Summary

The seventh US-workshop, held for the second time in Los Angeles was a very successful event. Finally, nearly 60 researches participated in this event and enjoyed 12 lectures that covered a broad range of current research topics and techniques ranging from nucleic acid-protein interactions at the single-molecule level to the usage of FCS to extract information about anisotropic rotational diffusion and lensfree microscopy and tomography on a chip."

Invited speakers

Please click on the title to see the full abstract.

• David Bensimon (ENS Paris)
  "Single Cell Physiology: Optical control of protein expression and activity at the single cell level; applications to morphogenesis in zebrafish"
  Living organisms are made of cells that are capable of responding to external signals by modifying their internal state (gene expression or protein phosphorylation patterns) and subsequently their external environment by the release of signaling molecules. In multicellular organisms in particular, cellular differentiation and signaling is essential for the development of the organism. While many of the key actors of these processes are known (morphogens in development, kinases in signal transduction) much less is known of the quantitative rules that govern their interaction with one another and with other cellular players I will present our results regarding the development of means to optically control the expression and activity of proteins at the single cell level in a live organism and its use as a tool to study morphogenesis in zebrafish. The idea is to use light to control the activity of biomolecules either through isomerisation or uncaging.
  Thus we have used a non-active isoform of Retinoic Acid (RA) to generate upon illumination the active all-trans form (or inactivate the trans-isomer). We have shown that RA is stored in the embryo until required at bud stage for proper development of the hindbrain. Using UV laser illumination we have been able to control its release locally in the precursor of the head region showing that it can then rescue the development of embryos whose endogenous RA synthetic pathway has been blocked by a drug.
  I will then present results using caged cyclofen to control the activity of various proteins (the recombinase Cre, GFP and some transcription factors) at the single cell level in a live embryo. These results suggest that single cell control of morphogen gradients is possible in a live organism which should open the way for a precise comparison between morphogenetic models and observations.
• Xavier Michalet (UCLA)
  "Parallel multispot smFRET analysis using an 8-pixel SPAD array"
  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 (a few minutes to an hour) to obtain sufficient statistics. It also prevents from 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.
• Robert Ros (Arizona State University)
  "Tip induced fluorescence quenching for nanometer optical and topographical resolution"
  Progress in nanosciences and life sciences is closely related to developments of high resolution imaging techniques. During the last decades the invention and improvement of scanning probe microscopy techniques like atomic force microscopy (AFM) have opened new views onto nanoscale materials. On the other side, optical microscopy has been pushed beyond the diffraction limit and the observation of single molecules has evolved into a standard technology. I will present a novel technique for simultaneous high resolution topographical and optical imaging. We are utilizing the effect that silicon AFM tips quench the fluorescence emission of fluorophores in a very localized manner. We were able to show spatially and temporally correlated optical and topographical imaging at sub-5nm resolution. In addition to measurements with individual fluorophores, we imaged DNA origami triangles with 120 nm side length into which two fluorophores are incorporated at a well defined position with a distance of 20.5 nm to each other. On this sample we were able to resolve the morphology of the triangle and to confirm the localization and the distance of the fluorophores. We anticipate that the simplicity of the method and the commercial availability of the tips and instrument will lead to the application of this technique in the field of nanobiology, nanophotonics, and nanoscience.
• Everett Lipman (UC Santa Barbara)
  "Tracking Molecular Motors with Single-Molecule Encoders "
  Devices such as inkjet printers and disk drives track position and velocity using optical encoders, which produce periodic signals precisely synchronized with mechanical motion. We have implemented this technique at the molecular scale by labeling DNA with fluorescent dyes at regular intervals. The resulting molecular encoders can be used in several ways for high-resolution continuous tracking of individual motor proteins. I will describe the synthesis of single-molecule encoders, data from and modeling of experiments on a helicase and a DNA polymerase, and some ideas for future work.
• Ingo Gregor (University of Göttingen)
  "FCS in Biophysics: Advanced Methods and Applications "
  Due to thermal motion, molecules and small particles suspended in solution show translational and rotational diffusion. Measurement of the respective diffusion constants give information about size and shape of the diffusing particle. Due to its third order dependence on the hydrodynamic radius, the rotational diffusion constant is a very sensitive measure for the size of an molecule. Typical rotational diffusion times of biological macromolecules such as globular proteins under physiological conditions are in the order of dozens to hundreds of nanoseconds.
  An excellent method to measure diffusion constants of biological samples is fluorescence correlation spectroscopy (FCS). In FCS one measures the correlation of the fluorescence signal coming from a sample of fluorescent or fluorescently labeled molecules (or particles) at very low concentration (typically pico- to nanomolar). One of its major applications is the measurement of the translational diffusion of molecules in solution. In recent years, fluorescence cross-correlation techniques have also gained enormous popularity for the measurement of binding and interaction between different molecular species.
