Matrisome AnalyzeR - a suite of tools to annotate and quantify ECM molecules in big datasets across organisms.

The extracellular matrix (ECM) is a complex meshwork of proteins that forms the scaffold of all tissues in multicellular organisms. It plays crucial roles in all aspects of life - from orchestrating cell migration during development, to supporting tissue repair. It also plays critical roles in the etiology or progression of diseases. To study this compartment, we have previously defined the compendium of all genes encoding ECM and ECM-associated proteins for multiple organisms. We termed this compendium the 'matrisome' and further classified matrisome components into different structural or functional categories. This nomenclature is now largely adopted by the research community to annotate '-omics' datasets and has contributed to advance both fundamental and translational ECM research. Here, we report the development of Matrisome AnalyzeR, a suite of tools including a web-based application and an R package. The web application can be used by anyone interested in annotating, classifying and tabulating matrisome molecules in large datasets without requiring programming knowledge. The companion R package is available to more experienced users, interested in processing larger datasets or in additional data visualization options.

B cell receptor-induced protein dynamics and the emerging role of SUMOylation revealed by proximity proteomics.

Successful B cell activation, which is critical for high-affinity antibody production, is controlled by the B cell antigen receptor (BCR). However, we still lack a comprehensive protein-level view of the very dynamic multi-branched cellular events triggered by antigen binding. Here, we employed APEX2 proximity biotinylation to study antigen-induced changes, 5-15 min after receptor activation, at the vicinity of the plasma membrane lipid rafts, wherein BCR enriches upon activation. The data reveals dynamics of signaling proteins, as well as various players linked to the subsequent processes, such as actin cytoskeleton remodeling and endocytosis. Interestingly, our differential expression analysis identified dynamic responses in various proteins previously not linked to early B cell activation. We demonstrate active SUMOylation at the sites of BCR activation in various conditions and report its functional role in BCR signaling through the AKT and ERK1/2 axes.

Accurate and rapid background estimation in single-molecule localization microscopy using the deep neural network BGnet.

Background fluorescence, especially when it exhibits undesired spatial features, is a primary factor for reduced image quality in optical microscopy. Structured background is particularly detrimental when analyzing single-molecule images for 3-dimensional localization microscopy or single-molecule tracking. Here, we introduce BGnet, a deep neural network with a U-net-type architecture, as a general method to rapidly estimate the background underlying the image of a point source with excellent accuracy, even when point-spread function (PSF) engineering is in use to create complex PSF shapes. We trained BGnet to extract the background from images of various PSFs and show that the identification is accurate for a wide range of different interfering background structures constructed from many spatial frequencies. Furthermore, we demonstrate that the obtained background-corrected PSF images, for both simulated and experimental data, lead to a substantial improvement in localization precision. Finally, we verify that structured background estimation with BGnet results in higher quality of superresolution reconstructions of biological structures.

In Situ Gelling Hydroxypropyl Cellulose Formulation Comprising Cannabidiol-Loaded Block Copolymer Micelles for Sustained Drug Delivery.

Cannabidiol (CBD) is a natural terpenophenolic compound with known pharmacological activities, but the poor solubility of CBD in water limits its widespread use in medicine and pharmacy. Polymeric (nano)carriers demonstrated high potential for enhancing the solubility and therapeutic activity of lipophilic drugs such as CBD. Here, we report the elaboration of a novel hydroxypropyl cellulose (HPC)-based in situ gelling formulation for controlled delivery of CBD. In the first stage, nanosized polymeric micelles from poly(ethylene oxide)-block-poly(α-cinnamyl-ε-caprolactone-co-ε-caprolactone) (PEO-b-P(CyCL-co-CL) diblock copolymers) were used to increase the solubility of CBD in water. Different copolymers were assessed, and the carrier with the highest encapsulation efficiency (EE) and drug loading capacity (DLC) was selected for further elaboration of nanocomposite in situ gel formulations. Next, the sol-to-gel transition behavior of HPC as a function of K2SO4 concentration in the aqueous solution was investigated by microcalorimetry and dynamic oscillatory rheology, and the optimal formulation capable of forming a physical gel under physiological conditions was determined. Finally, injectable nanocomposite hydrogels comprising cannabidiol were fabricated, and their drug release profile and cytotoxicity against human tumor cell lines were evaluated. The in situ gels exhibited prolonged drug release over 12 h, controlled by gel erosion, and the cytotoxicity of formulated cannabidiol was comparable with that of a free drug.

