scholarly journals Single-molecule spectroscopy and imaging over the decades

2015 ◽  
Vol 184 ◽  
pp. 9-36 ◽  
Author(s):  
W. E. Moerner ◽  
Yoav Shechtman ◽  
Quan Wang
Keyword(s):  
Single Molecule ◽  
Optical Switching ◽  
Fluorescent Protein ◽  
Single Molecules ◽  
Three Dimensional ◽  
Super Resolution ◽  
Laser Frequency ◽  
High Resolution Spectroscopy ◽  
Focal Volume ◽  
Ensemble Averaging

As of 2015, it has been 26 years since the first optical detection and spectroscopy of single molecules in condensed matter. This area of science has expanded far beyond the early low temperature studies in crystals to include single molecules in cells, polymers, and in solution. The early steps relied upon high-resolution spectroscopy of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral fine structure arising directly from the position-dependent fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 1990s, a variety of fascinating physical effects were observed for individual molecules, including imaging of the light from single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency. In the room temperature regime, researchers showed that bursts of light from single molecules could be detected in solution, leading to imaging and microscopy by a variety of methods. Studies of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. All of these early steps provided important fundamentals underpinning the development of super-resolution microscopy based on single-molecule localization and active control of emitting concentration. Current thrust areas include extensions to three-dimensional imaging with high precision, orientational analysis of single molecules, and direct measurements of photodynamics and transport properties for single molecules trapped in solution by suppression of Brownian motion. Without question, a huge variety of studies of single molecules performed by many talented scientists all over the world have extended our knowledge of the nanoscale and many microscopic mechanisms previously hidden by ensemble averaging.

scholarly journals 3D tracking of extracellular vesicles by holographic fluorescence imaging

2020 ◽  
Vol 6 (45) ◽  
pp. eabc2508
Author(s):  
Matz Liebel ◽  
Jaime Ortega Arroyo ◽  
Vanesa Sanz Beltrán ◽  
Johann Osmond ◽  
Ala Jo ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Fluorescent Detection ◽  
Live Cells ◽  
Fluorescent Light ◽  
3D Tracking ◽  
Anisotropic Motion ◽  
Lag Times

Fluorescence microscopy is the method of choice in biology for its molecular specificity and super-resolution capabilities. However, it is limited to a narrow z range around one observation plane. Here, we report an imaging approach that recovers the full electric field of fluorescent light with single-molecule sensitivity. We expand the principle of digital holography to fast fluorescent detection by eliminating the need for phase cycling and enable three-dimensional (3D) tracking of individual nanoparticles with an in-plane resolution of 15 nm and a z-range of 8 mm. As a proof-of-concept biological application, we image the 3D motion of extracellular vesicles (EVs) inside live cells. At short time scales (<4 s), we resolve near-isotropic 3D diffusion and directional transport. For longer lag times, we observe a transition toward anisotropic motion with the EVs being transported over long distances in the axial plane while being confined in the horizontal dimension.

scholarly journals PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking

2012 ◽  
Vol 20 (5) ◽  
pp. 4957 ◽  
Author(s):  
Ignacio Izeddin ◽  
Mohamed El Beheiry ◽  
Jordi Andilla ◽  
Daniel Ciepielewski ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Resolution Imaging

scholarly journals Obtaining 3D Super-resolution Information from 2D Super-resolution Images through a 2D-to-3D Transformation Algorithm

2017 ◽  
Author(s):  
Andrew Ruba ◽  
Wangxi Luo ◽  
Joseph Kelich ◽  
Weidong Yang
Keyword(s):  
Single Molecule ◽  
Rotational Symmetry ◽  
Three Dimensional ◽  
Super Resolution ◽  
Live Cells ◽  
Limited Information ◽  
Spatiotemporal Resolution ◽  
Superresolution Microscopy ◽  
Localization Analysis ◽  
Probability Information

AbstractCurrently, it is highly desirable but still challenging to obtain three-dimensional (3D) superresolution information of structures in fixed specimens as well as dynamic processes in live cells with a high spatiotemporal resolution. Here we introduce an approach, without using 3D superresolution microscopy or real-time 3D particle tracking, to achieve 3D sub-diffraction-limited information with a spatial resolution of ≤ 1 nm. This is a post-localization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability information. The method has been successfully applied to obtain structural and functional information for 25-300 nm sub-cellular organelles that have rotational symmetry. In this article, we will provide a comprehensive analysis of this method by using experimental data and computational simulations.

scholarly journals POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos

2021 ◽  
Vol 118 (11) ◽  
pp. e2019071118
Author(s):  
Ayana Sugizaki ◽  
Keisuke Sato ◽  
Kazuyoshi Chiba ◽  
Kenta Saito ◽  
Masahiko Kawagishi ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Molecular Orientation ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Super Resolution ◽  
Living Cells ◽  
Test Case ◽  
Polarization Microscopy ◽  
Universal Method ◽  
Early Embryos

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

scholarly journals Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching

2019 ◽  
Author(s):  
Elias A. Halabi ◽  
Dorothea Pinotsi ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Time Lapse ◽  
Switching Behavior ◽  
Spatiotemporal Resolution ◽  
New Information ◽  
Human Neurons ◽  
Long Time ◽  
Intracellular Organelles

Photoswitchable molecules have found multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allowed us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.

scholarly journals NanoJ-SQUIRREL: quantitative mapping and minimisation of super-resolution optical imaging artefacts

2017 ◽  
Author(s):  
Siân Culley ◽  
David Albrecht ◽  
Caron Jacobs ◽  
Pedro Matos Pereira ◽  
Christophe Leterrier ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Original Data ◽  
Focal Volume ◽  
Coated Pits ◽  
Superresolution Imaging ◽  
Cell Structures ◽  
Image Artefacts ◽  
Localisation Microscopy ◽  
Super Resolution Microscopy

