Biology subjects

van den Berg, B.

Publications and source records attributed to van den Berg, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Outer membrane 'PusCD' complexes coordinate phospho(inositol)lipid utilisation in plant Bacteroidota

Gram-negative bacteria belonging to the phylum Bacteroidota are prominent and ecologically important members of host-associated microbiomes. A feature of Bacteroidota species is the presence of diverse outer membrane TonB-dependent transporter and cognate surface exposed lipoprotein (TBDT-SLP) complexes. Many 'SusCD-like' TBDT-SLPs coordinate complex carbohydrate capture from the environment. Here, we show the model soil Bacteroidota species Flavobacterium johnsoniae synthesises two TBDT-SLP complexes (named PusCD1 and PusCD2) that are essential for growth on phosphatidylinositol, a member of an abundant lipid class within plant plasma membranes, as a phosphorus source. Structural characterisation of ligand-bound PusCD1 complex from F. johnsoniae membranes reveals a new binding mechanism for organophosphorus and broadens the known functional repertoire of this class of transporters. Diverse PusCD-like complexes are widespread among plant associated Bacteroidota, pointing to a major, previously unrecognised route for the recycling of organophosphorus compounds in the rhizosphere, with potential implications for engineering microbial-mediated phosphorus recovery from organic waste streams.

microbiology↗

bioRxiv2026

Structural and functional characterisation of the dextran utilisome from Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron (B. theta) is a model Bacteroidota of the healthy human gut microbiota and a specialist in glycan utilisation. Like other Bacteroides, B. theta has many highly regulated polysaccharide utilisation loci (PUL) that encode outer membrane (OM) TonB-dependent transporters (SusC), closely associated "lid" lipoproteins (SusD), and additional surface-exposed lipoproteins (SLPs) that bind and partially degrade specific glycans derived from host cells, diet, or other microbiota members. The canonical starch PUL products are thought to form a dynamic complex in the presence of starch. However, other PULs form stable complexes in the absence of substrate (recently named "utilisomes"), with additional surface lipoproteins tightly associated with the core SusCD complex. In this study, we characterised the B. theta dextran utilisome, with a SusCDdex core and an associated glycoside hydrolase (GHdex) and surface glycan binding protein (SBGPdex). Via X-ray crystallography we solved high-resolution structures of SBGPdex in isolation and SusDdex and GHdex bound to dextran oligosaccharides. We used isothermal titration calorimetry (ITC) to quantify ligand binding of wild type and mutant SLPs. We further used single particle cryo-EM of the catalytically inactive dextran utilisome to visualise open and closed states of the complex. Three occupied dextran binding sites were observed across SusCdex, SusDdex and GHdex, with substrate observed in both open and closed states of SusDdex. 3D variability analysis showed a minority of particles in the process of SusDdex lid closure. Together our work defines commonalities and differences across utilisomes dedicated to the import of simple glycans.

molecular biology↗

bioRxiv2025

Small siphophage binding to an open state of the LptDE outer membrane lipopolysaccharide translocon