Perspective: Emerging strategies for determining atomic-resolution structures of macromolecular complexes within cells.

In principle, electron cryo-tomography (cryo-ET) of thin portions of cells provides high-resolution images of the three-dimensional spatial arrangement of all members of the proteome. In practice, however, radiation damage creates a tension between recording images at many different tilt angles, but at correspondingly reduced exposure levels, versus limiting the number of tilt angles in order to improve the signal-to-noise ratio (SNR). Either way, it is challenging to read the available information out at the level of atomic structure. Here, we first review work that explores the optimal strategy for data collection, which currently seems to favor the use of a limited angular range for tilting the sample or even the use of a single image to record the high-resolution information. Looking then to the future, we point to the alternative of so-called "deconvolution microscopy", which may be applied to tilt-series or optically-sectioned, focal series data. Recording data as a focal series has the advantage that little or no translational alignment of frames might be needed, and a three-dimensional reconstruction might require only 2/3 the number of images as does standard tomography. We also point to the unexploited potential of phase plates to increase the contrast, and thus to reduce the electron exposure levels while retaining the ability align and merge the data. In turn, using much lower exposures per image could have the advantage that high-resolution information is retained throughout the full data-set, whether recorded as a tilt series or a focal series of images.

AutoCoEv-A High-Throughput In Silico Pipeline for Predicting Inter-Protein Coevolution.

Protein-protein interactions govern cellular processes via complex regulatory networks, which are still far from being understood. Thus, identifying and understanding connections between proteins can significantly facilitate our comprehension of the mechanistic principles of protein functions. Coevolution between proteins is a sign of functional communication and, as such, provides a powerful approach to search for novel direct or indirect molecular partners. However, an evolutionary analysis of large arrays of proteins in silico is a highly time-consuming effort that has limited the usage of this method for protein pairs or small protein groups. Here, we developed AutoCoEv, a user-friendly, open source, computational pipeline for the search of coevolution between a large number of proteins. By driving 15 individual programs, culminating in CAPS2 as the software for detecting coevolution, AutoCoEv achieves a seamless automation and parallelization of the workflow. Importantly, we provide a patch to the CAPS2 source code to strengthen its statistical output, allowing for multiple comparison corrections and an enhanced analysis of the results. We apply the pipeline to inspect coevolution among 324 proteins identified to be located at the vicinity of the lipid rafts of B lymphocytes. We successfully detected multiple coevolutionary relations between the proteins, predicting many novel partners and previously unidentified clusters of functionally related molecules. We conclude that AutoCoEv, can be used to predict functional interactions from large datasets in a time- and cost-efficient manner.

Efficient Synthesis of Fluorescent Coumarins and Phosphorous-Containing Coumarin-Type Heterocycles via Palladium Catalyzed Cross-Coupling Reactions.

Quantum-chemical calculations on the spectral properties of some aryl substituted 3-phosphonocoumarins were performed, and the effect of the substituents in the aryl moiety was evaluated. The structures possessing promising fluorescent properties were successfully synthesized via Suzuki and Sonogashira cross-coupling. The synthetic protocol was also applied for the phosphorous chemoisomer of 3-phosphonocoumarin, 1,2-benzoxaphosphorin, and their carboxylate analogues. The optical properties of the arylated and alkynylated products were experimentally determined. The obtained quantum-chemical and experimental results give the possibility for a fine tuning of the optical properties of phosphorous-containing coumarin systems by altering the substituent at its C-6 position.

B cells rapidly target antigen and surface-derived MHCII into peripheral degradative compartments.

In order to mount high-affinity antibody responses, B cells internalise specific antigens and process them into peptides loaded onto MHCII for presentation to T helper cells (TH cells). While the biochemical principles of antigen processing and MHCII loading have been well dissected, how the endosomal vesicle system is wired to enable these specific functions remains much less studied. Here, we performed a systematic microscopy-based analysis of antigen trafficking in B cells to reveal its route to the MHCII peptide-loading compartment (MIIC). Surprisingly, we detected fast targeting of internalised antigen into peripheral acidic compartments that possessed the hallmarks of the MIIC and also showed degradative capacity. In these vesicles, internalised antigen converged rapidly with membrane-derived MHCII and partially overlapped with cathepsin-S and H2-M, both required for peptide loading. These early compartments appeared heterogenous and atypical as they contained a mixture of both early and late endosomal markers, indicating a specialized endosomal route. Together, our data suggest that, in addition to in the previously reported perinuclear late endosomal MIICs, antigen processing and peptide loading could have already started in these specialized early peripheral acidic vesicles (eMIIC) to support fast peptide-MHCII presentation.

