scholarly journals Single Molecule Blinking and Photobleaching Separated by Wide-Field Fluorescence Microscopy

2005 ◽  
Vol 109 (30) ◽  
pp. 6652-6658 ◽  
Author(s):  
Thomas Gensch ◽  
Martin Böhmer ◽  
Pedro F. Aramendía
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Wide Field

scholarly journals Low-Light Photodetectors for Fluorescence Microscopy

2021 ◽  
Vol 11 (6) ◽  
pp. 2773
Author(s):  
Hiroaki Yokota ◽  
Atsuhito Fukasawa ◽  
Minako Hirano ◽  
Toru Ide
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
Science Studies ◽  
Single Photon ◽  
Laser Scanning Microscopy ◽  
Oxide Semiconductor ◽  
Wide Field ◽  
Single Molecule Fluorescence ◽  
Low Light

Over the years, fluorescence microscopy has evolved and has become a necessary element of life science studies. Microscopy has elucidated biological processes in live cells and organisms, and also enabled tracking of biomolecules in real time. Development of highly sensitive photodetectors and light sources, in addition to the evolution of various illumination methods and fluorophores, has helped microscopy acquire single-molecule fluorescence sensitivity, enabling single-molecule fluorescence imaging and detection. Low-light photodetectors used in microscopy are classified into two categories: point photodetectors and wide-field photodetectors. Although point photodetectors, notably photomultiplier tubes (PMTs), have been commonly used in laser scanning microscopy (LSM) with a confocal illumination setup, wide-field photodetectors, such as electron-multiplying charge-coupled devices (EMCCDs) and scientific complementary metal-oxide-semiconductor (sCMOS) cameras have been used in fluorescence imaging. This review focuses on the former low-light point photodetectors and presents their fluorescence microscopy applications and recent progress. These photodetectors include conventional PMTs, single photon avalanche diodes (SPADs), hybrid photodetectors (HPDs), in addition to newly emerging photodetectors, such as silicon photomultipliers (SiPMs) (also known as multi-pixel photon counters (MPPCs)) and superconducting nanowire single photon detectors (SSPDs). In particular, this review shows distinctive features of HPD and application of HPD to wide-field single-molecule fluorescence detection.

A Single-Molecule Homogeneous Immunoassay by Counting Spatially “Overlapping” Two-Color Quantum Dots with Wide-Field Fluorescence Microscopy

ACS Sensors ◽  
2018 ◽  
Vol 3 (12) ◽  
pp. 2644-2650 ◽  
Author(s):  
Xiaojun Liu ◽  
Conghui Huang ◽  
Chenghua Zong ◽  
Aiye Liang ◽  
Zhangjian Wu ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Wide Field ◽  
Homogeneous Immunoassay

scholarly journals Development of new photon-counting detectors for single-molecule fluorescence microscopy

2013 ◽  
Vol 368 (1611) ◽  
pp. 20120035 ◽  
Author(s):  
X. Michalet ◽  
R. A. Colyer ◽  
G. Scalia ◽  
A. Ingargiola ◽  
R. Lin ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Single Photon ◽  
Photon Counting ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Large Area ◽  
Single Molecule Fluorescence ◽  
Single Photon Counting ◽  
Single Molecule Fluorescence Microscopy

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

Direct observation of structural properties and fluorescent trapping sites in macrocyclic porphyrin arrays at the single-molecule level

2016 ◽  
Vol 18 (5) ◽  
pp. 3871-3877 ◽  
Author(s):  
Sujin Ham ◽  
Ji-Eun Lee ◽  
Suhwan Song ◽  
Xiaobin Peng ◽  
Takaaki Hori ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Structural Properties ◽  
Direct Observation ◽  
Single Molecule ◽  
Site Selection ◽  
Wide Field ◽  
Single Molecule Level ◽  
Selection For ◽  
Trapping Sites

By utilizing single-molecule defocused wide-field fluorescence microscopy, we have investigated the molecular structural properties and ascertained site selection for fluorescent trapping sites in multichromophoric systems.

scholarly journals Flat-field illumination for quantitative fluorescence imaging

2018 ◽  
Author(s):  
Ian Khaw ◽  
Benjamin Croop ◽  
Jialei Tang ◽  
Anna Möhl ◽  
Ulrike Fuchs ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Spatial Coherence ◽  
Optical Component ◽  
Wide Field ◽  
Gaussian Profile ◽  
Flat Field ◽  
Working Distance ◽  
Quantitative Fluorescence

