Shimon Weiss

Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy.

Dynamic structural changes of macromolecules undergoing biochemical reactions can be studied using novel single molecule spectroscopy tools. Recent advances in applying such distance and orientation molecular rulers to biological systems are reviewed, and future prospects and challenges are discussed.

Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)

Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.

Particle Size, Surface Coating, and PEGylation Influence the Biodistribution of Quantum Dots in Living Mice

This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6-9 and 2-3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

Ultrahigh resolution multicolor colocalization of single fluorescent probes

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 multicolor 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.

Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism.

Using fluorescence resonance energy transfer to monitor distances within single molecules of abortively initiating transcription initiation complexes, we show that initial transcription proceeds through a “scrunching” mechanism, in which RNA polymerase (RNAP) remains fixed on promoter DNA and pulls downstream DNA into itself and past its active center. We show further that putative alternative mechanisms for RNAP active-center translocation in initial transcription, involving “transient excursions” of RNAP relative to DNA or “inchworming” of RNAP relative to DNA, do not occur. The results support a model in which a stressed intermediate, with DNA-unwinding stress and DNA-compaction stress, is formed during initial transcription, and in which accumulated stress is used to drive breakage of interactions between RNAP and promoter DNA and between RNAP and initiation factors during promoter escape.

Time-gated biological imaging by use of colloidal quantum dots

The long (but not too long) fluorescence lifetime of CdSe semiconductor quantum dots was exploited to enhance fluorescence biological imaging contrast and sensitivity by time-gated detection. Significant and selective reduction of the autofluorescence contribution to the overall image was achieved, and enhancement of the signal-to-background ratio by more than an order of magnitude was demonstrated.

Double phase-conjugate mirror: analysis, demonstration, and applications

We report on the operation of the double phase-conjugate mirror (DPCM). Two inputs to opposite sides of a photorefractive barium titanate crystal, which may carry different spatial images, are shown to pump the same four-wave mixing process mutually and are self-refracted without any external or internal crystal surface. This results in the phase-conjugate reproduction of the two images simultaneously. This device is analyzed theoretically, and applications in image processing, interferometry, and rotation sensing are discussed. We also demonstrate the operation of a ring laser, using the DPCM, as well as a photorefractive resonator with two facing DPCMs that can support spatial information in its oscillations.

microPET-Based Biodistribution of Quantum Dots in Living Mice

This study evaluates the quantitative biodistribution of commercially available CdSe quantum dots (QD) in mice. Methods: 64Cu-Labeled 800- or 525-nm emission wavelength QD (21- or 12-nm diameter), with or without 2,000 MW (molecular weight) polyethylene glycol (PEG), were injected intravenously into mice (5.55 MBq/25 pmol QD) and studied using well counting or by serial microPET and region-of-interest analysis. Results: Both methods show rapid uptake by the liver (27.4–38.9 %ID/g) (%ID/g is percentage injected dose per gram tissue) and spleen (8.0–12.4 %ID/g). Size has no influence on biodistribution within the range tested here. Pegylated QD have slightly slower uptake into liver and spleen (6 vs. 2 min) and show additional low-level bone uptake (6.5–6.9 %ID/g). No evidence of clearance from these organs was observed. Conclusion: Rapid reticuloendothelial system clearance of QD will require modification of QD for optimal utility in imaging living subjects. Formal quantitative biodistribution/imaging studies will be helpful in studying many types of nanoparticles, including quantum dots.

EVIDENCE FOR A THERMAL CONTRIBUTION TO EMISSION INTERMITTENCY IN SINGLE CDSE/CDS CORE/SHELL NANOCRYSTALS

The on–off intermittent behavior of emission from single CdSe/CdS core/shell nanocrystals was investigated as a function of temperature and excitation intensity. Off times were found to be independent of excitation power and the temperature dependence reveals substantial reduction in the number of on–off cycles prior to final particle darkening at low temperatures. On times are found to vary linearly with excitation intensity over a broad range and the turn off rate shows activated Arrhenius behavior down to T=50 K. These observations are consistent with a darkening mechanism that is a combination of Auger photoionization and thermal trapping of charge. The inhomogeneity of various possible trap sites is discussed. A thermally activated neutralization process is required for the particle to return to the on state. The influence of shell composition on intermittency is compared for CdS and ZnS [M. Nirmal et al., Nature 383, 802 (1996)].

Dynamic partitioning of a glycosyl-phosphatidylinositol-anchored protein in glycosphingolipid-rich microdomains imaged by single-quantum dot tracking.

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‐inositol‐anchored 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 high‐resolution 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.