Brain Tissue Conductivity Measurements with MR-Electrical Properties Tomography: An In Vivo Study.

First in vivo brain conductivity reconstructions using Helmholtz MR-Electrical Properties Tomography (MR-EPT) have been published. However, a large variation in the reconstructed conductivity values is reported and these values differ from ex vivo conductivity measurements. Given this lack of agreement, we performed an in vivo study on eight healthy subjects to provide reference in vivo brain conductivity values. MR-EPT reconstructions were performed at 3 T for eight healthy subjects. Mean conductivity and standard deviation values in the white matter, gray matter and cerebrospinal fluid (σWM, σGM, and σCSF) were computed for each subject before and after erosion of regions at tissue boundaries, which are affected by typical MR-EPT reconstruction errors. The obtained values were compared to the reported ex vivo literature values. To benchmark the accuracy of in vivo conductivity reconstructions, the same pipeline was applied to simulated data, which allow knowledge of ground truth conductivity. Provided sufficient boundary erosion, the in vivo σWM and σGM values obtained in this study agree for the first time with literature values measured ex vivo. This could not be verified for the CSF due to its limited spatial extension. Conductivity reconstructions from simulated data verified conductivity reconstructions from in vivo data and demonstrated the importance of discarding voxels at tissue boundaries. The presented σWM and σGM values can therefore be used for comparison in future studies employing different MR-EPT techniques.

Single-Walled Carbon Nanotubes Modify Leaf Micromorphology, Chloroplast Ultrastructure and Photosynthetic Activity of Pea Plants.

Single-walled carbon nanotubes (SWCNTs) emerge as promising novel carbon-based nanoparticles for use in biomedicine, pharmacology and precision agriculture. They were shown to penetrate cell walls and membranes and to physically interact and exchange electrons with photosynthetic complexes in vitro. Here, for the first time, we studied the concentration-dependent effect of foliar application of copolymer-grafted SWCNTs on the structural and functional characteristics of intact pea plants. The lowest used concentration of 10 mg L-1 did not cause any harmful effects on the studied leaf characteristics, while abundant epicuticular wax generation on both leaf surfaces was observed after 300 mg L-1 treatment. Swelling of both the granal and the stromal regions of thylakoid membranes was detected after application of 100 mg L-1 and was most pronounced after 300 mg L-1. Higher SWCNT doses lead to impaired photosynthesis in terms of lower proton motive force generation, slower generation of non-photochemical quenching and reduced zeaxanthin content; however, the photosystem II function was largely preserved. Our results clearly indicate that SWCNTs affect the photosynthetic apparatus in a concentration-dependent manner. Low doses (10 mg L-1) of SWCNTs appear to be a safe suitable object for future development of nanocarriers for substances that are beneficial for plant growth.

Highly Elastic Super-Macroporous Cryogels Fabricated by Thermally Induced Crosslinking of 2-Hydroxyethylcellulose with Citric Acid in Solid State.

Biopolymer materials have been considered a "green" alternative to petroleum-based polymeric materials. Biopolymers cannot completely replace synthetic polymers, but their application should be extended as much as possible, exploiting the benefits of their low toxicity and biodegradability. This contribution describes a novel strategy for the synthesis of super-macroporous 2-hydroxyethylcellulose (HEC) cryogels. The method involves cryogenic treatment of an aqueous solution of HEC and citric acid (CA), freeze drying, and thermally induced crosslinking of HEC macrochains by CA in a solid state. The effect of reaction temperature (70-180 °C) and CA concentration (5-20 mass % to HEC) on the reaction efficacy and physico-mechanical properties of materials was investigated. Highly elastic cryogels were fabricated, with crosslinking carried out at ≥100 °C. The storage modulus of the newly obtained HEC cryogels was ca. 20 times higher than the modulus of pure HEC cryogels prepared by photochemical crosslinking. HEC cryogels possess an open porous structure, as confirmed by scanning electron microscopy (SEM), and uptake a relatively large amount of water. The swelling degree varied between 17 and 40, depending on the experimental conditions. The degradability of HEC cryogels was demonstrated by acid hydrolysis experiments.

