Conference 11650

Single Molecule Spectroscopy and Superresolution Imaging XIV

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  • Welcome and Introduction
  • BiOS Hot Topics
  • 1: Biological Applications of SM Spectroscopy and Superresolution Imaging
  • 2: Nanoscopy or Super-Resolution Fluorescence Imaging
  • 3: New Single Molecule or Superresolution Techniques
  • 4: Single Molecule or Particle Tracking
  • 5: Deep Learning, Computational and Theoretical Aspects of Single Molecule or Superresolution Techniques
  • Poster Session
2021-03-03T17:49:54-08:00
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Session R: Welcome and Introduction
11650-800
Author(s): Rainer Erdmann, PicoQuant GmbH (Germany); Ingo Gregor, Georg-August-Univ. Göttingen (Germany); Felix Koberling, PicoQuant GmbH (Germany)
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Introduction to SPIE Photonics West BiOS conference 11650: Single Molecule Spectroscopy and Superresolution Imaging XIV
Session LIVE: BiOS Hot Topics
11618-700
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Every year, attendees look forward to Saturday Night Hot Topics, an evening spent hearing highly engaged, world renowned speakers reveal the latest innovations in their areas of expertise. Don't miss this year's outstanding list of speakers.
11648-601
Author(s): Enrico Gratton, Univ. of California, Irvine (United States)
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11648-600
Author(s): Kevin K. Tsia, The Univ. of Hong Kong (Hong Kong, China)
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Session 1: Biological Applications of SM Spectroscopy and Superresolution Imaging
11650-1
Author(s): Anish R. Roy, Wei Zhang, Zeinab Jahed, Ching-Ting Tsai, Bianxiao Cui, W. E. Moerner, Stanford Univ. (United States)
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11650-2
Author(s): Ziqing Winston Zhao, National Univ. of Singapore (Singapore); Qian Peter Su, Univ. of Technology, Sydney (Australia); Sunney X. Xie, Yujie Sun, Peking Univ. (China)
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11650-4
Author(s): Gonfa Tesfaye Assefa, Tjaart P. J. Krüger, Michal Gwizdala, Univ. of Pretoria (South Africa)
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Phycobilisomes (PBs) are light-harvesting complexes in many strains of cyanobacteria. Using single molecule spectroscopy we have revealed the spectroscopic dynamics of PBs and discovered a novel type of photoprotective mechanism at physiologically relevant light intensities. This mechanism is light-activated and does not require interactions with other proteins. Moreover, by controlling the interaction between individual PBs and single orange carotenoid proteins (OCPs), we revealed an intermediate state of quenching signifying the docking of OCP on a PB. Finally, newly revealed light-harvesting states of PB rods are potentially related to energy transfer to different photosystems.
Session 2: Nanoscopy or Super-Resolution Fluorescence Imaging
11650-9
Author(s): Markus Sauer, Sebastian Reinhard, Julius-Maximilians-Univ. Würzburg (Germany); Toby Bell, Monash Univ. (Australia)
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Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.
11650-10
Author(s): Mike Heilemann, Johann Wolfgang Goethe-Univ Frankfurt am Main (Germany)
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We present fluorophore labels that transiently and repetitively bind to their targets as probes for various types of super-resolution fluorescence microscopy. Transient labels typically show a weak affinity to a target, and exchange constantly with the buffer that constitutes a reservoir with a large amount of intact probes, leading to repetitive binding events to the same target (we refer to these labels as “exchangeable labels”). This dynamic labeling approach is insensitive to common photobleaching and yields a constant fluorescence signal over time, which has been successfully exploited in SMLM, STED, single-particle tracking and super-resolution optical fluctuation imaging (SOFI). We discuss properties of suitable exchangeable labels and experimental parameters for optimal performance for the different super-resolution methods. In addition, we show how to combine different classes of exchangeable labels for high-quality multicolor super-resolution imaging.
