Young Investigator Award at BIOS Photonics/West

Since 2007 PicoQuant GmbH awards a "Young Investigator Award" at the session on "Single Molecule Spectroscopy and Imaging" during the BIOS / Photonics West show. Young scientists (age 35 or below) are encouraged to participate in this best paper competition which offers a cash award worth 750 USD. Participants must be both the primary author and presenter of an accepted abstract to be eligible. The proceedings paper also has to be submitted at least 1 week before the meeting starts for review purposes.

2010 award

As in the previous years it was impossible to nominate a single winner and the price was again increased to 1125 USD and split equally betwen Ryan A. Colyer (University of California), Matthew D. Lew (Stanford University) and Steffen J. Sahl (MPI Göttingen) for

High-throughput multispot single-molecule spectroscopy
Ryan A. Colyer, Giuseppe Scalia, Univ. of California, Los Angeles (United States); Taiho Kim, Nesher Technologies (United States); Ivan Rech, Daniele Resnati, Stefano Marangoni, Massimo Ghioni, Sergio Cova, Politecnico di Milano (Italy); Shimon Weiss, Xavier Michalet, Univ. of California, Los Angeles (United States)

Solution-based single-molecule spectroscopy and fluorescence correlation spectroscopy (FCS) are powerful techniques for accessing molecular properties such as size, brightness, conformation, and binding constants. We present a new parallelized approach with a multispot excitation and detection geometry using a combination of three powerful new technologies: (i) a liquid crystal spatial light modulator to produce multiple diffraction-limited excitation spots; (ii) a multipixel detector array matching the excitation pattern and (iii) a low-cost reconfigurable multichannel counting board. We demonstrate the capabilities of this technique by reporting FCS measurements of various calibrated samples as well as single-molecule burst measurements.

In vivo three-dimensional superresolution fluorescence tracking using a double-helix point spread function
Matthew D. Lew, Michael A. Thompson, Majid Badieirostami, W. E. Moerner, Stanford Univ. (United States)

We characterize the localization precision of a unique method for 3D superresolution imaging, which utilizes a double-helix point spread function (DH-PSF). We show that the DH-PSF has a higher and more uniform localization accuracy than the standard PSF throughout a 2 µm depth of field. The localization precision of our method, with 5000-7000 photons detected on top of background noise of ~5 photons/pixel, is 8-15 nm in the x-y directions and 10-20 nm in the axial direction. We finally use the DH-PSF to track the movement of ParB-fluorescent protein fusions in live Caulobacter crescentus cells in three dimensions with high accuracy.

STED nanoscopy and single molecule tracking map the nanoscale dynamics of plasma membrane lipids
Steffen J. Sahl, Marcel Leutenegger, Christian Ringemann, Veronika Müller, Stefan Hell, Christian Eggeling, Max Planck Institute for Biophysical Chemistry (Germany)

Far-field fluorescence microscopy of single molecules can provide detailed insights into molecular characteristics in biological and non-biological materials. However, due to its limited spatial resolution, conventional far-field microscopy can often not solve prominent problems in biology. For example, cholesterol-assisted lipid interactions such as the integration into lipid nanodomains ('rafts') are considered to play a functional part in a whole range of membrane-associated processes, but their direct and non-invasive observation in living cells is impeded by the resolution limit of >200nm. Using the superior spatial resolution of stimulated emission depletion (STED) microscopy as well as the exceeding spatial localization accuracy of single-molecule tracking, we report the direct and non-invasive detection of single diffusing lipid molecules in nano-sized areas in the plasma membrane of living cells and obtain new details of molecular membrane dynamics. Specifically, we combine a (tunable) resolution of down to 30nm with tools such as fluorescence correlation spectroscopy (FCS), or implement a simple optical method capable of tracking a fluorescent molecule in two dimensions, with high fidelity and currently unrivaled spatiotemporal resolution. As a result, we demonstrate that certain lipids or other 'raft'-associated molecules are transiently (~10ms) trapped on the nanoscale (<20nm areas) in cholesterol-mediated molecular complexes.

