| 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. |
| Download a PDF File of this article (779 KB) |
| 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. |
| Download a PDF File of this article (668 KB) |
| 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 |
| Download a PDF File of this chaper (2.4 MB) |
| 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. |
| Download a PDF File of this article (7.6 MB) |
| 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 |
| Download a PDF File of this chapter (21.1 MB) |
| 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. |
| Download a PDF File of this article (1.7 MB) |
| 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. |
| Download a PDF File of this article (1.5 MB) |
| 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. |
| Download a PDF File of this article (858 kB) |
| 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 |
| Download a PDF File of this chapter (31 Mb) |
| 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 |
| Download a PDF File of this article (615 kB) |
| 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. |
| Download a PDF File of this article (569 KB) |
| 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. |
| Download a PDF File of this article (1.0 MB) |
| 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. |
| Download a PDF File of this article (1.2 MB) |
| 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. |
| Download a PDF File of this article (3.2 MB) |
| 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. |
| Download a PDF File of this commentary (135 kB) |
| 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 |
| Download a PDF File of this correspondence (86 kB) and the original draft (64 kB) |
| 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. |
| Download a PDF File of this article (981 kB) |
| 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. |
| Link to a HTML version of this article |
| 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. |
| Download a PDF File of this article (26 kB) |
| 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. |
| Download a PDF File of this article (441 kB) |
| 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. |
| Download a PDF File of this letter (132 kB) |
| 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. |
| Link to an HTML version of this article or to a PDF file (1.9 MB) |
| 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). | Download a PDF File of this paper (138 kB) |
| 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 ;-) |
| Download a PDF File of this chapter (16.5 MB) |
| 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. |
| Download a PDF File of this article (8.6 MB) |
| 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. |
| Link to an HTML version of this article (in French) or to a PDF File (2.3 MB) |
| 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 |
| Download a PDF File of this article (2.9 MB) |