Development of new photon-counting detectors for single-molecule fluorescence microscopy

Michalet, X., Colyer, R. A., Scalia, G., Ingargiola, A., Lin, R., Millaud, J. E., Weiss, S., Siegmund, O. H. W., Tremsin, A. S., Vallerga, J. V., Cheng, A. , Levi, M., Aharoni, D., Arisaka, K., Villa, F., Guerrieri, F., Panzeri, F., Rech, I., Gulinatti, A., Zappa, F., Ghioni, M., Cova, S.

Philosophical Transactions of the Royal Society B 368: 20120035 (2013)

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

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Optimal diffusion coefficient estimation in single-particle tracking

Michalet, X., Berglund, A. J.

Physical Review E 85: 061916 (2012)

Single-particle tracking is increasingly used to extract quantitative parameters on single molecules and their environment, while advances in spatial and temporal resolution of tracking techniques inspire new questions and avenues of investigation. Correspondingly, sophisticated analytical methods are constantly developed to obtain more refined information from measured trajectories. Here we point out some fundamental limitations of these approaches due to the finite length of trajectories, the presence of localization error, and motion blur, focusing on the simplest motion regime of free diffusion in an isotropic medium (Brownian motion). We show that two recently proposed algorithms approach the theoretical limit of diffusion coefficient uncertainty. We discuss the practical performance of the algorithms as well as some important implications of these results for single-particle tracking.

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Phasor imaging with a widefield photon-counting detector

Colyer, R.A., Siegmund, O.H.W., Tremsin, A.S., Vallerga, J.V., Weiss, S., Michalet, X.

Journal of Biomedical Optics 17 (1): 016008 (2012)

Fluorescence lifetime can be used as a contrast mechanism to distinguish fluorophores for localization or tracking, for studying molecular interactions, binding, assembly, and aggregation, or for observing conformational changes via Förster resonance energy transfer (FRET) between donor and acceptor molecules. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful technique but its widespread use has been hampered by demanding hardware and software requirements. FLIM data is often analyzed in terms of multicomponent fluorescence lifetime decays, which requires large signals for a good signal-to-noise ratio. This confines the approach to very low frame rates and limits the number of frames which can be acquired before bleaching the sample. Recently, a computationally efficient and intuitive graphical representation, the phasor approach, has been proposed as an alternative method for FLIM data analysis at the ensemble and single-molecule level. In this article, we illustrate the advantages of combining phasor analysis with a widefield time-resolved single photon-counting detector (the H33D detector) for FLIM applications. In particular we show that phasor analysis allows real-time subsecond identification of species by their lifetimes and rapid representation of their spatial distribution, thanks to the parallel acquisition of FLIM information over a wide field of view by the H33D detector. We also discuss possible improvements of the H33D detector’s performance made possible by the simplicity of phasor analysis and its relaxed timing accuracy requirements compared to standard time-correlated single-photon counting (TCSPC) methods.

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High-throughput FCS using an LCOS spatial light modulator and an 8 × 1 SPAD array

Colyer, R.A., Scalia, G., Rech, I., Gulinatti, A., Ghioni, M., Cova, S., Weiss, S., Michalet, X.

Biomedical Optics Express 1: 1408-1431 (2010)

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.

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Mean square displacement analysis of single-particle trajectories with localization error: Brownian motion in an isotropic medium

Michalet, X.

Physical Review E 82: 041914 (2010)

We examine the capability of mean square displacement (MSD) analysis to extract reliable values of the diffusion coefficient D of a single particle undergoing Brownian motion in an isotropic medium in the presence of localization uncertainty. The theoretical results, supported by simulations, show that a simple unweighted least-square fit of the MSD curve can provide the best estimate of D provided an optimal number of MSD points are used for the fit. We discuss the practical implications of these results for data analysis in single-particle tracking experiments.

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Tracking Single Proteins in Live Cells Using Single-Chain Antibody Fragment-Fluorescent Quantum Dot Affinity Pair

Iyer, G., Michalet, X., Chang, Y.-P., Weiss, S.

