Johannes Hohlbein's research while affiliated with Wageningen University & Research and other places
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Publications (104)
CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the la...
In single-particle tracking, individual particles are localized and tracked over time to probe their diffusion and molecular interactions. Temporal crossing of trajectories, blinking particles, and false-positive localizations present computational challenges that have remained difficult to overcome. Here we introduce a robust, parameter-free alter...
CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the la...
Spatiotemporal assessment of lipid and protein oxidation is key for understanding quality deterioration in emulsified food products containing polyunsaturated fatty acids. In this work, we first mechanistically validated the use of the lipid oxidation-sensitive fluorophore BODIPY 665/676 as a semi-quantitative marker for local peroxyl radical forma...
The field of microscopy has been empowering humankind for many centuries by enabling the observation of objects that are otherwise too small to detect for the naked human eye. Microscopy techniques can be loosely divided into three main branches, namely photon-based optical microscopy, electron microscopy, and scanning probe microscopy with optical...
In single-particle tracking (spt), individual particles are localized and tracked over time to probe their diffusion and molecular interactions. Temporal crossing of trajectories, blinking particles, and false-positive localizations present computational challenges that have remained difficult to overcome. Here, we introduce a robust, parameter-fre...
The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative bindi...
Viscosity is a fundamental property of liquids. It determines transport and diffusion of particles in solution. Nonetheless, it is an open question how a gradient of viscosity - causing a gradient in diffusivity - can lead to viscophoretic transport, i.e., directed transport of particles and molecules in solution. Here, we determine viscophoretic d...
The signaling molecule auxin is pivotal in coordinating many growth and development processes in plants mainly through the modulation of gene expression. The transcriptional response to auxin is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif (TGTC[TC]/[GG]) called the auxin-response element (Au...
Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower ac...
In mayonnaise, lipid and protein oxidation are closely related and the interplay between them is critical for understanding the chemical shelf-life stability of mayonnaise. This is in particular the case for comprehending the role of low-density lipoprotein (LDL) particles as a main emulsifier. Here, we monitored oxidation and the concomitant aggre...
Light microscopy enables researchers to observe cellular mechanisms with high spatial and temporal resolution. However, the increasing complexity of current imaging technologies, coupled with financial constraints of potential users, hampers the general accessibility and potential reach of cutting-edge microscopy. Open microscopy can address this i...
Single-molecule localization microscopy (SMLM) is a powerful technique for elucidating structure and dynamics in the life- and material sciences with sub-50 nm spatial resolution. The simultaneous acquisition of spectral information (spectrally resolved SMLM, sSMLM) enables multiplexing using spectrally distinct fluorophores or enable the probing o...
In mayonnaise, lipid and protein oxidation are closely related and the interplay between them is critical for understanding the chemical shelf-life stability of mayonnaise. This is in particular the case for comprehending the role of low-density lipoprotein (LDL) particles as a main emulsifier. Here, we monitored oxidation and the concomitant aggre...
Turbidity poses a major challenge for the microscopic characterization of food systems. Local mismatches in refractive indices, for example, lead to significant image deterioration along sample depth. To mitigate the issue of turbidity and to increase the accessible optical resolution in food microscopy, we added adaptive optics (AO) and flat-field...
Lipid oxidation is one of the main causes of degradation in food emulsions. It leads to the generation of off-flavors and a concomitant reduction in shelf-life of the food products. The multiscale complexity of lipid oxidation reactions in food emulsions has raised the need to develop new methodological approaches. In this chapter, we will discuss...
Light microscopy allows observing cellular features and objects with sub-micrometer resolution. As such, light microscopy has been playing a fundamental role in the life sciences for more than a hundred years. Fueled by the availability of mass-produced electronics and hardware, publicly shared documentation and building instructions, open-source s...
Optical microscopy is an indispensable tool to characterize the microstructure of foods at ambient conditions. Depending on both the wavelength of light used to illuminate the sample and the opening angle of the microscope objective, the achievable resolution is limited to around 200 nm. This so-called classical diffraction limit implies that small...
Single‐molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, nanofluidic devices and camera‐based, single‐molecule Förster resonance energy transfer (smFRET) detection are combined to study the interactions between plant transcription factors of the auxin response factor (ARF) family and DN...
Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, we combine nanofluidic devices and camera-based, single-molecule Foerster resonance energy transfer (smFRET) detection to study the interactions between plant transcription factors of the Auxin response family (ARF) and DNA oligon...
Turbidity poses a major challenge for the microscopic characterization of many food systems. In these systems, local mismatches in refractive indices can cause reflection, absorption and scattering of incoming as well as outgoing light leading to significant image deterioration along sample depth. To mitigate the issue of turbidity and to increase...
Lipid oxidation in food emulsions is mediated by emulsifiers in the water phase and at the oil–water interface. To unravel the physico-chemical mechanisms and to obtain local lipid and protein oxidation rates, we used confocal laser scanning microscopy (CLSM), thereby monitoring changes in both the fluorescence emission of a lipophilic dye BODIPY 6...
Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. The analysis of apparent diffusion coefficients in vivo, for example, enables researchers to determine whether biomolecules are moving alone, as part of a larger complex, or are bound to large cellular components such as the membrane...
Significance
The plant hormone auxin controls many aspects of growth and development. It does so by changing the activity of AUXIN RESPONSE FACTOR (ARF) proteins that recognize specific DNA elements in plant genes and switch gene expression on or off. A major question in plant biology is how these ARF proteins bind unique DNA sequences and thus sel...
In single-molecule localization microscopy (SMLM), the use of engineered point spread function (PSF) provides access to three-dimensional localization information. The conventional approach of fitting PSFs with a single 2-dimensional Gaussian profile, however, often falls short in analyzing complex PSFs created by placing phase masks, deformable mi...
Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformati...
Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. Observed diffusion coefficients in vivo, for example, enable researchers to determine whether biomolecules are moving alone, as part of a larger complex or are bound to large cellular components such as the membrane or chromosomal DNA...
Hydrogels made of the polysaccharide κ-carrageenan are widely used in the food and personal care industry as thickeners or gelling agents. These hydrogels feature dense regions embedded in a coarser bulk network, but the characteristic size and behavior of these regions has remained elusive. Here, we use single-particle-tracking fluorescence micros...
The point spread function (PSF) of single molecule emitters can be engineered in the Fourier plane to encode three-dimensional localization information, creating double-helix, saddle-point or tetra-pod PSFs. Here, we describe and assess adaptations of the phasor-based single-molecule localization microscopy (pSMLM) algorithm to localize single mole...
CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here, we visualize individual Cascade complexes in a native ty...
DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific D...
Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity 1–3 . Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development...
CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching b...
Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-ac...
Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. However, probing biomolecular interactions and reactions with high throughput and time resolution remains challenging, often requiring surface-immobilized entities. Here, we introduce glass-made nanofluidic devices for th...
This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.
Super-resolution microscopy is frequently employed in the life sciences, but the number of freely accessible and affordable microscopy frameworks, especially for single particle tracking photo-activation localization microscopy (sptPALM), remains limited. To this end, we designed the miCube: a versatile super-resolution capable fluorescence microsc...
Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards ensuring the reproducibility and accuracy of measuring FRET efficiencies are currently lacking.
Here we report the results of a...
We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 × 10⁶ localizations per second to be calculated on a standard multi-core central processing unit with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a...
DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclea...
We introduce a nanofluidic mixing device entirely fabricated in glass for the fluorescence detection of single molecules. The design consists of a nanochannel T-junction and allows the continuous monitoring of chemical or enzymatic reactions of analytes as they arrive from two independent inlets. The fluorescently labeled molecules are tracked befo...
We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 million localizations per second on a standard multi-core CPU with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function (PSF) to two...
![Fig. 4 Mixing studies. (a) Mixing DNA hairpin (from the right, [NaCl] =...](publication/345694787/figure/fig4/AS:980668251119616@1610820939952/Mixing-studies-a-Mixing-DNA-hairpin-from-the-right-NaCl-0-M-with-a-high-salt_Q320.jpg)
Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. Probing chemical and biological interactions and reactions with high throughput and time resolution, however, remains challenging and often requires surface-immobilized entities. Here, utilizing camera-based fluorescence...
Single-molecule F\"orster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards ensuring both the reproducibility and accuracy of measuring FRET efficiencies are currently lacking. Here we report the resul...