Bacteriophages are bacterial viruses that provide alternatives to small-molecule drugs to combat infections by antibiotic-resistant bacteria. To infect a bacterial host, a phage needs to bind to the bacterial surface via receptor binding proteins (RBPs), which are critical for determining host specificity. For functionally important receptors, the RBP-receptor interaction could be exploited via phage steering, where emerging bacterial resistance due to receptor modification could make bacteria less fit or virulent. Despite this, relatively little is known about RBP-receptor interactions. Here we build on the recent discovery of coliphages that have the outer membrane (OM) lipopolysaccharide translocon LptDE as their terminal receptor and show via cryogenic electron microscopy (cryoEM) that, surprisingly, the RBP of the small siphophage Oekolampad binds to a hitherto unobserved, open state of LptDE. The open lateral gate of LptD is occupied by a {beta}-strand peptide originating from the degraded N-terminal jellyroll domain of LptD, suggesting the possibility of LptD inhibition via peptidomimetics. A structure of LptDE in complex with the superinfection exclusion (SE) protein Rtp45 of the Oekolampad-related phage Rtp shows a mechanism of SE where Rtp45-induced conformational changes in LptD resulting from steric clashes preclude RBP binding. Finally, analysis of spontaneous Oekolampad-resistant E. coli mutants identifies mutations in LptD that abolish the LptDE-RBP interaction in vitro. SDS-EDTA sensitivity assays of the mutants show no major defects in LptDE function, suggesting that phage steering via LptDE might be challenging. SignificanceThe outer membrane (OM) of Gram-negative bacteria is a protective barrier generated by lipopolysaccharide (LPS), a complex glycolipid that makes up the outer leaflet of the OM. Following its synthesis and transport from the inner membrane, LPS is inserted into the OM by LptDE, an OM protein complex essential for most Gram-negative bacteria. LPS insertion requires a hitherto unobserved, open state of LptDE. Here we report the unexpected finding that the receptor binding protein (RBP) of a bacteriophage binds to an open state of LptDE, allowing isolation and visualisation via cryogenic electron microscopy. The lateral gate of the LptDE-RBP complex is occupied by a {beta}-strand peptide, suggesting that the LptDE of pathogenic bacteria could be inhibited by peptidomimetics.

microbiology↗

bioRxiv2025

BtuJ1, a novel surface-exposed B12-binding protein in Bacteroidetes, functions as an extracellular vitamin reservoir that enhances fitness.

The acquisition of vitamin B12 and related cobamides is a key determinant for the fitness of Bacteroidetes in the gut. Depending on the species, this uptake process relies on one to four transport systems centered on conserved core outer membrane (OM) complexes composed of the TonB-dependent transporter BtuB and the surface-exposed lipoprotein BtuG. Additionally, the surface-exposed lipoprotein BtuH, although not tightly associated with the BtuBG complex, contributes to cobamide uptake and provides a fitness advantage. Here, we report the functional and structural characterization of BtuJ1 from Bacteroides thetaiotaomicron (B. theta), a novel B12-binding lipoprotein. Under limiting B12 conditions, BtuJ1 is the most abundant component among the three B12-transport systems encoded by B. theta. BtuJ1 is surface exposed and binds vitamin B12 and cobinamide (an intermediate in B12 biosynthesis) with low nM affinity, conferring a fitness advantage in B12-limited environments. In vitro B12 transfer experiments suggest a role for BtuJ1 as an extracellular reservoir for B12, expanding the functionalities of the diverse group of accessory OM proteins employed by Bacteroides to scavenge this essential cofactor in the competitive environment of the human gut. IMPORTANCEUnderstanding how key molecules support bacterial colonization of the human GI tract is essential to rationalize the structure of the complex microbial community inhabiting the distal gut. The Bacteroidetes are one of the dominant phyla in this environment. Given that most Bacteroidetes cannot make vitamin B12 but depend on it for growth, the fitness of many species likely depends on the acquisition of vitamin B12. Unlike the classical model bacterium Escherichia coli, which encodes a single OM B12 transporter, the genomes of Bacteroidetes often encode multiple uptake systems comprising a heterogeneous repertoire of surface-exposed lipoproteins. These proteins may assist OM assemblies for vitamin B12 transport that provide fitness advantages in vivo.

microbiology↗

bioRxiv2025

Discovery of a distinct BAM complex in the Bacteroidetes

The BAM ({beta}-barrel assembly machinery) complex is an evolutionarily conserved, multiprotein machine that catalyses the folding and membrane insertion of newly synthesised {beta}-barrel outer membrane (OM) proteins in Gram-negative bacteria. Based on Proteobacteria, bacterial BAM is also structurally conserved, with an essential BamAD core and up to three auxiliary periplasmic lipoproteins of poorly defined function. Here we show, using structural biology, quantitative proteomics and functional assays, that the BAM complex is radically different within the Bacteroidetes, a large and important phylum widely distributed within the environment and animal microbiomes. Cryogenic electron microscopy (cryo-EM) structures of BAM complexes from the human gut symbiont Bacteroides thetaiotaomicron and the human oral pathogen Porphyromonas gingivalis show similar, seven-component complexes of [~]325 kDa in size with most of the mass in the extracellular space. In addition to canonical BamA and BamD, the complexes contain an integral OM protein named BamF that is essential and intimately associated with BamA, as well as four surface-exposed lipoproteins (SLPs) named BamG-J. Together, BamF-J form a large, extracellular dome that likely serves as an assembly cage for the {beta}-barrel-SLP complexes that are a hallmark of the Bacteroidetes. Our data suggest that BAM functionality in Bacteroidetes is substantially expanded from that in Proteobacteria and underscores the importance of studying other phyla for a more complete understanding of fundamental biological processes.