11650-11
Author(s): Ming-Qiang Zhu, Huazhong Univ. of Science and Technology (China)
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Session 3: New Single Molecule or Superresolution Techniques
11650-12
Author(s): Mircea Cotlet, Brookhaven National Lab. (United States)
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Hybrid metal colloidal quantum dot nanoarchitectures are assembled to exhibit controllable emission intensity and polarization output via coupled plasmon-exciton interactions and by using DNA self-assembly methods. Using time-resolved single molecule optical modulation methods we demonstrate control of polarization output in a system who's components otherwise are polarization insensitive to incoming light. Reference Polarized Single-Particle Quantum Dot Emitters through Programmable Cluster Assembly, Honghu Zhang, et al. ACS Nano 2020, 14, 2, 1369–1378.
11650-13
Author(s): Phillip Charles McCann, The Univ. of Tokyo (Japan); Kotaro Hiramatsu, The Univ. of Tokyo (Japan), Japan Science and Technology Agency (Japan); Keisuke Goda, The Univ. of Tokyo (Japan), Univ. of California, Los Angeles (United States), Wuhan Univ. (China)
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Fluorescence-encoded Raman spectroscopy has become increasingly more popular by virtue of its high chemical specificity and sensitivity. However, current fluorescence-encoding methods are narrowband and lack sensitivity in the low wavenumber region which if addressed could further enhance these methods. To overcome these limitations, we propose and experimentally demonstrate a novel broadband method for fluorescence-encoded Raman spectroscopy, termed fluorescence-encoded time-domain coherent Raman spectroscopy (FLETCHERS), which is capable of probing molecular vibrations in the lower fingerprint region (200 – 750 cm-1 ) with sample concentrations as dilute as 100 nM and laser powers as low as 20 mW.
11650-14
Author(s): Lei Xu, Biqin Dong, Fudan Univ. (China)
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The field of view (FOV) of single-molecule localization microscopy (SMLM) is practically restricted to an area of approximately 50×50 μm^2 as only relatively perfect point spread functions (PSFs) in the center of the objective FOV can be used for accurate super-resolution image reconstruction. Here we present a systematical study to show that optical aberrations, such as spherical aberration, coma, field curvature, and chromatic aberration, commonly presented in the FOV periphery can significantly compromise the localization precision and accuracy and produce unreliable imaging results when using currently available localization algorithm for SMLM image reconstruction.
11650-15
Author(s): Dongeun Kim, Wonsang Hwang, Dug Young Kim, Yonsei Univ. (Korea, Republic of)
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Fluorescence anisotropy is a powerful tool for observing molecular rotational speed, which is widely applied for molecular conjugation measurements (quantitative assay, membrane tagging, and protein-protein interaction). Fluorescence anisotropy (r) can be obtained by subtracting horizontal polarized fluorescence (H) from vertical polarized fluorescence (V), and dividing by non-polarized fluorescence (F). Since F equals to V+2H, V and H can be thought of as weighted sum and subtraction of F and r∙F, respectively. Using phasor approach, which is graphical plotting technique based on fluorescence lifetime imaging microscopy, V and H are located on internal and external dividing points of F and r∙F. Considering those four phasor points (V, H, F, and r∙F) lie on a straight line, one can easily guess the value of the r. We first introduced phasor plot to fluorescence anisotropy, and confirmed that this method dramatically simplifies fluorescence anisotropy analysis with graphical intuition.
11650-18
Author(s): Fabrice Harms, Audrius Jasaitis, Cynthia Veilly, Xavier Levecq, Imagine Optic SA (France)
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PSF engineering has been widely used over the past years to enable 3D localization in Single Molecule Localization Microscopy (SMLM) techniques, such as PALM/STORM superresolution methods. It can make use of a cylindrical lens, a phase mask, a Spatial Light Modulator, a Deformable Mirror (DM) to encode depth on a spatial variation of the PSF along the Z axis. Among these techniques, we demonstrate that a DM-based approach – such as implemented in our MicAO-3DSR system - provides the best versatility/performance combination
Session 4: Single Molecule or Particle Tracking
11650-19
Author(s): Valentina Curcio, Institut Fresnel (France); Cesar Valades Cruz, Institut Curie (France); Luis A. Alemán Castañeda, Manos Mavrakis, Miguel Alonso, Sophie Brasselet, Institut Fresnel (France)
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Measuring single molecules’ 3D orientations in addition to their 3D spatial localization is still today a challenge, due to the intrinsic coupling of both spatial and orientational parameters in the point spread function (PSF) image formation. We present polarized fluorescence microscopy methods able to report both orientational and spatial information from single molecules with high precision. These methods are applied to STORM and PALM nanoscale imaging of actin filaments organization and membrane proteins’ conformational changes in 3D.
