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

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.

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Low-Light Photodetectors for Fluorescence Microscopy

Applied Sciences ◽

10.3390/app11062773 ◽

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.

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

ACS Sensors ◽

10.1021/acssensors.8b01092 ◽

2018 ◽

Vol 3 (12) ◽

pp. 2644-2650 ◽

Cited By ~ 5

Author(s):

Xiaojun Liu ◽

Conghui Huang ◽

Chenghua Zong ◽

Aiye Liang ◽

Zhangjian Wu ◽

...

Keyword(s):

Quantum Dots ◽

Fluorescence Microscopy ◽

Single Molecule ◽

Wide Field ◽

Homogeneous Immunoassay

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Single occupancy spectroelectrochemistry of freely diffusing flavin mononucleotide in zero-dimensional nanophotonic structures

Faraday Discussions ◽

10.1039/c5fd00072f ◽

2015 ◽

Vol 184 ◽

pp. 101-115 ◽

Cited By ~ 31

Author(s):

Lawrence P. Zaino ◽

Dane A. Grismer ◽

Donghoon Han ◽

Garrison M. Crouch ◽

Paul W. Bohn

Keyword(s):

Electron Transfer ◽

Single Molecule ◽

Light Emission ◽

Fluorescence Emission ◽

Flavin Mononucleotide ◽

Wide Field ◽

Potential Sweep ◽

Potential Control ◽

Fluorescence Measurements ◽

Electron Transfer Properties

Zero-mode waveguides (ZMW) have the potential to be powerful confinement tools for studying electron transfer dynamics at single molecule occupancy conditions. Flavin mononucleotide contains an isoalloxazine chromophore, which is fluorescent in the oxidized state (FMN) while the reduced state (FMNH2) exhibits dramatically lower light emission, i.e. a dark-state. This allows fluorescence emission to report the redox state of single FMN molecules, an observation that has been used previously to study single electron transfer events in surface-immobilized flavins and flavoenzymes, e.g. sarcosine oxidase, by direct wide-field imaging of ZMW arrays. Single molecule electron transfer dynamics have now been extended to the study of freely diffusing molecules using fluorescence measurements of Au ZMWs under single occupancy conditions. The Au in the ZMW serves both as an optical cladding layer and as the working electrode for potential control, thereby accessing single molecule electron transfer dynamics at μM concentrations. Consistent with expectations, the probability of observing single reduced molecules increases as the potential is scanned negative, Eappl < Eeq, and the probability of observing emitting oxidized molecules increases at Eappl > Eeq. Different single molecules exhibit different electron transfer properties as reflected in the position of Eeq and the distribution of Eeq among a population of FMN molecules. Two types of actively-controlled electroluminescence experiments were used: chronofluorometry experiments, in which the potential is alternately stepped between oxidizing and reducing potentials, and cyclic potential sweep fluorescence experiments, analogous to cyclic voltammetry, these latter experiments exhibiting a dramatic scan rate dependence with the slowest scan rates showing distinct intermediate states that are stable over a range of potentials. These states are assigned to flavosemiquinone species that are stabilized in the special environment of the ZMW nanopore.

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Electro-optic imaging enables efficient wide-field fluorescence lifetime microscopy

Nature Communications ◽

10.1038/s41467-019-12535-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 7

Author(s):

Adam J. Bowman ◽

Brannon B. Klopfer ◽

Thomas Juffmann ◽

Mark A. Kasevich

Keyword(s):