microbiology↗

bioRxiv2024

Glycoinformatic profiling of label-free intact heparan sulfate oligosaccharides

Heparan sulfates (HS) are a group of heterogenous linear, sulfated polysaccharides that play a role in in health and many diseases including cancer, cardiovascular, and kidney diseases. The structural variety of HS has greatly challenged the development and utility of HS analytics, particularly for native structures, leaving a significant gap in HS technologies for clinical application. Mass spectrometry (MS)-based profiling with bioinformatics offers a top-down approach that can retain variety in large data sets. Using healthy human plasmas, we developed an MS glycoprofiling approach for native HS oligosaccharides, which retains the structural complexity of each individual HS chain and generates an HS index (or Heparan-ome) for each patient. As a proof of concept, analysis of 56 plasma samples ranging from 6 groups of kidney disease patients revealed a new subset cluster (20%, 4/20) of membranous glomerulopathy (MG) patients with distinct HS profiles, highlighting the potential of HS glycoprofiling as a powerful new approach into clinical practice, which warrants future development into clinical diagnostics of kidney and other diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/613784v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@6dac34org.highwire.dtl.DTLVardef@449da1org.highwire.dtl.DTLVardef@c8eb88org.highwire.dtl.DTLVardef@df4deb_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Structural basis of iron piracy by a prominent human gut symbiont

Iron is an essential element that can be growth-limiting in microbial communities, particularly those present within host organisms. To acquire iron, many bacteria secrete siderophores, secondary metabolites that chelate ferric iron. These iron chelates can be transported back into the cell via TonB-dependent transporters in the outer membrane, followed by intracellular liberation of the iron. Pathogenic Escherichia coli and Salmonella produce siderophores during gut infection. In response to iron starvation, the human gut symbiont Bacteroides thetaiotaomicron upregulates an iron piracy system, XusABC, which steals iron-bound siderophores from the invading pathogens. Here, we investigated the molecular details of xenosiderophore uptake across the outer membrane by the XusAB complex. Our crystal and cryogenic electron microscopy structures explain how the XusB lipoprotein recognises iron-bound xenosiderophores and passes them on to the XusA TonB-dependent transporter. Moreover, we show that Xus homologues can transport a variety of siderophores with different iron-chelating functional groups.

microbiology↗

bioRxiv2023

Electrical brain activations in young children during a probabilistic reward-learning task are associated with behavioral strategy

Both adults and children learn through feedback which environmental events and choices are associated with higher probability of reward, an ability thought to be supported by the development of fronto-striatal reward circuits. Recent developmental studies have applied computational models of reward learning to investigate such learning in children. However, tasks and measures effective for assaying the cascade of reward-learning neural processes in children have been limited. Using a child-version of a probabilistic reward-learning task while recording event-related-potential (ERP) measures of electrical brain activity, this study examined key processes of reward learning in preadolescents (8-12 years old; n=30), namely: (1) reward-feedback sensitivity, as measured by the early-latency, reward-related, frontal ERP positivity, (2) rapid attentional shifting of processing toward favored visual stimuli, as measured by the N2pc component, and (3) longer-latency attention-related responses to reward feedback as a function of behavioral strategies (i.e., Win-Stay-Lose-Shift), as measured by the central-parietal P300. Consistent with our prior work in adults, the behavioral findings indicate preadolescents can learn stimulus-reward outcome associations, but at varying levels of performance. Neurally, poor preadolescent learners (those with slower learning rates) showed greater reward-related positivity amplitudes relative to good learners, suggesting greater reward-feedback sensitivity. We also found attention shifting towards to-be-chosen stimuli, as evidenced by the N2pc, but not to more highly rewarded stimuli as we have observed in adults. Lastly, we found the behavioral learning strategy (i.e., Win-Stay-Lose-Shift) reflected by the feedback-elicited parietal P300. These findings provide novel insights into the key neural processes underlying reinforcement learning in preadolescents.