Most super-resolution microscopy methods depend on steps that contribute to the formation of image artefacts. Here we present NanoJ-SQUIRREL, an ImageJ-based analytical approach providing a quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same focal volume, this approach generates a quantitative map of super-resolution defects, as well as methods for their correction. To illustrate its broad applicability to super-resolution approaches we apply our method to Localization Microscopy, STED and SIM images of a variety of in-cell structures including microtubules, poxviruses, neuronal actin rings and clathrin coated pits. We particularly focus on single-molecule localisation microscopy, where super-resolution reconstructions often feature imperfections not present in the original data. By showing the quantitative evolution of data quality over these varied sample preparation, acquisition and super-resolution methods we display the potential of NanoJ-SQUIRREL to guide optimization of superresolution imaging parameters.

Single-molecule spectroscopy

2017 ◽  
Author(s):  
Michel Orrit
Keyword(s):  
Nonlinear Optics ◽  
Single Molecule ◽  
Single Molecules ◽  
Super Resolution ◽  
Optical Methods ◽  
Single Molecule Spectroscopy ◽  
New Techniques ◽  
Single Nanoparticle ◽  
Cell Biologist ◽  
Sensitivity And Selectivity

This chapter gives an overview of the main optical methods used to detect and study single molecules and other small objects (nano-objects). Much of the work so far has exploited the excellent sensitivity and selectivity of fluorescence, but several new techniques, mostly based on nonlinear optics, have recently reached the single-molecule or single-nanoparticle regime. The chapter briefly discusses some results with reference to published reviews. Single-molecule techniques have now been incorporated into the arsenal of the physico-chemist and the cell biologist. However, the recent development of super-resolution techniques and of new labels suggests that further progress can be expected from measurements on single nano-objects in the next few years.

scholarly journals A bisected pupil for studying single-molecule orientational dynamics and its application to three-dimensional super-resolution microscopy

2014 ◽  
Vol 104 (19) ◽  
pp. 193701 ◽  
Author(s):  
Adam S. Backer ◽  
Mikael P. Backlund ◽  
Alexander R. von Diezmann ◽  
Steffen J. Sahl ◽  
W. E. Moerner
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Orientational Dynamics ◽  
Super Resolution Microscopy

scholarly journals Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) Microscopy for Ultra-superresolution Imaging of Dendra2-HA transfected NIH3T3 cells

2020 ◽  
Author(s):  
Partha Pratim Mondal
Keyword(s):  
Distribution Function ◽  
Single Molecule ◽  
Gaussian Function ◽  
Single Molecules ◽  
Super Resolution ◽  
Underlying Mechanism ◽  
Image Resolution ◽  
Size Spectrum ◽  
Nih3t3 Cells ◽  
Microscopy Technique

To be able to resolve molecular-clusters it is crucial to access vital informations (such as, molecule density and cluster-size) that are key to understand disease progression and the underlying mechanism. Traditional single-molecule localization microscopy (SMLM) techniques use molecules of variable sizes (as determined by its localization precisions (LPs)) to reconstruct super-resolution map. This results in an image with overlapping and superimposing PSFs (due to a wide size-spectrum of single molecules) that degrade image resolution. Ideally it should be possible to identify the brightest molecules (also termed as, the fortunate molecules) to reconstruct ultra-superresolution map, provided sufficient statistics is available from the recorded data. POSSIBLE microscopy explores this possibility by introducing narrow probability size-distribution of single molecules (narrow size-spectrum about a predefined mean-size). The reconstruction begins by presetting the mean and variance of the narrow distribution function (Gaussian function). Subsequently, the dataset is processed and single molecule filtering is carried out by the Gaussian distribution function to filter out unfortunate molecules. The fortunate molecules thus retained are then mapped to reconstruct ultra-superresolution map. In-principle, the POSSIBLE microscopy technique is capable of infinite resolution (resolution of the order of actual single molecule size) provided enough fortunate molecules are experimentally detected. In short, bright molecules (with large emissivity) holds the key. Here, we demonstrate the POSSIBLE microscopy technique and reconstruct single molecule images with an average PSF sizes of σ ± Δσ = 15 ± 10 nm, 30 ± 2 nm & 50 ± 2 nm. Results show better-resolved Dendra2-HA clusters with large cluster-density in transfected NIH3T3 fibroblast cells as compared to the traditional SMLM techniques.

scholarly journals 3D Multicolor Nanoscopy at 10,000 Cells a Day

2019 ◽  
Author(s):  
Andrew E S Barentine ◽  
Yu Lin ◽  
Miao Liu ◽  
Phylicia Kidd ◽  
Leonhard Balduf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
High Speed ◽  
Three Dimensional ◽  
Super Resolution ◽  
High Volume ◽  
Volume Data ◽  
Cell Nuclei ◽  
Fully Integrated ◽  
Resolution Imaging

ABSTRACTDiffraction-unlimited single-molecule switching (SMS) nanoscopy techniques like STORM /(F)PALM enable three-dimensional (3D) fluorescence imaging at 20-80 nm resolution and are invaluable to investigate sub-cellular organization. They suffer, however, from low throughput, limiting the output of a days worth of imaging to typically a few tens of mammalian cells. Here we develop an SMS imaging platform that combines high-speed 3D single-molecule data acquisition with an automated, fully integrated, high-volume data processing pipeline. We demonstrate 2-color 3D super-resolution imaging of over 10,000 mammalian cell nuclei in about 26 hours, connecting the traditionally low-throughput super-resolution community to the world of omics approaches.

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