11650-20
Author(s): Laura Marchetti, Scuola Normale Superiore (Italy), Ctr. per l'Innovazione delle Nanotecnologie, Istituto Italiano di Tecnologia (Italy), Univ. di Pisa (Italy); Fulvio Bonsignore, Scuola Normale Superiore (Italy); Rosy Amodeo, Scuola Normale Superiore (Italy), Ctr. per l'Innovazione delle Nanotecnologie, Istituto Italiano di Tecnologia (Italy); Chiara Schirripa Spagnolo, Aldo Moscardini, Scuola Normale Superiore (Italy); Francesco Gobbo, Scuola Normale Superiore (Italy), Ctr. for Discovery Brain Sciences, The Univ. of Edinburgh (United Kingdom); Antonino Cattaneo, Scuola Normale Superiore (Italy); Fabio Beltram, Stefano Luin, Scuola Normale Superiore (Italy), Istituto Nanoscienze, Consiglio Nazionale delle Ricerche (Italy)
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We developed a toolbox for independent single particle tracking of membrane receptors and their ligands, suitable for fast receptors like p75NTR, based on chemical tagging of recombinant proteins, TIRF microscopy, and automatized analyses of single particle trajectories. The superresolved localization of this technique allowed analyzing functions, interactions and stoichiometry of (pro)neurotrophin receptors p75NTR and TrkA in living cells, with particular attention on some of their existing or used mutants. The analysis also after treatments with ligands or drugs unraveled their mode of action in the first steps of sundry signaling pathways. We also optimized visualization and analysis of two moieties.
11650-21
Author(s): Anna-Karin Gustavsson, Stanford Univ. (United States), Rice Univ. (United States); Rajarshi P. Ghosh, Petar N. Petrov, Jan Liphardt, W. E. Moerner, Stanford Univ. (United States)
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The organization and dynamics of the genome regulate DNA replication, DNA repair, and gene expression. Here, we developed a methodology for studies of 3D organization and dynamics of the nucleome over time scales ranging from milliseconds to hours, and length scales ranging from tens of nanometers to micrometers. We achieve unprecedented 3D track lengths throughout the mammalian nucleus by combining a novel labeling scheme using nanobody ArrayG/N fusions of dSpCas9 targeted to specific DNA loci; light sheet illumination for gentle imaging of live cells with high contrast; and point spread function engineering for parallel detection of multiple loci in 3D.
11650-22
Author(s): Bertus van Heerden, Tjaart P. J. Krüger, Univ. of Pretoria (South Africa)
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One of the main challenges in studying single biomolecules in a native or near-native environment is their constant diffusion. An approach to solving this problem is real-time single particle tracking (SPT). In this study, we used statistical calculations and dynamic simulations to compare the orbital, Knight’s Tour and MINFLUX localization methods, in the context of fluorescence-based and interferometric scattering (iSCAT) approaches. While the Knight’s Tour method can track the fastest diffusion, MINFLUX achieves the greatest precision. The relative success of iSCAT vs fluorescence is strongly dependent on the particle size, and the photophysical properties and density of the fluorophores.