  The first successful FCS measurements of rotational diffusion were done the 1980s. However, FCS still is rarely applied to measure rotational diffusion. Experimentally, these measurements are more demanding than standard measurements of translational diffusion or cross-correlation. Additionally, the theory of FCS with rotational diffusion is demanding and there is no systematic study of how the measurements are influenced by experimental artifacts. Effects like depolarization when focusing/detecting light through objectives with large numerical aperture, label stoichiometry, non-zero angle between excitation/emission dipole of the label, overlap between the time scale of fluorescence decay and rotational diffusion, or insufficient co-rotation of the label with the labeled molecule have to be taken into account for a complete and correct analysis of the FCS data.
  We give an overview of using FCS for measuring rotational diffusion of macromolecules. After giving a brief review of the theoretical foundations, we systematically study the impact of different experimentally relevant parameters and present a measurement scheme to obtain all physically conceivable correlation curves of a polarization-sensitive FCS experiment. We demonstrate the possibility to extract information about anisotropic rotational diffusion. In conclusion we show that FCS is an attractive method for rotational diffusion measurements of macromolecules or colloids, exploiting its ability to measure at minute concentrations with highest sensitivity.
• Ryan Colyer (UCLA)
  "Phasors and FLIM with Widefield Photon Counting "
  Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique for distinguishing molecular species, studying molecular interactions or assembly, and observing Förster resonance energy transfer (FRET). The lifetime of the excited state is typically analyzed by timing photons very precisely and fitting one or more exponentials to a histogram of their arrival times. However, the fitting approach introduces significant difficulties for systems with many exponentials in a complex background, such as cellular imaging, or when attempting to fit to the low-count signals in single-molecule studies. By using a Fourier analysis of the photon arrival times, phasor analysis is able to reliably analyze lifetimes with no fitting, and using only computationally simple algebra. High sensitivity FLIM acquisition is typically done with a point detector and raster scanning. Because light is only collected from one spot of an image at a time, it typically takes many seconds or minutes to obtain a suitable image. However, with widefield photon counting light is collected from the entire image, enabling substantially faster frame rates. The combination of phasor analysis with widefield photon counting results in a flexible, robust, and fast approach to FLIM acquisition and analysis
• Serhan Isikman (UCLA)
  "Lensfree Microscopy and Tomography On a Chip "
  The ongoing revolution in digital technologies and information processing methods presents unique opportunities to transform the way optical imaging is performed, particularly toward improving the throughput of microscopes while at the same time reducing their size, cost and complexity. For this end, the field of lensfree computational imaging aims to discard lenses and other bulky optical components of conventional imaging systems, and provides a wide range of microscopy tools offering large space-bandwidth products, i.e., high-resolution imaging of ultra large imaging volumes. In this talk, our recent progress on brightfield and fluorescence lensfree on-chip microscopy and tomography modalities will be reviewed.
• Le A. Trinh (California Institute of Technology)
  "Probing the heterogeneity of protein kinetics during vertebrate organogenous"
  Fluorescence correlation spectroscopy (FCS) provides quantitative data on single protein molecules in living cells. Most FCS studies have been limited to analyzing fluorescently tagged proteins that are exogenously expressed. We have developed a gene trap approach, termed FlipTrap that enables the fluorescent tagging of proteins at their endogenous loci in zebrafish. The FlipTrap lines offer a unique opportunity to characterize the kinetics of single proteins during organogenesis. Focusing on the development of the inner ear, we show that FCS analyses of endogenously expressed FlipTrap proteins provide more accurate measurements of protein kinetics in the embryo. Importantly, FCS analyses of multiple proteins in different cell types and across developmental time identify developmental switches in protein kinetics that reflect the differentiation state of cell within the inner ear. The heterogeneities in protein dynamics revealed by these analyses highlights the complexity of the proteome and how comparative FCS coupled with the FlipTrap lines offers unprecedented quantitative data on proteins during organogenesis in living embryos.
• David Millar (Scripps Research Institute)
  "Nucleic Acid-Protein Interactions at the Single-Molecule Level"
  Single-molecule fluorescence spectroscopy is emerging as a powerful tool for detailed biophysical analyses of nucleic acid - protein interactions. Measurements at the single-molecule level can readily resolve and quantify different binding modes in complex interaction systems. Moreover, kinetic information on individual binding and dissociation steps or conformational changes can be obtained under conditions of thermodynamic equilibrium, without the need to synchronize a population of molecules. To illustrate these capabilities, I will describe two systems currently under study in my laboratory.