We developed a versatile DNA assay and framework for monitoring polymerization of DNA in real time and at the single-molecule level. The assay consists of an acceptor labelled DNA primer annealed to a DNA template that is labelled on its single stranded, downstream overhang with a donor fluorophore. Upon extension of the primer using a DNA polymera...
Movie of BRI1-GFP in presence of BL.
(AVI)
VAEM images of BRZ treated BRI1-GFP Line 2 epidermal root cells upon BL stimulation.
Live-cell VAEM imaging performed on 6 day old Arabidopsis seedling roots expressing BRI1-GFP (A) PM localized BRI1-GFP, (B) PM localized BRI1-GFP treated with 5 μM brassinazole for 3 days, (C) PM localized BRI1-GFP treated with 5 μM brassinazole for 3 days and subs...
Visualization of A. thaliana membrane compartments with VAEM.
Live-cell VAEM imaging performed on 6 day old Arabidopsis seedling roots expressing fluorescent markers for different membrane compartments. (A) PM localized LT16B-GFP, (B) ER localized WAVE6-mCherry, (C) Golgi localized WAVE18-mRFP, (D) TGN localized VHAa1-mRFP, (E) EE/LE localized ARA7...
Dynamics of ER marker (VMA21-GFP).
(AVI)
PM Receptor distribution of PIN2-GFP, BIR3-GFP, BRI1-GFP line 1 and Col0 Arabidopsis thaliana in live epidermal root cells using VAEM.
Live-cell VAEM imaging performed on 6 day old Arabidopsis seedling roots expressing (A) PM localized PIN2-GFP, (B) PM localized BIR3-GFP, (C) PM localized BRI1-GFP line 1, (D)
Arabidopsis thaliana ecotype Columbia....
Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epiflu...
VAEM fluorescence decay curves.
Shown are two representative decay curves of SERK/BAK1-mCherry clusters, and of BRI1-GFP line 1 clusters. As can be seen, the decay curves of SERK3-mCherry sometimes portrays almost a single molecule behaviour, but at other times, more receptors are present in a cluster. For both receptors, discreet decrease in fluor...
Gaussian fits of receptor molecules in PM surrounding the anticlinal cell wall before and after photobleaching.
The line represents the fit of a Gaussian distribution on the fluorescence intensity data across the anticlinal cell wall. Distance 0 is the midpoint of two adjacent plasma membranes in a confocal image. The cytoplasm is situated between...
Example of typical FRAP experiment.
In Fig S6 A and B, images of BRI1-GFP at different scanning iterations are shown. (A) shows images that undergo only scan bleaching whereas (B) contains features of FRAP region convoluted with scan bleaching. (C) Plots of the fluorescence intensity versus number of scans (top orange line: ROI in (A), middle grey...
FRAP data analysis.
(TIF)
Movie of BRI1-GFP in presence of BL.
(AVI)
Movie of BRI1-GFP in absence of BL.
(AVI)
Movie of SERK3-GFP.
(AVI)
Movie of SERK3-GFP.
(AVI)
Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open and closed conformations of DNA polymerases, our smFR...
Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-pro...
Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-pro...
Single molecule protein induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photo-induced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scale...
Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels. As the method is based on the detection of single...
DNA binding proteins utilise different recognition mechanisms to locate their DNA targets. While sequence-specific enzymes recognise a particular nucleotide sequence, structure-specific enzymes have no sequence specificity, and instead interact with particular DNA structures; for example, DNA polymerase I (Pol) binds gapped DNA and Flap endonucleas...
We present the design, preparation and characterization of two types of complex coacervate core micelles (C3Ms) with cross-linked cores and spectroscopic labels, and demonstrate their use as diffusional probes to investigate the microstructure of percolating biopolymer networks. The first type consists of poly(allylamine hydrochloride) (PAH) and po...
The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid ana...
The achievable time resolution of camera-based single-molecule detection is often limited by the frame rate of the camera. Especially in experiments utilizing single-molecule Förster resonance energy transfer (smFRET) to probe conformational dynamics of biomolecules, increasing the frame rate by either pixel-binning or cropping the field of view de...
DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intra-protein single-molecule FRET on diffusing molecules to observe f...
