Microfluidic flow-cell with passive flow control for microscopy applications

We present a fast, inexpensive and robust technique for constructing thin, optically transparent flow-cells with pump-free flow control. Using layers of glass, patterned adhesive tape and polydimethylsiloxane (PDMS) connections, we demonstrate the fabrication of planar devices with chamber height as low as 25 μm and with millimetre-scale (x,y) dimensions for wide-field microscope observation. The method relies on simple benchtop equipment and does not require microfabrication facilities, glass drilling or other workshop infrastructure. We also describe a gravity perfusion system that exploits the strong capillary action in the flow chamber as a passive limit-valve. Our approach allows simple sequential sample exchange with controlled flow rates, sub-5 μL sample chamber size and zero dead volume. We demonstrate the system in a single-molecule force spectroscopy experiment using magnetic tweezers.

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Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

10.1101/763706 ◽

2019 ◽

Author(s):

T. B. Brouwer ◽

N. Hermans ◽

J. van Noort

Keyword(s):

Diffraction Pattern ◽

Real Time ◽

Single Molecule ◽

Fourier Transforms ◽

Magnetic Tweezers ◽

Fast Fourier Transforms ◽

Tracking Algorithm ◽

Wide Field ◽

3D Tracking ◽

Chromatin Fibers

AbstractMany single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorentz Mie scattering theory yields the bead position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method. Here, we introduce 3D phasor tracking, a fast and robust bead tracking algorithm with nanometric localization accuracy in a z-range of over 10 µm. The algorithm is based on a 2D cross-correlation using Fast Fourier Transforms with computer-generated reference images, yielding a processing rate of up to 10.000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real-time on a generic CPU. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead tracking applications, especially where high throughput is required.SignificanceMicrobeads are often used in biophysical single-molecule manipulation experiments and accurately tracking their position in 3 dimensions is key for quantitative analysis. Holographic imaging of these beads allows for multiplexing bead tracking but image analysis can be a limiting factor. Here we present a 3D tracking algorithm based on Fast Fourier Transforms that is fast, has nanometric precision, is robust against common artifacts and is accurate over 10’s of micrometers. We show its real-time application for magnetic tweezers based force spectroscopy on more than 100 chromatin fibers in parallel, but we anticipate that many other bead-based biophysical essays can benefit from this simple and robust 3 phasor algorithm.

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Study of Passive Flow Control for Ship Air Wakes

10.21236/ada581864 ◽

2013 ◽

Cited By ~ 4

Author(s):

Nicholas R. LaSalle

Keyword(s):

Flow Control ◽

Passive Flow Control ◽

Passive Flow

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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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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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