  (1) The HIV-1 protein Rev mediates the nuclear export of unspliced (genomic RNA) and partially spliced mRNAs encoding viral structural proteins. Rev interacts with a highly conserved element within the viral pre-mRNA known as the Rev response element (RRE). This is a complex interaction in which multiple Rev monomers assemble on the RRE, mediated by a combination of RNA-protein and protein-protein interactions. In addition, while Rev is the central player, a number of host proteins, including the DEAD box helicase DDX1, are also required for efficient nuclear export activity. Single-molecule TIRF microscopy was used to monitor hundreds of individual Rev-RRE assembly reactions in parallel on the surface of a quartz slide, revealing the mechanism of oligomeric assembly and the microscopic rate constants for each step in the assembly pathway. Moreover, DDX1 was shown to promote oligomerization of Rev on the RRE, explaining how DDX1 acts as a cellular cofactor of HIV-1.
  (2) DNA polymerases replicate DNA substrates with extraordinarily high fidelity because of their ability to discriminate between cognate and non-cognate nucleotide substrates during each cycle of nucleotide incorporation. Correct nucleotide selection is thought to arise from an induced-fit mechanism, whereby a correct incoming nucleotide induces a large conformational change of the polymerase. By monitoring the interaction between a single polymerase molecule (Klenow fragment) and a single DNA molecule by means of Förster resonance energy transfer (FRET), it is possible to identify different conformational states of the polymerase and to correlate these with specific steps in the replication cycle. The single-molecule FRET measurements reveal that the polymerase naturally fluctuates between three distinct conformations (open, closed and a novel half-closed conformation) in the absence of any nucleotide substrates. However, in the presence of a correct incoming nucleotide (complementary to the template base), the polymerase remains in the closed conformation for an extended period, in readiness for the ensuing phosphoryl transfer reaction. In contrast, when an incorrect nucleotide is supplied instead, the polymerase repeatedly accesses the half-closed conformation but rarely progresses to the fully-closed conformation, suggesting that the half-closed state acts as a fidelity checkpoint to test the incoming nucleotide for complementarity with the template base. These observations provide new insights into the important role of enzyme conformational dynamics during the process of nucleotide substrate selection.
• Samuel Burri (EPFL)
  "Towards Large Format, High Performance CMOS SPAD Image Sensors"
  In this short presentation we will outline the recent developments in the field of solid-state single-photon image sensors designed in standard CMOS technologies in our group. We outline the development that led to these results, our state-of-the-art, and some the applications we are currently pursuing in the field of biomedical imaging and space exploration.
• Felix Koberling (PicoQuant GmbH)
  "Next Generation TCSPC Detection for Confocal Microscopy "
  More than 20 years ago single photon counting based single molecule detection started with cooled photomultiplier tubes (PMT). Already 5 years later the single photon avalanche photodiode (SPAD) starts to replace the PMT especially due to its higher detection efficiency and became the workhorse in confocal single molecule microscopy. In the last decade step by step SPAD technology improvements enabled to meet most of the requirements of modern Time-Correlated Single Photon Counting (TCSPC) for ultrasensitive detection.
  Recently a new detector module became available which allows to merge the still remaining timing performance advantages of the PMT with the photon processing efficiency of the SPAD. We incorporated this novel hybrid photomultiplier detector module (PMA hybrid) in a cooled, self-contained housing and will present its outstanding performance like narrow and ultrastable IRF, low darkcounts and almost negligible afterpulsing. Our experiments will demonstrate the advantages for several applications like FLIM and FCS.
  Beneath this new single point detector we will show recent results for different types of TCSPC detector arrays for highly parallel and / or spectrally resolved detection together with high throughput counting electronics and new approaches towards a robust and efficient multidimensional data analysis.
• Rainer Erdmann (PicoQuant GmbH)
  "Fluorescence Lifetime: a new dimension for confocal imaging and FCS"
  Confocal laser scanning microscopes (CLSM) are an essential tool in biological and biomedical research. Their functionality can be further enhanced by adding sensitive time-resolved data acquisition capabilities, enabling Fluorescence Lifetime Imaging (FLIM), lifetime based Förster Resonance Energy Transfer (FRET) and Fluorescence (Lifetime) Correlation Spectroscopy (F(L)CS) down to the single molecule level. By using the non-descanned (NDD) internal detectors of the laser scanning microscope, also NDD FLIM acquisition becomes easy and powerful. Based on a recently added network interface, the FLIM and FCS data acquisition can now be directly accessed from the CLSM computer. This unique integration enables a seamless work flow.
  Förster Resonance Energy Transfer (FRET) studies provide a very powerful tool for a broad range of biological applications since this technique enables to measure intra- and intermolecular distances down to several nanometres. Other than intensity-based FRET measurements, FLIM can further reveal sub-populations; thus, allowing to determine the fraction of free donors compared to associated donor molecules within a complex. The result of such an analysis yields not only the FRET efficiency distribution of FLIM-FRET images, but also the fraction and distribution of complete to incomplete FRET complexes. In addition, so-called lifetime sensitive sensors allow the monitoring of environmental conditions such as pH and ion concentration.
  In order to achieve fast spectral FLIM measurements, up to eight parallel TCSPC detection channels can be employed. To overcome data transfer restrictions of the USB 2.0 interface, we have evaluated and developed new host interface solutions for our multichannel TCSPC device HydraHarp 400.

Previous Workshops

For further details about each year's event, please select the year from the list below.

Information and Correspondence