neuroscience↗

bioRxiv2023

An ancestral dual function of OmpM as outer membrane tether and nutrient uptake channel in diderm Firmicutes

The outer membrane (OM) in diderm, or Gram-negative, bacteria must be tethered to peptidoglycan (PG) for mechanical stability and to maintain cell morphology. Most diderm phyla from the Terrabacteria group lack well-characterised OM attachment systems such as Brauns lipoprotein, Pal and OmpA, but instead have the OmpM protein. OmpM has a periplasmic region containing an S-layer homology domain and a {beta}-barrel region that is embedded in the OM. Here, we have determined the structure of the most abundant OmpM protein from the diderm firmicute Veillonella parvula by single particle cryogenic electron microscopy (cryo-EM). We reveal an OM portion similar to well-characterised trimeric general porins and a mobile periplasmic PG-binding region. Combining the cryo-EM data with crystal structures, structure predictions and molecular dynamics simulations, we show that the periplasmic region of OmpM likely adopts multiple conformations. Single-channel electrophysiology demonstrates ion conductance properties similar to E. coli OmpF. Finally, we demonstrate via functional assays that the four OmpM paralogues are likely to be the only general porins in V. parvula. Together, our results show that OM tethering and nutrient acquisition are genetically linked in V. parvula (and other diderm Firmicutes), suggesting that concurrent loss of two key OM-related functions, tethering and nutrient uptake, promoted the multiple OM loss events that have been inferred in the Terrabacteria.

biochemistry↗

bioRxiv2022

BtuB TonB-dependent transporters and BtuG surface lipoproteins form stable complexes for vitamin B12 uptake in gut Bacteroides.

Vitamin B12 (cobalamin) is the most complex vitamin and essential for many human gut microbes. However, cobalamin is synthesised only by a limited number of bacteria, making many gut microbes dependent on scavenging to meet their cobalamin requirements. Since bacterial densities in the gut are extremely high, competition for cobalamin is severe, making it a keystone micronutrient that shapes human gut microbial communities. Contrasting with Enterobacteria like Escherichia coli which only have one outer membrane (OM) transporter dedicated to B12 uptake (BtuB), members of the dominant genus Bacteroides often encode several vitamin B12 OM transporters together with a conserved array of surface-exposed B12-binding lipoproteins. Here we show, via X-ray crystallography, cryogenic electron microscopy (cryoEM) and molecular dynamics (MD) simulations, that the BtuB1 and BtuB2 transporters from the prominent human gut bacterium Bacteroides thetaiotaomicron form stable complexes with the surface-exposed lipoproteins BtuG1 and BtuG2. The lipoproteins cap the external surface of their cognate BtuB transporter and, when open, capture B12 via electrostatic attraction. After B12 capture, the BtuG lid closes, with concomitant transfer of the vitamin to the BtuB transporter and subsequent transport. We propose that TonB-dependent, lipoprotein-assisted small molecule uptake is a general feature of Bacteroides spp. that is important for the success of this genus in colonising the human gut.

microbiology↗

bioRxiv2022

Outer membrane utilisomes mediate oligosaccharide uptake in gut Bacteroidetes

Bacteroidetes are abundant members of the human microbiota, with species occupying the distal gut capable of utilising a myriad of diet- and host-derived glycans. Transport of glycans across the outer membrane (OM) of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, that are thought to operate via a pedal-bin mechanism in which the lids open and close to facilitate substrate binding. However, additional cell surface-exposed lipoproteins, namely surface glycan binding proteins and glycoside hydrolases, play critical roles in the capture and processing of large glycan chains into transport-competent substrates. Despite constituting a crucial mechanism of nutrient acquisition by our colonic microbiota, the interactions between these components in the OM are poorly understood. Here we show that for the levan and dextran utilisation systems of Bacteroides thetaiotaomicron, the additional OM components assemble on the core SusCD transporter, forming stable glycan utilising machines which we term utilisomes. Single particle electron cryogenic electron microscopy (cryo-EM) structures in the absence and presence of substrate reveal concerted conformational changes that rationalise the role of each component for efficient nutrient capture, as well as providing a direct demonstration of the pedal bin mechanism of substrate capture in the intact utilisome.