2009 award

The large amount of excellent talks this year made it impossible to pick a single winner. In the end it was necessary to increase the award amount to 1125 USD and split it equally between Sigrun Henkenjohann (University of Bielefeld), Nathan P. Wells (Los Alamos National Laboratories) and Jonas Fölling (MPI Göttingen) for

Photoinduced electron transfer probes for the observation of enzyme activities
Sigrun Henkenjohann, Markus Sauer, University of Bielefeld (Germany)

In our work we demonstrate the general applicability of Photoinduced Electron Transfer (PET) Probes for the observation of various hydrolases at the ensemble and single molecule level. The rapid response time of the probes enables real-time monitoring of enzyme activities and provides quantitative data which are compared to those of commonly available and recently published, more complex probes like those based on Fluorescence Resonance Energy Transfer (FRET).

Going beyond 2D: following membrane diffusion and topography in the IgE-Fc[epsilon]RI system using 3 dimensional tracking microscopy
Nathan P. Wells, Guillaume A. Lessard, Mary E. Phipps, Peter M. Goodwin, Los Alamos National Lab. (United States); Diane S. Lidke, The Univ. of New Mexico (United States); Bridget S. Wilson, Univ. of New Mexico (United States); James H. Werner, Los Alamos National Lab. (United States)

The ability to follow and observe single molecules as they function in live cells would represent a major milestone for molecular-cellular biology. Here we present a tracking microscope that is able to track quantum dots in 3 dimensions and simultaneously record time-resolved emission statistics from a single dot. This innovative microscopy approach is based on four spatial filters and closed loop feedback to constantly keep a single quantum dot in the focal spot. Using this microscope, we demonstrate the ability to follow quantum dot-labeled IgE antibodies bound to Fc[epsilon]RI membrane receptors in live RBL-2H3 cells. The results are consistent with prior studies of 2 dimensional membrane diffusion (Andrews et al., Nat. Cell Biol., 10, 955, 2008).
In addition, the microscope captures motion in the axial (Z) direction, which permits tracking of diffusing receptors relative the "hills and valleys" of the dynamically changing membrane landscape. Our novel approach is uniquely capable of following single-molecule dynamics on live cells with 3 dimensional spatial resolution.

Multi-parameter far-field fluorescence nanoscopy based on photoswitching single molecules
Andreas Schönle, Ilaria Testa, Jonas Fölling, Claas von Middendorff, Claudia Geisler, Max-Planck-Institut für Biophysikalische Chemie (Germany); Mariano Bossi, Univ. de Buenos Aires (Argentina); Vladimir N. Belov, Christian Eggeling, Alexander Egner, Stefan W. Hell, Max-Planck-Institut für Biophysikalische Chemie (Germany)

By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single molecule level, we demonstrate multi-parameter spectroscopic imaging with spatial resolutions down to 15nm. Because ensemble averaging is avoided by the sequential switching and consecutive detection of single emitters, the method allows application of all techniques known from single-molecule spectroscopy in sparse samples to the imaging of densely labeled specimen. This opens up a new class of functional imaging techniques.

2008 award

All talks presented in that session were of high quality so that it proved extremely difficult for the jury to nominate the winner. In the end the price was awarded to Andrea M. Armani from the California Institute of Technology (CalTech) for

Label-free detection of cytokines using optical microcavities
Andrea M. Armani, Scott E. Fraser, California Institute of Technology

Ultra-high-Q microresonators have demonstrated sensitive and specific chemical and biological detection.[1, 2] The sensitivity is derived from the long photon lifetime inside the cavity which amplifies small signals. Specificity is achieved through surface functionalization. Recently, microtoroid resonators demonstrated quality factors greater than 100 million. Here, these ultra-high-Q microcavities are shown to be capable of performing label-free, single molecule studies [3, 4]. The detection mechanism is new and relies upon a thermo-optic mechanism to enhance resonant wavelength shifts induced through binding of a molecule.
In the present work, planar arrays of ultra-high-Q microtoroid resonators were used to detect Interleukin-2 (IL-2) in fetal bovine serum (FBS). IL-2 is a cytokine released in response to immune system activation by extrinsic and intrinsic stimuli. The surface of the microtoroids was sensitized for detection using anti-IL-2 and a series of "doped" serum solutions were prepared containing [300, 600, 900]aM of IL-2. The label-free, single molecule detection data and the theoretical basis for detection will be presented.
[1] A. M. Armani, R. P. Kulkarni, S. E. Fraser, R. C. Flagan, and K. J. Vahala, Science, 2007.
[2] A. M. Armani and K. J. Vahala, Optics Letters, vol. 31, pp. 1896-1898, 2006.
[3] A. M. Armani, D. K. Armani, B. Min, K. J. Vahala, and S. M. Spillane, Applied Physics Letters, vol. 87, pp. 151118, 2005.
[4] D. K. Armani, T. J. Kippenberg, S. M. Spillane, and K. J. Vahala, Nature, vol. 421, pp. 925-928, 2003.