Methods in Enzymology 475: 61-79 (2010)

Quantum dots (QDs) are extremely bright fluorescent imaging probes that are particularly useful for tracking individual molecules in living cells. Here, we show how a two-component system composed of a high-affinity single-chain fragment antibody and its cognate hapten (fluorescein) can be utilized for tracking individual proteins in various cell types. The single-chain fragment antibody against fluorescein is genetically appended to the protein of interest, while the hapten fluorescein is attached to the end of the peptide that is used to coat the QDs. We describe (i) the method used to functionalize QDs with fluorescein peptides; (ii) the method used to control the stoichiometry of the hapten on the surface of the QD; and (iii) the technical details necessary to observe single molecules in living cells.

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Single-quantum dot imaging with a photon counting camera

Michalet, X., Colyer, R. A., Antelman, J., Siegmund, O. H. W., Tremsin, A., Vallerga, J. V., Weiss, S.

Current Pharmaceutical Biotechnology 10: 543-557 (2009)

The expanding spectrum of applications of single-molecule fluorescence imaging ranges from fundamental in vitro studies of biomolecular activity to tracking of receptors in live cells. The success of these assays has relied on progresses in organic and non-organic fluorescent probe developments as well as improvements in the sensitivity of light detectors. We describe a new type of detector developed with the specific goal of ultra-sensitive single-molecule imaging. It is a wide-field, photon-counting detector providing high temporal and high spatial resolution information for each incoming photon. It can be used as a standard low-light level camera, but also allows access to a lot more information, such as fluorescence lifetime and spatio-temporal correlations. We illustrate the single-molecule imaging performance of our current prototype using quantum dots and discuss on-going and future developments of this detector.

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Nanometer Distance Measurements between Multicolor Quantum Dots

Antelman, J., Wilking-Chang, C., Weiss, S., Michalet, X.

Nano Letters 9: 2199-2205 (2009)

Quantum dot dimers made of short double-stranded DNA molecules labeled with different color quantum dots at each end were imaged using multicolor stage-scanning confocal microscopy. This approach eliminates chromatic aberration and color registration issues usually encountered in other multicolor imaging techniques. We demonstrate nanometer accuracy in individual distance measurement by suppression of quantum dot blinking and thoroughly characterize the contribution of different effects to the variability observed between measurements. Our analysis opens the way to accurate structural studies of biomolecules and biomolecular complexes using multicolor quantum dot labeling.

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Lighting Up Individual DNA Binding Proteins with Quantum Dots

Ebenstein, Y., Gassman, N., Kim, S., Antelman, J., Kim, Y., Ho, S., Samuel, R., Michalet, X., Weiss, S.

Nano Letters 9: 1598-1603 (2009)

The ability to determine the precise loci and occupancy of DNA-binding proteins is instrumental to our understanding of cellular processes like gene expression and regulation. We propose a single-molecule approach for the direct visualization of proteins bound to their template DNA. Fluorescent quantum dots (QD) are used to label proteins bound to DNA, allowing multicolor, nanometer-resolution localization. Protein-DNA complexes are linearly extended and imaged to determine the precise location of the protein binding sites. The method is demonstrated by detecting individual QD-labeled T7-RNA polymerases on the T7 bacteriophage genome. This work demonstrates the potential of this approach to precisely read protein binding position or, alternatively, “write” such information on extended DNA with QDs via sequence-specific molecular recognition.

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Dynamic Partitioning of a Glycosyl-Phosphatidylinositol-Anchored Protein in Glycosphingolipid-Rich Microdomains Imaged by Single-Quantum Dot Tracking

Pinaud, F., Michalet, X., Iyer, G., Margeat, E., Moore, H.-P., Weiss, S.

Traffic 10: 691-712 (2009)

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositolanchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed highresolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane.

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Suppression of Quantum Dot Blinking in DTT-Doped Polymer Films

Antelman, J., Ebenstein,Y.,Dertinger, T., Michalet, X., Weiss, S.