Polymer-Modified Single-Walled Carbon Nanotubes Affect Photosystem II Photochemistry, Intersystem Electron Transport Carriers and Photosystem I End Acceptors in Pea Plants.

Single-walled carbon nanotubes (SWCNT) have recently been attracting the attention of plant biologists as a prospective tool for modulation of photosynthesis in higher plants. However, the exact mode of action of SWCNT on the photosynthetic electron transport chain remains unknown. In this work, we examined the effect of foliar application of polymer-grafted SWCNT on the donor side of photosystem II, the intersystem electron transfer chain and the acceptor side of photosystem I. Analysis of the induction curves of chlorophyll fluorescence via JIP test and construction of differential curves revealed that SWCNT concentrations up to 100 mg/L did not affect the photosynthetic electron transport chain. SWCNT concentration of 300 mg/L had no effect on the photosystem II donor side but provoked inactivation of photosystem II reaction centres and slowed down the reduction of the plastoquinone pool and the photosystem I end acceptors. Changes in the modulated reflection at 820 nm, too, indicated slower re-reduction of photosystem I reaction centres in SWCNT-treated leaves. We conclude that SWCNT are likely to be able to divert electrons from the photosynthetic electron transport chain at the level of photosystem I end acceptors and plastoquinone pool in vivo. Further research is needed to unequivocally prove if the observed effects are due to specific interaction between SWCNT and the photosynthetic apparatus.

Addressing systematic errors in axial distance measurements in single-emitter localization microscopy.

Nanoscale localization of point emitters is critical to several methods in optical fluorescence microscopy, including single-molecule super-resolution imaging and tracking. While the precision of the localization procedure has been the topic of extensive study, localization accuracy has been less emphasized, in part due to the challenge of producing an experimental sample containing unperturbed point emitters at known three-dimensional positions in a relevant geometry. We report a new experimental system which reproduces a widely-adopted geometry in high-numerical aperture localization microscopy, in which molecules are situated in an aqueous medium above a glass coverslip imaged with an oil-immersion objective. We demonstrate a calibration procedure that enables measurement of the depth-dependent point spread function (PSF) for open aperture imaging as well as imaging with engineered PSFs with index mismatch. We reveal the complicated, depth-varying behavior of the focal plane position in this system and discuss the axial localization biases incurred by common approximations of this behavior. We compare our results to theoretical calculations.

Epistane, an anabolic steroid used for recreational purposes, causes cholestasis with elevated levels of cholic acid conjugates, by upregulating bile acid synthesis (CYP8B1) and cross-talking with nuclear receptors in human hepatocytes.

Anabolic-androgenic steroids are testosterone derivatives, used by body-builders to increase muscle mass. Epistane (EPI) is an orally administered 17α-alkylated testosterone derivative with 2a-3a epithio ring. We identified four individuals who, after EPI consumption, developed long-lasting cholestasis. The bile acid (BA) profile of three patients was characterized, as well the molecular mechanisms involved in this pathology. The serum BA pool was increased from 14 to 61-fold, basically on account of primary conjugated BA (cholic acid (CA) conjugates), whereas secondary BA were very low. In in vitro experiments with cultured human hepatocytes, EPI caused the accumulation of glycoCA in the medium. Moreover, as low as 0.01 µM EPI upregulated the expression of key BA synthesis genes (CYP7A1, by 65% and CYP8B1, by 67%) and BA transporters (NTCP, OSTA and BSEP), and downregulated FGF19. EPI increased the uptake/accumulation of a fluorescent BA analogue in hepatocytes by 50-70%. Results also evidenced, that 40 µM EPI trans-activated the nuclear receptors LXR and PXR. More importantly, 0.01 µM EPI activated AR in hepatocytes, leading to an increase in the expression of CYP8B1. In samples from a human liver bank, we proved that the expression of AR was positively correlated with that of CYP8B1 in men. Taken together, we conclude that EPI could cause cholestasis by inducing BA synthesis and favouring BA accumulation in hepatocytes, at least in part by AR activation. We anticipate that the large phenotypic variability of BA synthesis enzymes and transport genes in man provide a putative explanation for the idiosyncratic nature of EPI-induced cholestasis.