Single-molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool for elucidating biological structure and mechanisms on the molecular level. Here, we focus on applications of smFRET to study interactions between DNA and enzymes such as DNA and RNA polymerases. SmFRET, used as a nanoscopic ruler, allows for the detection and...
The alternating-laser excitation (ALEX) scheme continues to expand the possibilities of fluorescence-based assays to study biological entities and interactions. Especially the combination of ALEX and single-molecule Förster Resonance Energy Transfer (smFRET) has been very successful as ALEX enables the sorting of fluorescently labelled species base...
The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and fr...
Supplementary Figures S1-S6, Supplementary Tables S1- S2, Supplementary Methods, Supplementary Data and Supplementary References
This paper introduces a novel fluidic device based on syringe-driven flow of fluorescent species through a parallel array of nanochannels, in which the geometrical confinement enables long observation times of non-immobilized molecules. Extremely low flow rates are achieved by operating the array of nanochannels in parallel with a larger microchann...
Dark quenchers are chromophores that primarily relax from the excited state to the ground state nonradiatively (i.e., are dark). As a result, they can serve as acceptors for Förster resonance energy transfer experiments without contributing significantly to background in the donor-emission channel, even at high concentrations. Although the advantag...
Histograms of single-molecule Förster resonance energy transfer (FRET) efficiency are often used to study the structures of biomolecules and relate these structures to function. Methods like probability distribution analysis analyze FRET histograms to detect heterogeneities in molecular structure, but they cannot determine whether this heterogeneit...
Single-molecule FRET (smFRET) has long been used as a molecular ruler for the study of biology on the nanoscale (∼2-10 nm); smFRET in total-internal reflection fluorescence (TIRF) Förster resonance energy transfer (TIRF-FRET) microscopy allows multiple biomolecules to be simultaneously studied with high temporal and spatial resolution. To operate a...
Single-molecule fluorescence microscopy is currently one of the most popular methods in the single-molecule toolbox. In this review, we discuss recent advances in fluorescence instrumentation and assays: these methods are characterized by a substantial increase in complexity of the instrumentation or biological samples involved. Specifically, we de...
Third-generation DNA sequencing technologies are expected to transform biomedical research and health care. Although powerful single-molecule DNA sequencing methods are available, they suffer from significant limitations, including the need for sophisticated instrumentation, microfluidics, or nanofabrication. Here, we introduce a simpler approach b...
Histograms of single-molecule FRET efficiency (E) are frequently employed to characterize macromolecular dynamics, and have been been used to better understand the structure and function of proteins such as E.coli RNA Polymerase, Calmodulin and LacY. While such plots are useful for characterizing simple distance changes represented by shifts in mea...
The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early...
The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early...
Since the first single-molecule fluorescence resonance energy transfer (FRET) measurement in 1996, the technique has contributed
substantially to our understanding of biological molecules and processes by probing the structure and dynamics of nucleic
acids, protein molecules, and their complexes with other molecules. This review discusses basic con...
The power of three-dimensional orientation detection of single emitting dipoles using a sophisticated scheme with three detectors in a confocal microscope is quantitatively explored by means of Monte Carlo simulations. We show that several hundreds of photons are sufficient for a reliable orientation determination. In typical single-molecule experi...
The confined diffusion behavior of fluorescent probe molecules inside nanoporous alumina membranes was studied by using nanopores-confinement technique of fluorescence correlation spectroscopy (FCS). The ordered porous alumina membranes were prepared with a pore diameter of 35-40 nm, porosity of 20-25%, and thickness of 35 μm. The alumina membranes...
Citations
... 9405020) were used without any intense pipetting of the droplet suspension. Single-molecule fluorescence microscopy imaging of such samples was performed using a home-built super-resolution microscope -the miEye 45,46 . To visualize the individual glass surface-attached α-syn droplets, TIRF microscopy was employed 47 , whereas imaging of larger α-syn formations, such as droplet aggregates, was conducted under HILO illumination. ...
... However, relatively little is known of the precise nature of the cis-elements. Although their position weight matrices (PWM) have been characterized, their real, in vivo occurring regulatory functions are usually performed by imperfectly matched, weak, and cooperative DNA-protein interactions dependent on DNA or protein modification [43][44][45][46][47]. Interactions between cis-active elements and trans-factors are also strongly regulated by the local 3D shape of DNA [48]. ...