Journal of Physical Chemistry C 113: 11541-11545 (2009)

In this report we evaluate the emission properties of single quantum dots embedded in a thin, thiol-containing polymer film. We report the suppression of quantum dot blinking leading to a continuous photon flux from both organic and water soluble quantum dots and demonstrate their application as robust fluorescent point sources for ultrahigh resolution localization. In addition, we apply the polymer coating to cell samples immunostained with antibody conjugated QDs and show that fluorescence intensity from the polymer embedded cells shows no sign of degradation after 67 h of continuous excitation. The reported thin polymer film coating may prove advantageous for immuno-cyto/histo-chemistry as well as for the fabrication of quantum dot containing devices requiring a reliable and stable photon source (including a single photon source) or stable charge characteristics while maintaining intimate contact between the quantum dot and the surrounding matrix.

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High Affinity scFv-Hapten Pair as a Tool for Quantum Dot Labeling and Tracking of Single Proteins in Live Cells

Iyer, G., Michalet, X., Chang, Y.-P., Matyas, S. E., Payne, G., Weiss, S.

Nano Letters 8: 4618-4623 (2008)

We describe a general approach to label cell surface proteins using quantum dots (QD) for single-molecule tracking. QDs coated with small-hapten modified peptides are targeted to cell surface fusion proteins containing the corresponding single-chain fragment antibody (scFv). The approach is illustrated with the small hapten fluorescein (FL) and a high-affinity anti-FL scFv fused to two different proteins in yeast and murine neuronal cell line N2a.

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Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy

Korlann, Y., Dertinger, T., Michalet, X., Weiss, S., Enderlein, J.

Optics Express 16: 14609-14616 (2008)

We present a new technique, polarization-modulation dual-focus fluorescence correlation spectroscopy (pmFCS), based on the recently introduced dual-focus fluorescence correlation spectroscopy (2fFCS) to measure the absolute value of diffusion coefficients of fluorescent molecules at pico- to nanomolar concentrations. Analogous to 2fFCS, the new technique is robust against optical saturation in yielding correct values of the diffusion coefficient. This is in stark contrast to conventional FCS where optical saturation leads to an apparent decrease in the determined diffusion coefficient with increasing excitation power. However, compared to 2fFCS, the new technique is simpler to implement into a conventional confocal microscope setup and is compatible with cw-excitation, only needing as add-ons an electro-optical modulator and a differential interference contrast prism. With pmFCS, the measured diffusion coefficient (D) for Atto655 maleimide in water at 25oC is determined to be equal to (4.09 ± 0.07)×10-6cm2/s, in good agreement with the value of 4.04×10-6cm2/s as measured by 2fFCS.

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Equilibrium shape degeneracy in starfish vesicles

Michalet, X.

Physical Review E 76: 021914 (2007)

Phospholipid dispersed in aqueous buffer above their critical micellar concentration form bilayers, which can spontaneously adopt closed metastable shapes with sizes ranging from a few hundred nanometers to few tens of micrometers. The equilibrium shapes of these vesicles are well described by the Canham-Evans-Helfrich curvature elastic energy. Their floppiness allows for thermal fluctuations to be easily detected, but no spontaneous shape transformation is usually observed for vesicles of spherical topological genus (i.e. shapes with no holes) because of strict geometrical constraints and/or energy barriers. This report shows that for a particular class of shapes with spherical topological genus (starfish vesicles), dramatic spontaneous shape transformations can occur due to the degeneracy of the shape solutions, as demonstrated by numerical calculations. These predictions are supported by experimental observations of a 3-arm starfish vesicle undergoing spontaneous shape transformations similar to those predicted numerically.

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Periodic acceptor excitation spectroscopy of single molecules

Doose, S., Heilemann, M., Michalet, X., Weiss, S., Kapanidis, A. N.

European Biophysics Journal 36: 669-674 (2007)

Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.

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Detectors for single-molecule fluorescence imaging and spectroscopy

Michalet, X., Siegmund, O.H.W., Vallerga, J.V., Jelinsky, P., Millaud, J.E., Weiss, S.

Journal of Modern Optics 54: 239-282 (2007)

Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomeriza- tion, cellular signaling, protein–protein or protein–nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. Here, the necessary performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy, are investigated.

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Shot-noise limited single-molecule FRET histogram: comparison between theory and experiments

Nir, E., Michalet, X., Hamadani, K., Laurence, T. A., Neuhauser, D., Kovchegov, Y., Weiss, S.

