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 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.

scholarly journals Light-Sheet Fluorescence Microscopy with Scanning Non-diffracting Beams

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hosein Kafian ◽  
Meelad Lalenejad ◽  
Sahar Moradi-Mehr ◽  
Shiva Akbari Birgani ◽  
Daryoush Abdollahpour
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Coherence ◽  
Three Dimensional ◽  
Light Sheet ◽  
Tissue Imaging ◽  
Wide Field ◽  
Self Healing ◽  
Main Challenge ◽  
Cancer Tumor ◽  
Light Sheet Fluorescence Microscopy

Abstract Light-sheet fluorescence microscopy (LSFM) has now become a unique tool in different fields ranging from three-dimensional (3D) tissue imaging to real-time functional imaging of neuronal activities. Nevertheless, obtaining high-quality artifact-free images from large, dense and inhomogeneous samples is the main challenge of the method that still needs to be adequately addressed. Here, we demonstrate significant enhancement of LSFM image qualities by using scanning non-diffracting illuminating beams, both through experimental and numerical investigations. The effect of static and scanning illumination with several beams are analyzed and compared, and it is shown that scanning 2D Airy light-sheet is minimally affected by the inhomogeneities in the samples, and provides higher contrasts and uniform resolution over a wide field-of-view, due to its reduced spatial coherence, self-healing feature and longer penetration depth. Further, the capabilities of the illumination scheme is utilized for both single-and double-wavelength 3D imaging of large and dense mammospheres of cancer tumor cells as complex inhomogeneous biological samples.

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 Light-Sheet Fluorescence Microscopy with Scanning Non-diffracting Beams

2019 ◽  
Author(s):  
Hosein Kafian ◽  
Meelad Lalenejad ◽  
Sahar Moradi-Mehr ◽  
Shiva Akbari Birgani ◽  
Daryoush Abdollahpour
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Coherence ◽  
Three Dimensional ◽  
Light Sheet ◽  
Tissue Imaging ◽  
Wide Field ◽  
Self Healing ◽  
Main Challenge ◽  
Cancer Tumor ◽  
Light Sheet Fluorescence Microscopy

AbstractLight-sheet fluorescence microscopy (LSFM) has now become a unique technique in different fields ranging from three-dimensional (3D) tissue imaging to real-time functional imaging of neuronal activities. Nevertheless, obtaining high-quality artifact-free images from large, dense and inhomogeneous samples is the main challenge of the method that still needs to be adequately addressed. Here, we demonstrate significant enhancement of LSFM image qualities by using scanning non-diffracting illuminating beams, both through experimental and numerical investigations. The effect of static and scanning illumination with several beams are analyzed and compared, and it is shown that scanning 2D Airy light sheet is minimally affected by the artifacts, and provide higher contrasts and uniform resolution over a wide field-of-view, due to its reduced spatial coherence, self-healing feature and higher penetration depth. Further, the capabilities of the illumination scheme is utilized for both single and double wavelength 3D imaging of a large and dense mammospheres of cancer tumor cells as complex inhomogeneous biological samples.

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 Progress in Wide Field CCD Astrometry

1998 ◽  
Vol 179 ◽  
pp. 386-388
Author(s):  
N. Zacharias
Keyword(s):  
The Other ◽  
Field Of View ◽  
Wide Field ◽  
Real Image ◽  
Model Function ◽  
The Real ◽  
Gaussian Profile ◽  
Flat Field

Instrumental parameters and astrometric results from five telescopes are summarized in Table 1. The KPNO 0.9m has field-corrector optics. The CTIO 0.9m is a classical Cassegrain. The 4-meter telescopes both have doublet field-correctors. The USNO 0.2m (8-inch) astrograph has a 5-element lens which is designed for a 9° flat field of view for photographic plates. A bandpass of 570-650 nm is used at the USNO 0.2m while most frames with the other telescopes have been taken through a Gunn r (600-710 nm) filter. Stellar images on the CCD frames have been fitted with a 2-D circular symmetric Gaussian profile, giving the centroiding error σfit. Plots of σfit vs. instrumental magnitude look similar in shape for all telesopes. The asymptotic fit precision, σafp, is the limit in σfit achieved for bright stars, given in milli pixels (mpx) in Table 1. This centering error is overestimated by an amount depending on the deviation of the real image profile from the model function (Winter 1997).

scholarly journals Tuneable wide-field illumination and single-molecule photoswitching with a single MEMS mirror

2021 ◽  
Author(s):  
Lucas Herdly ◽  
Paul Janin ◽  
Ralf Bauer ◽  
Sebastian van de Linde
Keyword(s):  
Single Molecule ◽  
Microelectromechanical Systems ◽  
Super Resolution ◽  
Cost Effective ◽  
Field Of View ◽  
Wide Field ◽  
Field Mode ◽  
Flat Field ◽  
Kinetics Of ◽  
Single Molecule Localization Microscopy

Homogeneous illumination in single-molecule localization microscopy (SMLM) is key for the quantitative analysis of super-resolution images. Therefore, different approaches for flat-field illumination have been introduced as alternative to the conventional Gaussian illumination. Here, we introduce a single microelectromechanical systems (MEMS) mirror as a tuneable and cost-effective device for adapting wide-field illumination in SMLM. In flat-field mode the MEMS allowed for consistent SMLM metrics across the entire field of view. Employing single-molecule photoswitching, we developed a simple yet powerful routine to benchmark different illumination schemes on the basis of local emitter brightness and ON-state lifetime. Moreover, we propose that tuning the MEMS beyond optimal flat-field conditions enables to study the kinetics of photoswitchable fluorophores within a single acquisition.

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