2007 award

The quality of the talks presented in that session were very high so that it proved extremely difficult for the jury to nominate the winner. In the end the price was split between H.R.C. Dietrich (TU Delft) and M.I. Rudenko (University of California/Santa Cruz) for

A new optical method for characterizing single molecule interactions
H. R. C. Dietrich, B. Vermolen, B. Rieger, I. T. Young, Y. Garini, Technische Univ. Delft (Netherlands)

In recent years, fundamental biochemical and biophysical research has started to apply single molecule techniques rather than techniques that look at a general population of molecules. This allows one to gain detailed insight into biological processes and functions of biomolecules. Therefore, the improvement and development of new single molecular methods are of increasing importance. DNA-protein interactions are of interest due to their importance in the control of cellular processes. These interactions occur, for example, in transcription regulation, DNA replication and DNA repair. We have developed an optical method to study dynamic behaviors related to DNA-protein interactions. This technique is based on sub-micron gold beads that scatter polychromatic light. One end of the DNA molecule is immobilized onto a gold support; the other end is attached to a gold bead (d = 80 nm). In solution, the DNA/bead system is moving within a volume that depends on the length of the DNA polymer. The precise determination of this action volume gives information on the effective length of the DNA tether and its variation due to the interaction with other proteins or with changes in the environment (e.g. pH and salt concentration). This method allows the detection and study of single molecular interactions in a very simple and robust way. We use a darkfield microscope equipped with a sensitive CCD camera in order to image the DNA/bead system. We can record about 20 images per second. We then analyze the motion of each individual bead/molecule over time intervals ranging up to two hours as our system setup does not suffer from bleaching. From the change in the bead position due to Brownian motion, we can deduce diffusion characteristics and other relevant parameters. We will present results of experiments based on the analysis of the DNA/bead system under varying conditions of fluid viscosity, salt concentration, addition of recA and naked DNA.

Integration and characterization of SiN nanopores for single-molecule detection in liquid-core ARROW waveguides
M. I. Rudenko, D. Yin, Univ. of California/Santa Cruz; M. Holmes, A. R. Hawkins, Brigham Young Univ.; H. Schmidt, Univ.of California/Santa Cruz

Single molecule detection using microfluidic waveguide channels provides researchers with valuable information for drug development and promotes fascinating discoveries regarding molecular dynamics on the nanoscale. We have developed a novel device (“lab on chip”) based on low loss liquid-core antiresonant reflecting optical (ARROW) waveguides with single molecule fluorescence sensitivity and SiN nanopores, integrated on one platform. Analysis of concurrent optical (fluorescence) and electrical (ionic current blockade) signals provides new single molecule detection capabilities. The ARROW waveguide consists of alternating PECVD-deposited SiN and SiO2 layers. The channel is formed by a sacrificial etch. We measured ARROW channel resistance with solution of 0.1M KCl and Hepes at pH 7.6 as a ribosome buffer and also different concentrations of KCl using Ag/AgCl electrodes, and found impedance to be in GOhm range. In order to integrate a nanopore, a micropore is first dry etched through the top layers of the waveguide leaving only a SiN layer covering the core. A focused ion beam machine is used to form a 60-120 nm (top diameter) conical nanopore in the SiN membrane. The pore size can be reduced further to 20-40 nm using a scanning electron microscope. Detailed studies of the nanopore fabrication, size determination, electrical properties and particle translocation will be presented. In summary, we have shown that nanopores can be integrated with liquid-core optical waveguides to pave the way for electrical and optical single molecule detection on a single chip.