Journal of Physical Chemistry B 110: 22103-22124 (2006)

We describe a simple approach and present a straightforward numerical algorithm to compute the best fit shot-noise limited proximity ratio histogram (PRH) in single-molecule fluorescence resonant energy transfer diffusion experiments. The key ingredient is the use of the experimental burst size distribution, as obtained after burst search through the photon data streams. We show how the use of an alternated laser excitation scheme and a correspondingly optimized burst search algorithm eliminates several potential artifacts affecting the calculation of the best fit shot-noise limited PRH. This algorithm is tested extensively on simulations and simple experimental systems. We find that dsDNA data exhibits a wider PRH than expected from shot noise only, and hypothetically account for it by assuming a small Gaussian distribution of distances with an average standard deviation of 1.6 Å. Finally, we briefly mention the results of a future publication, and illustrate them with a simple two-state model system (DNA hairpin), for which the kinetic transition rates between the open and close conformations are extracted.

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Photon-counting H33D detector for biological fluorescence imaging

Michalet, X., Siegmund, O.H.W., Vallerga, J.V., Jelinsky, P., Millaud, J.E., Weiss, S.

Nuclear Instruments and Methods in Physics Research A 567: 133-166 (2006)

We have developed a photon-counting High-temporal and High-spatial resolution, High-throughput 3-Dimensional detector (H33D) for biological imaging of fluorescent samples. The design is based on a 25mm diameter S20 photocathode followed by a 3-microchannel plate stack, and a cross-delay line anode. We describe the bench performance of the H33D detector, as well as preliminary imaging results obtained with fluorescent beads, quantum dots and live cells and discuss applications of future generation detectors for single molecule imaging and high-throughput study of biomolecular interactions.

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Single-Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

Michalet, X., Weiss, S., Jäger, M.

Chemical Reviews 106: 1785-1813 (2006)

Single-molecule measurements provide unique information on heterogeneous populations of molecules: They give access to the complete distribution of observables (rather than only their first moments), allow discrimination between static and dynamic heterogeneity of their properties, and enable the detection of rare events or a succession of events hidden by ensemble averaging and the impossibility to synchronize molecules. Single-molecule methods have now pervaded several disciplines, in particular chemistry, evolving from stage of proof-of-principle experiments to decisive research and discovery tools...

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Advances in fluorescence imaging with quantum dot bio-probes
Pinaud, F., Michalet, X., Bentolila, L. A., Tsay, J. M., Li, J. J., Iyer, G., Weiss, S.
Biomaterials 27: 1679-1687 (2006)
After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed, the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo.
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Wavefunction engineering: From quantum wells to near-infrared type-II colloidal quantum dots synthesized by layer-by-layer colloidal epitaxy

Li, J. J., Tsay, J. M., Michalet, X., Weiss, S.

Chemical Physics 318: 82-90 (2005)

We review the concept and the evolution of bandgap and wavefunction engineering, the seminal contributions of Dr. Chemla to the understanding of the rich phenomena displayed in epitaxially grown quantum confined systems, and demonstrate the application of these concepts to the colloidal synthesis of high quality type-II CdTe/CdSe quantum dots using successive ion layer adsorption and reaction chemistry. Transmission electron microscopy reveals that CdTe/CdSe can be synthesized layer by layer, yielding particles of narrow size distribution. Photoluminescence emission and excitation spectra reveal discrete type-II transitions, which correspond to energy lower than the type-I bandgap. The increase in the spatial separation between photoexcited electrons and holes as a function of successive addition of CdSe monolayers was monitored by photoluminescence lifetime measurements. Systematic increase in lifetimes demonstrates the high level of wavefunction engineering and control in these systems.

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Protein-protein interactions as a tool for site-specific labeling of proteins

Jäger, M., Michalet, X., Weiss, S.

