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

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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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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Phasor-based single-molecule fluorescence lifetime imaging using a wide-field photon-counting detector

10.1117/12.809496 ◽

2009 ◽

Cited By ~ 7

Author(s):

R. Colyer ◽

O. Siegmund ◽

A. Tremsin ◽

J. Vallerga ◽

S. Weiss ◽

...

Keyword(s):

Single Molecule ◽

Fluorescence Lifetime ◽

Photon Counting ◽

Wide Field ◽

Fluorescence Lifetime Imaging ◽

Single Molecule Fluorescence ◽

Photon Counting Detector ◽

Lifetime Imaging

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Solid State High Throughput Screening Microscopy

10.1101/602011 ◽

2019 ◽

Author(s):

M. Ashraf ◽

S. Mohanan ◽

B Sim ◽

A. Tam ◽

D. Brousseau ◽

...

Keyword(s):

Solid State ◽

High Throughput ◽

High Throughput Screening ◽

Imaging System ◽

Focal Point ◽

Collection Efficiency ◽

Frame Rate ◽

Single Sample ◽

Wide Field ◽

Led Array

We introduce a solid state high throughput screening (ssHTS) imaging modality that uses a novel Newtonian telescope design to image multiple spatially separated samples without moving parts or robotics. Conventional high-throughput imaging modalities either require movement of the sample to the focal plane of the imaging system1–3 or movement of the imaging system itself4,5, or use a wide-field approach to capture several samples in one frame. Schemes which move the sample or the imaging system can be mechanically complex and are inherently slow, while wide-field imaging systems have poor light collection efficiency and resolution compared to systems that image a single sample at a given time point. Our proposed ssHTS system uses a large parabolic reflector and an imaging lenses positioned at their focal distances above each sample. A fast LED array sequentially illuminate samples to generate images that are captured with a single camera placed at the focal point of the reflector. This optical configuration allows each sample to completely fill a sensors field of view. Since each LED illuminates a single sample and LED switch times are very fast, images from spatially separated samples can be captured at rates limited only by the camera’s frame rate. The system is demonstrated by imaging cardiac monolayer and Caenorhabditis elegans (C. elegans) preparations.

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Wide-field subsecond temporal resolution optical monitoring systems for detection and study of cosmic hazards

Uspekhi Fizicheskih Nauk ◽

10.3367/ufnr.0183.201308i.0888 ◽

2013 ◽

Vol 183 (8) ◽

pp. 888-894

Author(s):

G.M. Beskin ◽

S.V. Karpov ◽

V.L. Plokhotnichenko ◽

S.F. Bondar ◽

A.V. Perkov ◽

...

Keyword(s):

Temporal Resolution ◽

Monitoring Systems ◽

Wide Field ◽

Optical Monitoring

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Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

International Journal of Molecular Sciences ◽

10.3390/ijms22041903 ◽

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.

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

ACS Photonics ◽

10.1021/acsphotonics.1c00843 ◽

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

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Ultrawide-angle and high-efficiency metalens in hexagonal arrangement

Scientific Reports ◽

10.1038/s41598-020-72668-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Chun-Yuan Fan ◽

Chia-Ping Lin ◽

Guo-Dung J. Su

Keyword(s):

High Efficiency ◽

Numerical Aperture ◽

Vital Role ◽

Optical Systems ◽

Wide Field ◽

Wide Angle ◽

Camera Systems ◽

Single Lens ◽

Hexagonal Arrangement ◽

Virtual And Augmented Reality

Abstract Wide-angle optical systems play a vital role in imaging applications and have been researched for many years. In traditional lenses, attaining a wide field of view (FOV) by using a single optical component is difficult because these lenses have crucial aberrations. In this study, we developed a wide-angle metalens with a numerical aperture of 0.25 that provided a diffraction-limited FOV of over 170° for a wavelength of 532 nm without the need for image stitching or multiple lenses. The designed wide-angle metalens is free of aberration and polarization, and its full width of half maximum is close to the diffraction limit at all angles. Moreover, the metalens which is designed through a hexagonal arrangement exhibits higher focusing efficiency at all angles than most-seen square arrangement. The focusing efficiencies are as high as 82% at a normal incident and 45% at an incident of 85°. Compared with traditional optical components, the proposed metalens exhibits higher FOV and provides a more satisfactory image quality because of aberration correction. Because of the advantages of the proposed metalens, which are difficult to achieve for a traditional single lens, it has the potential to be applied in camera systems and virtual and augmented reality.

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