microbiology↗

bioRxiv2022

Structural basis for host recognition and superinfection exclusion by bacteriophage T5

A key but poorly understood stage of the bacteriophage life cycle is the binding of phage receptor binding proteins (RBPs) to receptors on the host cell surface, leading to injection of the phage genome and, for lytic phages, host cell lysis. To prevent a secondary viral infection by the same or a closely related phage, superinfection exclusion (SE) proteins can prevent the binding of RBPs via modulation of the host receptor structure in ways that are also unclear. Here we present the cryo-EM structure of the phage T5 outer membrane (OM) receptor FhuA in complex with the T5 RBP pb5, and the crystal structure of FhuA complexed to the OM SE lipoprotein Llp. Pb5 inserts four loops deeply into the extracellular lumen of FhuA and contacts the plug, but does not cause any conformational changes in the receptor, supporting the view that DNA translocation does not occur through the lumen of OM channels. The FhuA-Llp structure reveals that Llp is periplasmic and binds to a non-native conformation of the plug of FhuA, causing the inward folding of two extracellular loops via "reverse" allostery. The inward-folded loops of FhuA overlap with the pb5 binding site, explaining how Llp binding to FhuA abolishes further infection of E. coli by phage T5, and suggesting a mechanism for SE via the jamming of TonB-dependent transporters by small phage lipoproteins.

microbiology↗

bioRxiv2021

Influence of Response Criterion on Nociceptive Detection Thresholds and Evoked Potentials

Pain scientists and clinicians search for objective measures of altered nociceptive processing to study and stratify chronic pain patients. Nociceptive processing can be studied by observing a combination of nociceptive detection thresholds and evoked potentials. However, it is unknown whether the nociceptive detection threshold measured using a Go-/No-Go (GN) procedure can be biased by a response criterion. In this study, we compared nociceptive detection thresholds, psychometric slopes and central evoked potentials obtained during a GN procedure with those obtained during a 2-interval forced choice (2IFC) procedure to determine 1) if the nociceptive detection threshold during a GN procedure is biased by a criterion and 2) to determine if nociceptive evoked potentials observed in response to stimuli around the detection threshold are biased by a criterion. We found that the detection threshold can be higher when assessed using a GN procedure in comparison with the 2IFC procedure. The average P2 component in the central evoked potential showed on-off behavior with respect to stimulus detection and increased proportionally with the detection probability during a GN procedure. These data suggest that nociceptive detection thresholds estimated using a GN procedure are subject to a response criterion.

neuroscience↗

bioRxiv2021

Where is the money? Dynamics in feedback processing and attention during spatial probabilistic learning

Learned feature-based stimulus-reward-associations can modulate behavior and the underlying neural processing of information. In our study, we investigated the neurocognitive mechanisms underlying learning of spatial stimulus-reward-associations. Participants performed a probabilistic spatial reward-learning task that required participants, within 40 trials, to learn which out of four locations on a computer screen yielded the most gain-feedback when chosen. Our behavioral findings show that participants learned to choose which location was most rewarding. Those findings were paralleled by significant amplitude differences in event-related potentials (ERPs) elicited by the presentation of loss and gain feedback; the amplitude of the feedback-related negativity (FRN) was more negative in response to loss feedback compared to gain feedback, but showed no modulation by trial-number. On the other hand, the late positive component (LPC), became larger in response to losses as the learning-set progressed, but smaller in response to gains. Additionally, immediately following feedback presentation, brain activity in the visual cortex - read out through alpha frequency oscillations measured over occipital sites - was predictive of the amplitude of the N2pc ERP component, a marker of spatial attention orienting, observed on the next trial. Taken together, we elucidated neurocognitive dynamics underlying feedback processing in spatial reward learning, and the subsequent effects that spatial stimulus-reward association learning have on spatial attention.