Protein Science 14: 2059-2068 (2005)

Probing structures and dynamics within biomolecules using ensemble and single-molecule fluorescence resonance energy transfer requires the conjugation of fluorophores to proteins in a site-specific and thermodynamically nonperturbative fashion. Using single-molecule fluorescence-aided molecular sorting and the chymotrypsin inhibitor 2–subtilisin BPN0 complex as an example, we demonstrate that protein–protein interactions can be exploited to afford site-specific labeling of a recombinant double-cysteine variant of CI2 without the need for extensive and time-consuming chromatography. The use of protein–protein interactions for site-specific labeling of proteins is compatible with and complementary to existing chemistries for selective labeling of N-terminal cysteines, and could be extended to label multiple positions within a given polypeptide chain.

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Accurate FRET Measurements Within Single Diffusing Biomolecules Using Alternating-Laser Excitation

Lee, N. K., Kapanidis, A. N., Wang, Y., Michalet, X., Mukhopadhyay, J., Ebright, R. H., Weiss, S.

Biophysical Journal 88: 2939-2953 (2005)

Fluorescence resonance energy transfer (FRET) between a donor (D) and an acceptor (A) at the single-molecule level currently provides qualitative information about distance, and quantitative information about kinetics of distance changes. Here, we used the sorting ability of confocal microscopy equipped with alternating-laser excitation (ALEX) to measure accurate FRET efficiencies and distances from single molecules, using corrections that account for crosstalk terms that contaminate the FRET-induced signal, and for differences in the detection efficiency and quantum yield of the probes. ALEX yields accurate FRET independent of instrumental factors, such as excitation intensity or detector alignment. Using DNA fragments, we showed that ALEX-based distances agree well with predictions from a cylindrical model of DNA; ALEX-based distances fit better to theory than distances obtained at the ensemble level. Distance measurements within transcription complexes agreed well with ensemble-FRET measurements, and with structural models based on ensemble-FRET and X-ray crystallography. ALEX can benefit structural analysis of biomolecules, especially when such molecules are inaccessible to conventional structural methods due to heterogeneity or transient nature.

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Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics

Michalet, X., Pinaud, F. F., Bentolila, L. A., Tsay, J. M., Doose, S., Li, J. J., Sundaresan, G., Wu, A. M., Gambhir, S. S., Weiss, S.

Science 307: 538 -544 (2005)

Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.

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Femtomole Mixer for Microsecond Kinetic Studies of Protein Folding

Hertzog, D. E., Michalet, X., Jäger, M., Kong, X., Santiago, J. G., Weiss, S. and Bakajin, O.

Analytical Chemistry 76 (24): 7169 -7178 (2004)

We have developed a microfluidic mixer for studying protein folding and other reactions with a mixing time of 8 s and sample consumption of femtomoles. This device enables us to access conformational changes under conditions far from equilibrium and at previously inaccessible time scales. In this paper, we discuss the design and optimization of the mixer using modeling of convective diffusion phenomena and a characterization of the mixer performance using microparticle image velocimetry, dye quenching, and Förster resonance energy-transfer (FRET) measurements of single-stranded DNA. We also demonstrate the feasibility of measuring fast protein folding kinetics using FRET with acyl-CoA binding protein.

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The power and prospects of fluorescence microscopies and spectroscopies

Michalet, X., Kapanidis, A.N., Laurence, T., Pinaud, F., Doose, S., Pflughoefft, M., and Weiss, S.

Annual Reviews in Biophysics and Biomolecular Structures 32: 161-182 (2003)

Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory’s interest in single-molecule microscopy and spectroscopy.

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Single-molecule spectroscopy and microscopy

Michalet, X., Weiss. S.

Comptes rendus Physique 3: 619-644 (2002)

Advances in detector sensitivity and improvements in instrument design have recently provided scientists with tools to probe single molecules with light, and monitor their photophysical properties with exquisite sensitivity and spatial as well as temporal resolution. Spectroscopic and temporal information is used to explore molecular structure, conformational dynamics, local environment and intermolecular interactions of individual species. High-resolution single-molecule microscopy allows these methods to be used for in vitro or in vivo molecular colocalization with nanometer precision. The collected data have generated a wealth of new information in domains ranging from chemistry and biology to solid state physics.