Reference: Plant Synthetic Promoters
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... Spectro-microscopic systems have been developed for simultaneously acquiring highresolution images and spectral features at the single-molecule level. [10][11][12] These techniques make use of dichroic filter, [13][14][15] PSF engineering, 16,17 and spectral dispersion. [10][11][12][18][19][20][21] First, ratiometric detection employed one or more appropriate dichroic mirrors (DCMs) to distinguish fluorophores based on their respective emission intensity in two channels. ...
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... Biomolecules 2023, 13, 1291 2 of 33 radical attack and because it produces the majority of biomarkers commonly used to monitor such damage but because such biomarkers-the LP secondary products-are also major effectors of the damage itself [16], owing to their toxicity and to their ability to alter other biomolecules such as proteins [17], thereby extending the radical/oxidative damage [18]. ...
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... In recent years, these have led to 'open science' practices, including openly archived data, openly licensed code and open access to publications describing key results [1][2][3]. However, instrumentation often lags behind data and results in terms of openness: there are, as yet, no widely adopted conventions requiring the designs of a novel instrument to be shared alongside manuscripts describing its use [4], though there is an increasing number of projects that do share plans for replication [5]. This special issue collects several articles that discuss examples of projects endeavouring to adopt open hardware as a means to better reproducibility, or greater accessibility, of cutting-edge microscopy. ...
Reference: Open, reproducible hardware for microscopy
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... We achieved such flat illumination profile using a refractive optical element, which was also presented in [27]. Yet, a similar flat illumination profile performance should also be realizable with other (sometimes more complex) approaches, as realized before for single-molecule based fluorescence microscopy (for example [49][50][51][52]). Second, we decoupled the TIRF illumination from the focus locking (as it was previously realized in [18]), thus making it easier to add more excitation wavelength for fluorescence and generally simplifying the device setup. ...
... While All chemicals and solvents were commercially available (Merk, Sigma-Aldrich, Milan, Italy). Para-methoxyphenol (1), 2-tert-butyl-4-methoxyphenol (2), 2,6-di-tert-butyl-4-methylphenol (BHT) (3), 2,4,6-trimethylphenol (4), 2-methoxy-4-ethylphenol (5), 2,6dimethoxy-4-allylphenol (6), (±)-α-Tocopherol (7), 2,2,5,7,8-pentamethyl-6-chromanol (8), para-hydroquinone (9), tert-butylhydroquinone (10), 2,5-di-tert-butylhydroquinone (11), para-benzoquinone (12), tert-butylbenzoquinone (13), 2,5-di-tert-butylbenzoquinone (14), catechol (15), 4-methylcatechol (16), 4-tert-butylcatechol (17), caffeic acid (18), catechin (19), quercetin (20), propyl gallate (21), triacetin were of the highest available purity and were used as received. Antioxidant concentrations are % w/w. ...
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... 23 This means that SRM exceeds the resolution limit of light microscopy (200 nm) imposed by Abbe diffraction limit, 24 and nanometric and molecular structures of biological materials can be resolved below 100 nm using SRM or light nanoscopy. 25,26 In addition, SRM techniques require epi-illumination, while total internal reflection fluorescence (TIRF) microscopy is necessary to capture SRM images. 27 The use of TIRF microscopy ( Figure 1B) offers the possibility of obtaining valuable information about a sample's surface that conventional CLSM cannot. ...
... Micro-nano uidic chip is a multifunctional chip integrating micro-channels and nano-channels, which has become established as tools in biomedical studies. Due to the unique scale effect of nano-channels, micro-nano uidic chips have many irreplaceable functions, such as detection and screening of DNA molecules [1,2], drug delivery and release [3,4], sample separation and collection [5,6], and uid transistor [7,8], etc. Therefore, how to realize micro-nano uidic chip manufacturing has become an important research topic. ...
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... Acquired stacks were pre-processed using the Faster Temporal Median ImageJ plugin (https:// github.com/HohlbeinLab/FTM2; Jabermoradi et al., 2021) with a window size of 100 frames. These stacks were then analyzed using Detection of Molecules (DoM) plugin v.1.2.1 for ImageJ (https:// github.com/ekatrukha/DoM_Utrecht), ...