11650-23
Author(s): Shangguo Hou, Jack Exell, Kevin Welsher, Duke Univ. (United States)
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Measuring the behavior of single molecules enables the discovery of states and dynamics obscured by bulk measurements. However, molecules in solution rapidly diffuse in three dimensions, precluding long-duration and high-temporal resolution measurement. To overcome this hurdle, we have developed 3D single-molecule active real-time tracking (3D-SMART) which enables active feedback tracking of rapidly diffusing (exceeding 10 μm2/sec) and lowly emitting fluorescent (rates of 10 kHz or less) particles. Here we demonstrate the application of 3D-SMART to a range targets, from single virus-like particles and quantum dots in water, all the way down to single fluorophores in viscous solution. This new single molecule tracking can be applied to continuously monitor single proteins and nucleic acids, including real-time measurement of transcription on a freely diffusing, single-dye labelled DNA strand. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.
Session 5: Deep Learning, Computational and Theoretical Aspects of Single Molecule or Superresolution Techniques
11650-5
Author(s): Xiaotong Yuan, Varun Mannam, Univ. of Notre Dame (United States); Yide Zhang, Univ. of Notre Dame (United States), Caltech (United States); Scott Howard, Univ. of Notre Dame (United States)
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Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool in biomedical studies for its capability to provide an additional molecular contrast in fluorescence microscopy. To investigate sub-cellular molecular dynamics, super-resolution FLIM has been developed. However, statistical localization based techniques have not been demonstrated. To address this, we present a theoretical model that demonstrates how frequency-domain (FD) FLIM resolution is fundamentally different from diffraction-limited fluorescence microscopy. From this understanding, we perform localization-based super-resolution FD-FLIM using super-resolution radial fluctuations (SRRF) technique. The approach is demonstrated both numerically and experimentally.
11650-6
Author(s): Gili Dardikman-Yoffe, Yonina C. Eldar, Weizmann Institute of Science (Israel)
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The use of photo-activated fluorescent molecules to create long sequences of low emitter-density diffraction-limited images enables high-precision emitter localization, but at the cost of low temporal resolution. We suggest combining a recent high-performing classical method with model-based deep learning, using the algorithm unfolding approach, to design a compact neural network considering domain knowledge. Our results show that we can obtain super-resolution imaging from a small number of high emitter density frames without knowledge of the optical system and across different test sets. Thus, we believe LSPARCOM will find broad use in single molecule localization microscopy of biological structures, and pave the way to interpretable, efficient live-cell imaging in a broad range of settings.
11650-7
Author(s): Varun Mannam, Univ. of Notre Dame (United States); Yide Zhang, Univ. of Notre Dame (United States), Caltech (United States); Xiaotong Yuan, Scott Howard, Univ. of Notre Dame (United States)
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Fluorescence microscopy in modern biology enables us to visualize biological organisms with micrometer scale resolution, but difficult to recognize sub-micron/nm. Super-resolution techniques allow for sub-diffraction imaging, but frequently require expensive optical-setup or specialized fluorophores. To lower the technical barrier, deep learning has been previously demonstrated generating super-resolution (SR) from diffraction-limited (DL) images. Such approaches require thousands of training images, which is prohibitively difficult in many biological samples. We demonstrate a new approach to train SR CNNs using small (800 images to create 15 training targets) that are impossible using conventional CNN training approaches.
11650-8
Author(s): Abhijit Marar, Peter Kner, The Univ. of Georgia (United States)
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Localization based microscopy using self-interference digital holography (SIDH) provides three-dimensional (3D) positional information about point sources with nanometer scale precision. One of the most basic questions in localization-based microscopy concerns the accuracy with which the location of a point source can be determined. Here, using the Fisher information matrix, we calculate the theoretical limit to localization precision for SIDH and compare the precision bounds to existing methods for depth localization over a 20 µm depth of field. Further, we also discuss the effects of background noise on the localization precision when imaging point emitters using SIDH.