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Properties of Fluorescent Semiconductor Nanocrystals and their Application to Biological Labeling

Michalet, X., Pinaud, F., Lacoste, T. D., Dahan, M., Bruchez, M. P., Alivisatos, A. P., Weiss. S.

Single Molecules 2 (4): 261-276 (2001)

We review recent advances in the development of colloidal fluorescent semiconductor nanocrystals (a class of quantum dots) for biological labeling. Although some of the photophysical properties of nanocrystals are not fully understood and are still actively investigated, researchers have begun developing bioconjugation schemes and applying such probes to biological assays. Nanocrystals possess several qualities that make them very attractive for fluorescent tagging: broad excitation spectrum, narrow emission spectrum, precise tunability of their emission peak, longer fluorescence lifetime than organic fluorophores and negligible photobleaching. On the down side, their emission is strongly intermittent ("blinking”) and their size is relatively large for many biological uses. We describe how to take advantage of nanocrystals’ spectral properties to increase the resolution of fluorescence microscopy measurements down to the nanometer level. We also show how their long fluorescence lifetime can be used to observe molecules and organelles in living cells without interference from background autofluorescence, a pre-requisite for single molecule detectability. Finally, their availability in multicolor species and their single molecule sensitivity open up interesting possibilities for genomics applications.

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Ultrahigh-Resolution Colocalization of Spectrally Separable Point-like Fluorescent Probes

Michalet, X., Lacoste, T. D., Weiss. S.

Methods 25: 87-102 (2001)

An ultrahigh-resolution colocalization method based on the simultaneous acquisition and analysis of spectrally separated images of the excitation point-spread function of point-like fluorescent probes is reviewed. It is shown that molecular distances can be measured with accuracy better than 10 nm using conventional far-field optics. A detailed account of the methodology, theoretical considerations, signal processing, and data fitting algorithms is given.

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Ultrahigh-resolution multicolor colocalization of single fluorescent probes

Lacoste, T. D., Michalet, X., Pinaud, F., Chemla, D. S., Alivisatos, A. P., Weiss. S.

Proceedings of the National Academy of Sciences USA 97 (17): 9461-9466 (2000)

An optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described in this paper. It relies on the use of two unique families of fluorophores, namely energy-transfer fluorescent beads (TransFluoSpheres) and semiconductor nanocrystal quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A multi-color sample-scanning confocal microscope was constructed that allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps with a piezo-scanner. The resulting spots are accurately localized by fitting them to the known shape of the excitation point-spread function of the microscope. We present results of two-dimensional colocalization of TransFluoSpheres (40 nm in diameter) and of nanocrystals (3–10 nm in diameter) and demonstrate distance-measurement accuracy of better than 10 nm using conventional far-field optics. This ruler bridges the gap between fluorescence resonance energy transfer, near- and far-field imaging, spanning a range of a few nanometers to tens of micrometers.

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Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA

Herrick, J., Michalet, X., Conti, C., Schurra, C., Bensimon, A.

Proceedings of the National Academy of Sciences USA 97 (1): 222-227 (2000)

An approach was developed for the quantification of subtle gains and losses of genomic DNA. The approach relies on a process called molecular combing. Molecular combing consists of the extension and alignment of purified molecules of genomic DNA on a glass coverslip. It has the advantage that a large number of genomes can be combed per coverslip, which allows for a statistically adequate number of measurements to be made on the combed DNA. Consequently, a high-resolution approach to mapping and quantifying genomic alterations is possible. The approach consists of applying fluorescence hybridization to the combed DNA by using probes to identify the amplified region. Measurements then are made on the linear hybridization signals to ascertain the region’s exact size. The reliability of the approach first was tested for low copy number amplifications by determining the copy number of chromosome 21 in a normal and trisomy 21 cell line. It then was tested for high copy number amplifications by quantifying the copy number of an oncogene amplified in the tumor cell line GTL-16. These results demonstrate that a wide range of amplifications can be accurately and reliably quantified. The sensitivity and resolution of the approach likewise was assessed by determining the copy number of a single allele (160 kb) alteration.

