Biology subjects

Vandenberg, W.

Publications and source records attributed to Vandenberg, W..

12 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

Per-pixel unmixing of spectrally overlapping fluorophores using intra-exposure excitation modulation

Multilabel fluorescence imaging is essential for the visualization of complex systems, though a major challenge is the limited width of the usable spectral window. Here, we present a new method, exNEEMO, that enables per-pixel quantification of spectrally-overlapping fluorophores based on their light-induced dynamics, in a way that is compatible with a very broad range of timescales over which these dynamics may occur. Our approach makes use of intra-exposure modulation of the excitation light to distinguish the different emitters given their reference responses to this modulation. We use approach to simultaneously image four green photochromic fluorescent proteins at the full spatial resolution of the imaging. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/538742v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1f49247org.highwire.dtl.DTLVardef@1af85c2org.highwire.dtl.DTLVardef@768a92org.highwire.dtl.DTLVardef@83b81b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

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

Photochromic fluorophores enable imaging of lowly-expressed proteins in the autofluorescent fungus Candida albicans

Fluorescence microscopy is a standard research tool in many fields, though collecting reliable images can be difficult in systems characterized by low expressions levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly- or endogenously-expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungals agents in the fungal pathogen C. albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by a high background fluorescence. ImportanceUnderstanding the spatial and temporal organization of proteins-of-interest is key to unravel cellular processes and identify novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans and resistance mechanisms against these therapeutic agents is rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable imaging of lowly-expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems allowing the visualization of intricate processes.

cell biology↗

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↗

bioRxiv2021

Simultaneous readout of multiple FRET pairs using photochromism

Forster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral band-width required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra. We develop the theory underlying this method and validate our approach using numerical simulations. To apply our system, we develop rsAKARev, a photochromic biosensor for cAMP-dependent kinase (PKA), and combine it with the spectrally-identical biosensor EKARev, a reporter for ERK kinase activity, to deliver simultaneous readout of both activities in the same cell. We further perform multiplexed PKA, ERK, and calcium measurements by including a third, spectrally-shifted biosensor. Our work demonstrates that exploiting donor photochromism in FRET can be a powerful approach to simultaneously read out multiple associations within living cells.

biophysics↗

bioRxiv2020

Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins

Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

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

SOFIevaluator: a strategy for the quantitative quality assessment of SOFI data

AbstractSuper-resolution fluorescence imaging techniques allow optical imaging of specimens beyond the diffraction limit of light. Super-resolution optical fluctuation imaging (SOFI) relies on computational analysis of stochastic blinking events to obtain a super-resolved image. As with some other super-resolution methods, this strong dependency on computational analysis can make it difficult to gauge how well the resulting images reflect the underlying sample structure. We herein report SOFIevaluator, an unbiased and parameter-free algorithm for calculating a set of metrics that describes the quality of super-resolution fluorescence imaging data for SOFI. We additionally demonstrate how SOFIevaluator can be used to identify fluorescent proteins that perform well for SOFI imaging under different imaging conditions.

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↗