AbstractThe uneven illumination of a Gaussian profile makes quantitative analysis highly challenging in laser-based wide-field fluorescence microscopy. Here we present flat-field illumination (FFI) where the Gaussian beam is reshaped into a uniform flat-top profile using a high-precision refractive optical component. The long working distance and high spatial coherence of FFI allows us to accomplish uniform epi and TIRF illumination for multi-color single-molecule imaging. In addition, high-throughput borderless imaging is demonstrated with minimal image overlap.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

scholarly journals Recurrent neural network-based volumetric fluorescence microscopy

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Luzhe Huang ◽  
Hanlong Chen ◽  
Yilin Luo ◽  
Yair Rivenson ◽  
Aydogan Ozcan
Keyword(s):  
Neural Network ◽  
Image Reconstruction ◽  
Fluorescence Microscopy ◽  
Recurrent Network ◽  
Sample Volume ◽  
Depth Of Field ◽  
Objective Lens ◽  
Wide Field ◽  
Volumetric Imaging ◽  
3D Image Reconstruction

AbstractVolumetric imaging of samples using fluorescence microscopy plays an important role in various fields including physical, medical and life sciences. Here we report a deep learning-based volumetric image inference framework that uses 2D images that are sparsely captured by a standard wide-field fluorescence microscope at arbitrary axial positions within the sample volume. Through a recurrent convolutional neural network, which we term as Recurrent-MZ, 2D fluorescence information from a few axial planes within the sample is explicitly incorporated to digitally reconstruct the sample volume over an extended depth-of-field. Using experiments on C. elegans and nanobead samples, Recurrent-MZ is demonstrated to significantly increase the depth-of-field of a 63×/1.4NA objective lens, also providing a 30-fold reduction in the number of axial scans required to image the same sample volume. We further illustrated the generalization of this recurrent network for 3D imaging by showing its resilience to varying imaging conditions, including e.g., different sequences of input images, covering various axial permutations and unknown axial positioning errors. We also demonstrated wide-field to confocal cross-modality image transformations using Recurrent-MZ framework and performed 3D image reconstruction of a sample using a few wide-field 2D fluorescence images as input, matching confocal microscopy images of the same sample volume. Recurrent-MZ demonstrates the first application of recurrent neural networks in microscopic image reconstruction and provides a flexible and rapid volumetric imaging framework, overcoming the limitations of current 3D scanning microscopy tools.

scholarly journals Tunable Wide-Field Illumination and Single-Molecule Photoswitching with a Single MEMS Mirror

ACS Photonics ◽  
2021 ◽  
Vol 8 (9) ◽  
pp. 2728-2736
Author(s):  
Lucas Herdly ◽  
Paul Janin ◽  
Ralf Bauer ◽  
Sebastian van de Linde
Keyword(s):  
Single Molecule ◽  
Wide Field

scholarly journals Total Internal Reflection Fluorescence Microscopy (TIRFM) – novel techniques and applications

2020 ◽  
Vol 8 (11) ◽  
Author(s):  
Verena Richter ◽  
Michael Wagner ◽  
Herbert Schneckenburger
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Plasma Membranes ◽  
Focal Adhesions ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Thin Layers ◽  
Structured Illumination ◽  
Structured Illumination Microscopy

Total Internal Reflection Fluorescence Microscopy (TIRFM) has been established almost 40 years ago for studies of plasma membranes or membrane proximal sites of living cells. The method is based on light incidence at an angle above the critical angle of total internal reflection and generation of an evanescent electromagnetic field penetrating about 100 nm into a sample and permitting selective excitation of membrane proximal fluorophores. Two techniques are presented here: prism-type TIRFM and objective-type TIRFM with high aperture microscope objective lenses. Furthermore, numerous applications are summarized, e.g. measurement of focal adhesions, cell-substrate topology, endocytosis or exocytosis of vesicles as well as single molecule detection within thin layers. Finally, highly innovative combinations of TIRFM with Förster Resonance Energy Transfer (FRET) measurements as well as with Structured Illumination Microscopy (SIM) and fluorescence reader technologies are presented.

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