Destruction of Pseudomonas aeruginosa pre-formed biofilms by cationic polymer micelles bearing silver nanoparticles.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen often associated with biofilm infections. This study evaluated the capacity for biofilm destruction of a novel combination of cationic polymer micelles formed from poly(2-(dimethylamino)ethyl methacrylate)-b-poly(ε-caprolactone)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA-PCL-PDMAEMA) triblock copolymer either alone, or loaded with silver nanoparticles (M_AgNPs). Pre-formed P. aeruginosa biofilms were incubated with either blank micelles, AgNO3, or M_AgNPs. Biofilm biomass (crystal violet assay), metabolic activity (Alamar blue reduction), structure (SEM) and viability (CLSM after Live/Dead staining, or plating for CFU) were checked. The results showed that the micelles alone loosened the biofilm matrix, and caused some alterations in the bacterial surface. AgNO3 killed the bacteria in situ leaving dead biofilm bacteria on the surface. M_AgNPs combined the two types of activities causing significant biofilm reduction, and alteration and death of biofilm bacteria. Therefore, the applied PDMAEMA-based micelles appear to be a successful candidate for the treatment of P. aeruginosa biofilm infections.

Design and Evaluation of a Rodent-Specific Transcranial Magnetic Stimulation Coil: An In Silico and In Vivo Validation Study.

BACKGROUND: Rodent models are fundamental in unraveling cellular and molecular mechanisms of transcranial magnetic stimulation (TMS)-induced effects on the brain. However, proper translation of human TMS protocols to animal models have been restricted by the lack of rodent-specific focal TMS coils. OBJECTIVE: We aimed to improve TMS focalization in rodent brain with a novel small, cooled, and rodent-specific TMS coil. METHODS: A rodent-specific 25-mm figure-of-eight TMS coil was developed. Stimulation focalization was simulated in silico for the rodent coil and a commercial human 50-mm figure-of-eight TMS coil. Both coils were also compared in vivo by electromyography measurements of brachialis motor evoked potential (MEP) responses to TMS at different brain sites in anesthetized rats (n = 6). Focalization was determined from the coils' level of stimulation laterality. Differences in MEPs were statistically analyzed with repeated-measures, within-subjects, ANOVA. RESULTS: In silico simulation results deemed the human coil insufficient for unilateral stimulation of the rat motor cortex, whereas lateralized electrical field induction was projected attainable with the rodent coil. Cortical, in vivo MEP amplitude measurements from multiple points in each hemisphere, revealed unilateral activation of the contralateral brachialis muscle, in absence of ipsilateral brachialis activation, with both coils. CONCLUSION: Computer simulations motivated the design of a smaller rodent-specific TMS coil, but came short in explaining the capability of a larger commercial human coil to induce unilateral MEPs in vivo. Lateralized TMS, as demonstrated for both TMS coils, corroborates their use in translational rodent studies, to elucidate mechanisms of action of therapeutic TMS protocols.

Angiopoietin-Like Protein 8 Is a Novel Vitamin D Receptor Target Gene Involved in Nonalcoholic Fatty Liver Pathogenesis.

Hepatic vitamin D receptor (VDR) expression is increased in patients with nonalcoholic fatty liver (NAFL) and is required for liver steatosis in an NAFL mouse model. However, how hepatocyte VDR is involved in setting up steatosis remains unclear. The authors transduced human hepatocyte-derived cells with an adenoviral vector encoding human VDR and found that angiopoietin-like protein 8 (ANGPTL8) expression was increased upon VDR activation by vitamin D or lithocholic acid. The mRNA levels of hepatic VDR- and vitamin D-related genes [cytochrome P450 (CYP) 2R1, CYP27A1, and CYP3A4] were higher in NAFL patients compared with normal liver subjects. Noteworthy, hepatic ANGPTL8 mRNA and protein levels were elevated in NAFL patients, and its mRNA correlated with VDR mRNA and with the steatosis grade. Moreover, increases in serum conjugated bile acids, including the VDR agonist glycine-lithocholic acid, were observed in NAFL patients. Additionally, free fatty acids and insulin were able to up-regulate both VDR and ANGPTL8 mRNA in human hepatocytes, whereas ANGPTL8 gene knockdown attenuated free fatty acids-induced triglyceride accumulation in these cells. In conclusion, activated VDR up-regulates ANGPTL8 expression, contributing to triglyceride accumulation in human hepatocytes. Moreover, hepatic ANGPTL8 mRNA positively correlates with VDR mRNA content and the grade of steatosis in NAFL patients, suggesting that this novel pathway may play a key role in the pathogenesis of hepatosteatosis.