Session P: Poster Session
11650-24
Author(s): Siddhi Kediya, Central University of Gujarat (India); Anu Manhas, Pandit Deendayal Petroleum Univ. (India); Prakash C. Jha, Central Univ. of Gujarat (India)
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The excessive intake of fluoride anion could cause the chronic disease in human beings. Therefore, the detection of fluoride anion is a dire need of the recent era. However, to treat the problem, the small molecular sensors have been used as fluorescent probe to detect the fluoride anion. In this contribution, the fluoride anion sensing mechanism of molecular sensor N-(2-(benzothiazole-2-yl) phenyl)-4-methoxybenzamide (BTBA-OCH3) has been revealed using Density Functional Theory(DFT)/Time Dependent-Density Functional Theory (TD-DFT) methods. The feasibility of ESIPT process in the molecular probe and the inhibition of the same process on the fluoride addition was cross checked using computational methods. The UV-Vis absorption and emission spectra with and without anion were reproduced theoretically.
11650-25
Author(s): Daiki Hara, Yuki Shimaoka, Shin-nosuke Uno, Masaya Okada, Shigeki Iwanaga, Sysmex Corp. (Japan)
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Abnormal deposition of amyloid-β (Aβ) causes the formation of senile plaques, which is one of the main pathological features of Alzheimer disease. To visualize the plaques at nanometer resolution, single-molecule localization microscopy (SMLM) is promising. It is also essential to develop a method for suppressing autofluorescence especially under high-magnifications used for detecting single molecules. Here, we report a novel method to reduce autofluorescence in mouse brains which is applicable to visualize the structure of Aβ plaques by SMLM. The super-resolution images of Aβ plaques showed fibrous structures that were not able to be discerned by conventional fluorescence imaging.
11650-26
Author(s): Tim Hellwig, Maximilian Brinkmann, Refined Laser Systems GmbH (Germany)
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We present a novel multi-color light source with more than 100mW average output power that can be switched between four fixed output wavelengths selectable during construction of the source in the range of 350 – 1600nm. The light-source is based on a cost-effective and robust fiber optical parametric oscillator (OPO) and emits clean 25ps pulses at 40MHz without any tails or ringing and with the low timing jitter of a mode-locked laser. This development constitutes an important step for replacing bulky and expensive Ti:sapphire lasers and free-space OPOs in power demanding FLIM applications like widefield or light sheet FLIM.
11650-27
Author(s): Anna Zhikhoreva, Andrey Belashov, Ioffe Institute (Russian Federation); Mark Gelfond, National Medical Research Ctr of Oncology named after N. N. Petrov (Russian Federation); Irina V. Semenova, Oleg Vasyutinskii, Ioffe Institute (Russian Federation)
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Photodynamic inactivation is known to be effective for treatment of various viral and bacterial infections. In view of COVID-19 pandemic the search for therapeutic modalities efficient against this virus is of high demand. We present experimental results on detection of singlet oxygen (SO) generated using Radachlorin photosensitizer in a nebulizer aerosol jet and on different biological surfaces modeling, in particular, mucous membranes. The lifetimes of SO and photosensitizer triplet state were shown to depend noticeably on the surface type. Moreover the surface type was found to be strongly affecting the photosensitizer photobleaching kinetics, with mucous samples providing much slower bleaching.
Conference Chair
Georg-August-Univ. Göttingen (Germany)
Conference Chair
PicoQuant GmbH (Germany)
Conference Chair
PicoQuant GmbH Berlin (Germany)
Program Committee
The Univ. of Southern California (United States)
Program Committee
Friedrich-Schiller-Univ. Jena (Germany)
Program Committee
Univ. of Oxford (United Kingdom), Friedrich-Schiller Univ. Jena (Germany)
Program Committee
Georg-August-Univ. Göttingen (Germany)
Program Committee
Imperial College London (United Kingdom)
Program Committee
Ewa M. Goldys
The Univ. of New South Wales (Australia)
Program Committee
Univ. of North Texas Health Science Ctr. at Fort Worth (United States), Texas Christian Univ. at Fort Worth (United States)
Program Committee
Goethe-Univ. Frankfurt am Main (Germany)
Program Committee
KU Leuven (Belgium)
Program Committee
Zhen-Li Huang
Huazhong Univ. of Science and Technology (China)
Program Committee
Univ. Bielefeld (Germany)
Program Committee
Univ. of California, Los Angeles (United States)
Program Committee
Institute of Chemistry (China)
Additional Information
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