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High-resolution mapping of the X-linked lymphoproliferative syndrome region by FISH on combed DNA

Monier, K., Michalet, X., Lamartine, J., Schurra, C., Heitzmann, F., Yin, L., Cinti, R., Sylla,  B. S., Creaven, M., Porta, G., Vourc'h, C., Robert-Nicoud, M., Bensimon, A., Romeo, G.

Cytogenetics and Cell Genetics 81 (3-4): 259-264 (1998)

X-linked lymphoproliferative syndrome is an inherited immunodeficiency for which the responsible gene is currently unknown. Several megabase-sized deleted regions mapping to Xq25 have been identified in XLP patients, and more recently a 130-kb deletion has been reported (Lamartine et al., 1996; Lanyi et al., 1996). To establish a physical map of this deleted region and to identify the XLP gene, two cosmid contigs were established (Lamartine et al., 1996). However, the physical map of this region is still uncompleted and controversial and three points remain unsolved: (1) the centromeric-telomeric orientation of the whole region, (2) the relative orientation of the two contigs, and (3) the size of the gap between the two contigs. To provide a definitive answer to these questions, high-resolution mapping by fluorescence in situ hybridization on combed DNA and molecular approaches were combined to establish the physical map of the XLP region over 600 kb. Our results identified a gap of 150 kb between the two contigs, established the relative orientation of one contig to the other, and determine the centromeric-telomeric orientation of the whole region. Our results show that the order of the marker over this region is:

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Dynamic Molecular Combing: Stretching the Whole Human Genome for High-Resolution Studies

Michalet, X., Ekong, R., Fougerousse, F., Rousseaux, S., Schurra, C., Hornigold, N., van Slegtenhorst, M., Wolfe, J., Povey, S., Beckmann, J. S., Bensimon, A.

Science 277: 1518-1523 (1997)

DNA in amounts representative of hundreds of eukaryotic genomes was extended on silanized surfaces by dynamic molecular combing. The precise measurement of hybridized DNA probes was achieved directly without requiring normalization. This approach was validated with the high-resolution mapping of cosmid contigs on a yeast artificial chromosome (YAC) within yeast genomic DNA. It was extended to human genomic DNA for precise measurements ranging from 7 to 150 kilobases, of gaps within a contig and of microdeletions in the tuberous sclerosis 2 gene on patients DNA. The simplicity, reproducibility and precision of this approach, makes it a powerful tool for a variety of genomic studies.

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Vesicles of Toroidal Topology: Observed Morphogy and Shape Transformations

Michalet, X., Bensimon, D.

Journal de Physique II France 5: 263-287 (1995)

We report observations of toroidal vesicles with circular and noncircular cross sections, axisymmetric and nonaxisymmetric. Shape transformations induced by temperature changes are also described, which permit a connection to a mathematical conjecture due to Willmore. Our observations are analysed using a numerical procedure which allows the determination of the relevant geometrical parameters of the shapes. We compare these observations with recent theoretical predictions and point out some unexpected properties of the observed equilibrium shapes.

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Observation of Stable Shapes and Conformal Diffusion in Genus 2 Vesicles

Michalet, X., Bensimon, D.

Science 269: 666-668 (1995)

The observed equilibrium shapes of phospholipid vesicles of topological genus 2 (shapes with two holes) are found to be in agreement with theoretical predictions on the basis of a minimization of the elastic curvature energy for fluid membranes under the constraints of constant area, volume and area difference (between the inner and outer layers of the membrane). For some particular geometrical characteristics, the shapes of the vesicles change continuously and randomly on a slow time scale (tens of seconds) and thus exhibit conformal diffusion. This phenomenon is a reflection of the conformal degeneracy of the elastic curvature energy. Its observation sets a limit (three constraints) on the number of physical constraints relevant to the determination of the shapes of vesicles.

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Fluctuating Vesicles of Nonspherical Topology

Michalet, X., Bensimon, D., Fourcade, B.

Physical Review Letters 72 (1): 168-171 (1994)

We report the observation of phospholipid vesicles of high topology, 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 two nearby concentric membranes. Using boudary layer methods we determine tha shape and elastic energy of the necks and corroborate this analysis by a numerical solution of the minimization problem. This approach leads to a qualitatively correct description of our experiments.

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