Comparative analysis of nutrient content and energy of eggs from different chicken genotypes.

BACKGROUND: Eggs are important foods in the daily diet of humans and have great biological activity and a high digestibility. Egg yolk is a good source of biologically active substances such as fatty acids, phospholipids, sterols and tocopherols. The eggs of seven chicken genotypes were analyzed for their chemical composition, and a detailed study of the lipids in egg yolk was conducted. RESULTS: Energy composition of the egg yolk and egg albumen was 29.06-30.51 MJ kg-1 and 19.77-20.93 MJ kg-1 respectively. Regarding their chemical composition: water ranged from 471.7 to 515.4 g kg-1 and 878.3-885.9 g kg-1 ; fat content in dry matter ranged from 607 to 647 g kg-1 and 6.7-11.6 g kg-1 ; protein varied from 302 to 331.7 g kg-1 and 823.6-892.5 g kg-1 ; ash ranged from 33.7 to 37.7 g kg-1 and 63.8-74.0 g kg-1 ; and nitrogen-free extracts ranged from 12.7 to 36.5 g kg-1 and 35.0-96.2 g kg-1 . The sterols and phospholipids in the yolk lipids were 16-26 g kg-1 and 59-127 g kg-1 . The main fatty acids in the lipids were oleic (39.1-47.3%) and palmitic (26.0-35.5%) acids. Cholesterol in the yolk lipids ranged from 15.9 to 25.9 g kg-1 . Phosphatidylcholine (389-573 g kg-1 ), phosphatidylethanolamine (219-355 g kg-1 ) and phosphatidylinositol (112-284 g kg-1 ) were the main phospholipids. The content of saturated fatty acids in the phospholipids was significantly higher than that in triacylglycerols. CONCLUSION: Small variations in the chemical composition of eggs from seven different genotypes were observed. Significant differences in the fatty acid compositions of the main classes of phospholipids and the triacylglycerol fraction were established. © 2019 Society of Chemical Industry.

A novel concurrent TMS-fMRI method to reveal propagation patterns of prefrontal magnetic brain stimulation.

Major depressive disorder (MDD) is a severe mental disorder associated with high morbidity and mortality rates, which remains difficult to treat, as both resistance and recurrence rates are high. Repetitive transcranial magnetic stimulation (TMS) of the left dorsolateral prefrontal cortex (DLPFC) provides a safe and effective treatment for selected patients with treatment-resistant MDD. Little is known about the mechanisms of action of TMS provided to the left DLPFC in MDD and we can currently not predict who will respond to this type of treatment, precluding effective patient selection. In order to shed some light on the mechanism of action, we applied single pulse TMS to the left DLPFC in 10 healthy participants using a unique TMS-fMRI set-up, in which we could record the direct effects of TMS. Stimulation of the DLPFC triggered activity in a number of connected brain regions, including the subgenual anterior cingulate cortex (sgACC) in four out of nine participants. The sgACC is of particular interest, because normalization of activity in this region has been associated with relief of depressive symptoms in MDD patients. This is the first direct evidence that TMS pulses delivered to the DLPFC can propagate to the sgACC. The propagation of TMS-induced activity from the DLPFC to sgACC may be an accurate biomarker for rTMS efficacy. Further research is required to determine whether this method can contribute to the selection of patients with treatment resistant MDD who will respond to rTMS treatment.

Light sheet approaches for improved precision in 3D localization-based super-resolution imaging in mammalian cells [Invited].

The development of imaging techniques beyond the diffraction limit has paved the way for detailed studies of nanostructures and molecular mechanisms in biological systems. Imaging thicker samples, such as mammalian cells and tissue, in all three dimensions, is challenging due to increased background and volumes to image. Light sheet illumination is a method that allows for selective irradiation of the image plane, and its inherent optical sectioning capability allows for imaging of biological samples with reduced background, photobleaching, and photodamage. In this review, we discuss the advantage of combining single-molecule imaging with light sheet illumination. We begin by describing the principles of single-molecule localization microscopy and of light sheet illumination. Finally, we present examples of designs that successfully have married single-molecule super-resolution imaging with light sheet illumination for improved precision in mammalian cells.