystals for biomedical applications
Michalet, X., Pinaud, F. F., Bentolila, L.A., Tsay, J. M., Doose, S., Li, J. J., Iyer, G., Weiss, S.
Proceedings of SPIE 5704: 57-68 (2005)
We have developed a new functionalization approach for semiconductor nanocrystals based on a single-step exchange of surface ligands with custom-designed peptides. This peptide-coating technique yield small, monodisperse and very stable water-soluble NCs that remain bright and photostable. We have used this approach on several types of core and core-shell NCs in the visible and near-infrared spectrum range and used fluorescence correlation spectroscopy for rapid assessment of the colloidal and photophysical properties of the resulting particles. This peptide coating strategy has several advantages: it yields probes that are immediately biocompatible; it is amenable to improvements of the different properties (solubilization, functionalization, etc) via rational design, parallel synthesis, or molecular evolution; it permits the combination of several functions on individual NCs. These functionalized NCs have been used for diverse biomedical applications. Two are discussed here: single-particle tracking of membrane receptor in live cells and combined fluorescence and PET imaging of targeted delivery in live animals.
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Single-Molecule Spectroscopy Comes of Age
Kelley, A. M., Michalet, X., Weiss, S.
Science 292: 1671-1672 (2001)
A summary of the March 2001 (San Diego) ACS Meeting on Single Molecule Spectroscopy, coorganized by Anne Myers Kelley and Shimon Weiss.
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Stretching Single-Stranded DNA on a Surface
Michalet, X.
Nanoletters 1 (7): 341-343 (2001)
A sufficiently long strand of DNA bends and balls up, like any other polymer longer than its persistence length. The ability to stretch a DNA molecule lengthwise on a substrate has garnered a great deal of attention in the past several years. There has been notable success in stretching double-stranded DNA (dsDNA), but it is more difficult to stretch single-stranded DNA (ssDNA), both because of the very short persistence length and because it is very “sticky,” and attaches nonspecifically to surfaces. In this issue of Nano Letters, Woolley and Kelly present a new method for stretching single-stranded DNA strands on surfaces.
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Ultrahigh Resolution Multicolor Colocalization of Single Fluorescent Nanocrystals
Michalet, X., Lacoste, T. D., Pinaud, F., Chemla. D. S., Alivisatos, A. P., Weiss. S.
Proceedings of SPIE 4258: 8-15 (2001)
A new method for in vitro and possibly in vivo ultrahigh-resolution colocalization and distance measurement between biomolecules is described, based on semiconductor nanocrystal probes. This ruler bridges the gap between FRET and far-field (or near-field scanning optical microscope) imaging and has a dynamic range from few nanometers to tens of micrometers. The ruler is based on a stage–scanning confocal microscope that allows the simultaneous excitation and localization of the excitation point-spreadfunction (PSF) of various colors nanocrystals while maintaining perfect registry between the channels. Fit of the observed diffraction and photophysics-limited images of the PSFs with a two-dimensional Gaussian allows one to determine their position with nanometer accuracy. This new high-resolution tool opens new windows in various molecular, cell biology and biotechnology applications.
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Correspondence: French research needs
Michalet, X.
Nature 388 120 (1997)
As far as I remember, I wrote this letter as a testimony of my own experience as (i) a pure product of the French "grandes écoles" system, (ii) having had the possibility to accept a permanent civil servant position in the CNRS, (iii) having worked at the Pasteur Institute for over 4 years while this was in principle impossible legally, (iv) having discovered an almost complete inexistence of venture capital in France, while I was trying to start a company with my former colleagues. In summary, I was venting out some serious frustrations about the French system. Whereas this has any relevance about yesterday's (and today's) actual situation is anybody's guess... It definitely had a polemic appeal to Nature's editors.
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Peignage moléculaire d'ADN. Cartographie physique du génome et diagnostic génétique
Michalet, X., Bensimon, A.
Médecine/Sciences 13 (11): 1299-1305 (1997)
Le peignage moléculaire permet la préparation reproductible de surfaces couvertes d’une haute densité de molécules d’ADN parallèles les unes aux autres et étirées avec un taux constant et uniforme. L’utilisation de méthodes d’hybridation fluorescentes classiques en fait un outil performant pour la cartographie physique haute résolution de clones, permettant notamment de lever toutes les ambiguïtés subsistant dans les cartes de restriction, mais aussi de se dispenser de l’établissement de telles cartes. Enfin, dans le domaine du diagnostic génétique, la détection de micro-délétions de quelques dizaines de kilobases à partir d’ADN génomique de patients illustre les potentialités de la technique pour l’étude du génome humain.
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Le peignage moléculaire: un outil à haute résolution
Michalet, X., Bensimon, A.
Biofutur 165 Technoscope, Cahier 90: 8 (1997)
A very brief report on molecular combing (in French).
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Vesicles of Complex Topology
Fourcade, B., Michalet, X., Bensimon, D.
in Nonmedical Applications of Liposomes. Theory and Basic Sciences. Lasic, D.D., Barenholz, Y., Editors.
Vol. I, CRC Press (1996)
This is merely an abridged version of my Ph D thesis translated by Bertrand Fourcade ;-)
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Toroidal vesicles: observed morphology and numerical modelisation
Michalet, X., Bensimon, D.
in Interplay of Genetic and Physical Processes in the Develoment of Biological Form. At the frontier of Physics and Biology. Beysens, D., Forgacs, G., Gaill, F., Editors. Les Houches Series, p 23-37, World Scientific (1995)
This is an incomplete report (due to a file transmission error) on the modelisation of vesicles of complex topology. More details to be found in my Ph D thesis.
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La physique des liposomes
Michalet, X., Jülicher, F., Fourcade, B., Seifert, U., Bensimon, D.
La Recherche 25 (269): 1012-1018 (1994)
Liposomes are known for their use in some cosmetic products or potentially to deliver drugs. But these small and soft vesicles, whose membrane is comprised of a double layer of lipid molecules, are also a model system for the physics of fluid membranes or even of biological membranes. In particular, researchers are interested in the morphology of liposomes, which can adopt shapes such as that of a pear, an annulus, a two-hole button, switch from one shape to another, fuse or split in two, etc. How is the geometry of liposomes determined? What are the possible shapes and shape transformations? These are some of the questions that physicists have succesfully answered in the past few years.
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Vesicles of high topological genus
Michalet, X., Bensimon, D.
in Soft Order in Physical Systems
Rabin, Y., Bruinsma, R., Editors. Proceedings of a NATO Advanced Research Workshop, Les Houches. p 199-202, Plenum Press (1994)
We report the observation of phospholipid vesicles of high topological genus, exhibiting strong thermal fluctuations. By deeply affecting the global shape of the vesicle, these differ from the usual local thermal undulations of the membrane. They can be described as positional fluctuations of necks linking 2 nearby concentric membranes. We then present the results of a simple model for the shape of the necks based on an electrostatic analogy. This approach is corroborated by a numerical solution of the minimization problem
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