Biology subjects

Van den Eynde, R.

Publications and source records attributed to Van den Eynde, R..

8 recordsLinked to original sources

bioRxiv2025

A non-toxic, user-friendly buffer that enhances green fluorophore performance in conventional and super-resolution imaging

Fluorophore brightness and photostability continue to be limiting factors in fluorescence microscopy. Several buffers that enhance these properties have been identified to date, though they do not work for all dyes, may require harmful components, or have limited storage lives. We have identified a simple, in-expensive, and non-toxic buffer that strongly enhances the brightness and resistance to photodestruction of green emissing synthetic fluorophores, using potassium iodide as a main photostabilizing additive and MgCl2 as enhancer for binding kinetics in DNA-PAINT super resolution imaging. We show that this AN-ice buffer can deliver an up to 30% improvement in fluorescence brightness and 5x more detected events in DNA-PAINT imaging. Furthermore, the same effect can be used to strongly increase the photobleaching resistance of all the green dyes tested for conventional imaging.

biochemistry↗

bioRxiv2023

The TriScan: fast and sensitive 3D confocal fluorescence imaging using a simple optical design

We present the TriScan, a compact and inexpensive fluorescence microscope that can combine the speed of widefield microscopy with the 3D-sectioning capabilities of confocal microscopy. The optical layout is based on a module that combines line-scan confocal imaging with a sensitive camera detector, realized using a simple optical layout that permits the use of arbitrarily fast scanning mirrors. The resulting design is theoretically capable of full field-of-view acquisition rates in the kilohertz regime combined with a diffraction-limited resolution and single-molecule sensitivity. In doing so, the system provides the ease-of- use and speed of widefield imaging combined with the optical sectioning of one-photon confocal imaging. The simple and inexpensive design is suitable for a broad variety of settings ranging from research to diagnostics and screening.

biophysics↗

bioRxiv2023

Fast and artifact-free excitation multiplexing using synchronized image scanning

We present the Resonator, a simple optical device that provides quasi-simultaneous fluorescence imaging with multiple excitation wavelengths. The device uses a resonant scanning mirror to periodically displace the sample image on a camera sensor at a rate that is much faster than the image acquisition rate. The excitation light is synchronized with the scanner motion to create two laterally shifted copies of the image, each containing the fluorescence excited by a single wavelength. The additional information is then encoded either into the point-spread function of the imaging or as multiple distinct images. Since this multiplexing is performed at very high rates, our design can eliminate or mitigate artifacts caused by temporal aliasing in conventional sequential imaging. We demonstrate the use of our system for the monitoring of fast light-induced dynamics in single quantum dots and for the imaging of Ca2+ signalling in hippocampal neurons.

biophysics↗

bioRxiv2022

Simultaneous multicolor fluorescence imaging using duplication-based PSF engineering

We present a way to encode more information in fluorescence imaging by splitting the emission into copies of the original point-spread function (PSF), which offers broadband operation and compatibility with other PSF engineering modalities and existing analysis tools. We demonstrate the approach using the Circulator, an add-on that encodes the fluorophore emission band into the PSF, enabling simultaneous multicolor super-resolution and single-molecule microscopy using essentially the full field of view.

biophysics↗

bioRxiv2021

Self-contained and modular structured illumination microscope

We present a modular implementation of structured illumination microscopy (SIM) that is fast, largely self-contained and that can be added onto existing fluorescence microscopes. Our instrument, which we call HIT-SIM, can theoretically deliver well over 50 super-resolved images per second and is readily compatible with existing acquisition software packages. We provide a full technical package consisting of schematics, a list of components and an alignment scheme that provides detailed specifications and assembly instructions. We illustrate the performance of the instrument by imaging optically large samples containing sequence-specifically stained DNA fragments.

biophysics↗

bioRxiv2020

Active focus stabilization for fluorescence microscopy by tracking actively generated infrared spots.

Fluorescent time-lapse experiments often suffer from focus drift, regularly rendering long measurements partially unusable. Frequently, this instability can be traced back to the specific mechanical components of the setup, but even in highly robust implementations z-drift occurs due to small temperature fluctuations which are hard to avoid. To resolve this issue, microscope manufacturers often offer their own interpretation of out-of-focus correction modules for their flagship instruments. However, self-assembled or older systems typically have to fend for their own or adapt their measurements to circumvent drift effects. In this manuscript, we propose a cost-efficient z-drift detection- and correction system that, due to its modular design, can be attached to any fluorescence microscope with an actuated stage or objective, be it in a custom or commercial setup. The reason for this wide applicability is specific to the design, which has a straightforward alignment procedure and allows sharing optics with the fluorescent emission path. Our system employs an infrared (IR) laser that is passed through a double-hole mask to achieve two parallel beams which are made to reflect on the coverslip and subsequently detected on an industrial sCMOS camera. The relative position of these beams is then uniquely linked to the z-position of a microscope-mounted sample. The system was benchmarked by introducing temperature perturbations, where it was shown to achieve a stable focus, and by scanning different positions while simulating a perturbation in the z-position of the stage, where we show that a lost focus can be recovered within seconds.

biochemistry↗

bioRxiv2019

High speed multi-plane super-resolution structured illumination microscopy of living cells using an image-splitting prism

Super-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work we present a multi-plane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multi-plane image splitting prism combined with high-speed SR-SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally super-resolved three-dimensional image stack. We demonstrate the performance of multi-plane SR-SIM by applying this instrument to the dynamics of live mitochondrial network.

biophysics↗

bioRxiv2018

A cost-effective approach to Super-resolution Optical Fluctuation (SOFI) microscopy using an industry-grade CMOS camera

Super-resolution (SR) fluorescence microscopy, especially at high speeds, is typically carried out on high-performance research microscopes. The substantial cost of such equipment, combined with the limited distribution of such instruments in imaging facilities, have complicated access to super-resolution imaging for research groups in the biosciences. In this work we promote the accessibility of Super-resolution Optical Fluctuation Imaging (SOFI) to the scientific community by demonstrating its flexibility in terms of the minimal required performance of the imaging system. We show that SOFI data can be acquired using a very cost-efficient industrial-grade detector on both a standard research microscope and an entirely home-built wide-field system. This enables more scientists to enter the field of live-cell super-resolution imaging, as it provides access to a robust and fast SR imaging modality at comparatively modest cost.

biophysics↗