Temporal Resolution ◽

Single Molecule ◽

Fluorescence Lifetime ◽

Collection Efficiency ◽

Frame Rate ◽

Optical Systems ◽

Wide Field ◽

Pockels Cell ◽

Low Light ◽

Optical Efficiency

Abstract Nanosecond temporal resolution enables new methods for wide-field imaging like time-of-flight, gated detection, and fluorescence lifetime. The optical efficiency of existing approaches, however, presents challenges for low-light applications common to fluorescence microscopy and single-molecule imaging. We demonstrate the use of Pockels cells for wide-field image gating with nanosecond temporal resolution and high photon collection efficiency. Two temporal frames are obtained by combining a Pockels cell with a pair of polarizing beam-splitters. We show multi-label fluorescence lifetime imaging microscopy (FLIM), single-molecule lifetime spectroscopy, and fast single-frame FLIM at the camera frame rate with 103–105 times higher throughput than single photon counting. Finally, we demonstrate a space-to-time image multiplexer using a re-imaging optical cavity with a tilted mirror to extend the Pockels cell technique to multiple temporal frames. These methods enable nanosecond imaging with standard optical systems and sensors, opening a new temporal dimension for wide-field low-light microscopy.

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Versatile Uniform Illumination for Quantitative Wide-Field Fluorescence and Single-Molecule Localization Microscopies

Biophysical Journal ◽

10.1016/j.bpj.2019.11.938 ◽

2020 ◽

Vol 118 (3) ◽

pp. 150a

Author(s):

Adrien Mau ◽

Nicolas Bourg ◽

Sandrine Lévêque-Fort

Keyword(s):

Single Molecule ◽

Wide Field ◽

Uniform Illumination

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

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2012.0035 ◽

2013 ◽

Vol 368 (1611) ◽

pp. 20120035 ◽

Cited By ~ 79

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.

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Local Structural Flexibility of Nucleic Acid Probed by a Wide Field Single Molecule FRET Imaging Technique

Frontiers in Optics 2009/Laser Science XXV/Fall 2009 OSA Optics & Photonics Technical Digest ◽

10.1364/ls.2009.lswd5 ◽

2009 ◽

Cited By ~ 1

Author(s):

Tae-Hee Lee

Keyword(s):

Nucleic Acid ◽

Single Molecule ◽

Imaging Technique ◽

Structural Flexibility ◽

Wide Field ◽

Single Molecule Fret

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

The Journal of Physical Chemistry A ◽

10.1021/jp0510847 ◽

2005 ◽

Vol 109 (30) ◽

pp. 6652-6658 ◽

Cited By ~ 29

Author(s):

Thomas Gensch ◽

Martin Böhmer ◽

Pedro F. Aramendía

Keyword(s):

Fluorescence Microscopy ◽

Single Molecule ◽

Wide Field

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Assessment of Gold Bio-Functionalization for Wide-Interface Biosensing Platforms

Sensors ◽

10.3390/s20133678 ◽

2020 ◽

Vol 20 (13) ◽

pp. 3678

Author(s):

Lucia Sarcina ◽

Luisa Torsi ◽

Rosaria Anna Picca ◽

Kyriaki Manoli ◽

Eleonora Macchia

Keyword(s):

Single Molecule ◽

Thin Film Transistor ◽

Cost Effective ◽

Gold Surface ◽

Wide Field ◽

Technical Potential ◽

Multiple Biomarkers ◽

Wide Interface ◽

High Analytical Performance

The continuous improvement of the technical potential of bioelectronic devices for biosensing applications will provide clinicians with a reliable tool for biomarker quantification down to the single molecule. Eventually, physicians will be able to identify the very moment at which the illness state begins, with a terrific impact on the quality of life along with a reduction of health care expenses. However, in clinical practice, to gather enough information to formulate a diagnosis, multiple biomarkers are normally quantified from the same biological sample simultaneously. Therefore, it is critically important to translate lab-based bioelectronic devices based on electrolyte gated thin-film transistor technology into a cost-effective portable multiplexing array prototype. In this perspective, the assessment of cost-effective manufacturability represents a crucial step, with specific regard to the optimization of the bio-functionalization protocol of the transistor gate module. Hence, we have assessed, using surface plasmon resonance technique, a sustainable and reliable cost-effective process to successfully bio-functionalize a gold surface, suitable as gate electrode for wide-field bioelectronic sensors. The bio-functionalization process herein investigated allows to reduce the biorecognition element concentration to one-tenth, drastically impacting the manufacturing costs while retaining high analytical performance.

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