neuroscience↗

bioRxiv2021

Reward and expectancy effects on neural signals of motor preparation and execution

The prospect of rewards can have strong modulatory effects on response preparation. Importantly, selection and execution of movements in real life happens under an environment characterized by uncertainty and dynamic changes. The current study investigated how the brains motor system adapts to the dynamic changes in the environment in pursuit of rewards. In addition, we studied how the prefrontal cognitive control system contributes in this adaptive control of motor behavior. To this end, we tested the effect of rewards and expectancy on the hallmark neural signals that reflect activity in motor and prefrontal systems, the lateralized readiness potential (LRP) and the mediofrontal (mPFC) theta oscillations, while participants performed an expected and unexpected action to retrieve rewards. To better capture the dynamic changes in neural processes represented in the LRP waveform, we decomposed the LRP into the preparation (LRPprep) and execution (LRPexec) components. The overall pattern of LRPprep and LRPexec confirmed that they each reflect motor preparation based on the expectancy and motor execution when making a response that is either or not in line with the expectations. In the comparison of LRP magnitude across task conditions, we found a greater LRPprep when large rewards were more likely, reflecting a greater motor preparation to obtain larger rewards. We also found a greater LRPexec when large rewards were presented unexpectedly, suggesting a greater motor effort placed for executing a correct movement when presented with large rewards. In the analysis of mPFC theta, we found a greater theta power prior to performing an unexpected than expected response, indicating its contribution in response conflict resolution. Collectively, these results demonstrate an optimized motor control to maximize rewards under the dynamic changes of real-life environment.

neuroscience↗

bioRxiv2021

EEG-based visual deviance detection in freely behaving mice

The mouse is widely used as an experimental model to study visual processing. To probe how the visual system detects changes in the environment, functional paradigms in freely behaving mice are strongly needed. We developed and validated the first EEG-based method to investigate visual deviance detection in freely behaving mice. Mice with EEG implants were exposed to a visual deviant detection paradigm that involved changes in light intensity as standard and deviant stimuli. By subtracting the standard from the deviant evoked waveform, deviant detection was evident as bi-phasic negativity (starting around 70 ms) in the difference waveform. Additionally, deviance-associated evoked (beta/gamma) and induced (gamma) oscillatory responses were found. We showed that the results were stimulus-independent by applying a "flip-flop" design and the results showed good repeatability in an independent measurement. Together, we put forward a validated, easy-to-use paradigm to measure visual deviance processing in freely behaving mice.

neuroscience↗

bioRxiv2021

Structural basis for silicon uptake by higher plants

Metalloids are elements with physical and chemical properties that are intermediate between metals and non-metals. Silicon (Si) is the most abundant metalloid in the Earths crust and occurs at high levels in many plants, especially those belonging to the Poaceae (grasses). Most of the worlds staple food crops such as rice, barley and maize accumulate silicon to high levels, resulting in resistance to abiotic and biotic stresses and consequently better plant growth and crop yields. The first step in silicon accumulation is the uptake of silicic acid (Si), the bioavailable from of silicon, by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins. Here we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). While the OsNIP2;1 channel is closed in the crystal by intracellular loop D, unbiased molecular dynamics (MD) simulations reveal a rapid channel opening on sub-microsecond time scales. MD simulations further show how Si interacts with an extracellular five-residue selectivity filter that provides the main barrier for transmembrane diffusion. Our data provide a foundation for understanding and potential manipulation of metalloid selectivity of an important and understudied aquaporin subfamily. SignificanceMany of the worlds most important food crops such as rice, barley and maize accumulate silicon to high levels, resulting in better plant growth and crop yields. The first step in silicon accumulation is the uptake of silicic acid (Si) by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins. Here, we present the X-ray crystal structure and molecular dynamics simulations of the archetypal NIP family member from Oryza sativa (OsNIP2;1) to visualise Si uptake. Our data provide a platform for improved understanding of Si uptake by plants that could be utilised, e.g., in silicon biofortification of important crops and potential alleviation of arsenic accumulation in the rice grain.

plant biology↗

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↗