Optical Fluorescence Microscopy Research Articles

Nanoscale elemental and morphological imaging of nitrogen-fixing cyanobacteria.

Nitrogen-fixing cyanobacteria bind atmospheric nitrogen and carbon dioxide using sunlight. This experimental study focused on a laboratory-based model system, Anabaena sp., in nitrogen-depleted culture. When combined nitrogen is scarce, the filamentous procaryotes reconcile photosynthesis and nitrogen fixation by cellular differentiation into heterocysts. To better understand the influence of micronutrients on cellular function, 2D and 3D synchrotron X-ray fluorescence mappings were acquired from whole biological cells in their frozen-hydrated state at the Bionanoprobe, Advanced Photon Source. To study elemental homeostasis within these chain-like organisms, biologically relevant elements were mapped using X-ray fluorescence spectroscopy and energy-dispersive X-ray microanalysis. Higher levels of cytosolic K+, Ca2+, and Fe2+ were measured in the heterocyst than in adjacent vegetative cells, supporting the notion of elevated micronutrient demand. P-rich clusters, identified as polyphosphate bodies involved in nutrient storage, metal detoxification and osmotic regulation, were consistently co-localized with K+ and occasionally sequestered Mg2+, Ca2+, Fe2+, and Mn2+ ions. Machine-learning based k-mean clustering revealed that P/K clusters were associated with either Fe or Ca, with Fe and Ca clusters also occurring individually. In accordance with XRF nanotomography, distinct P/K-containing clusters close to the cellular envelope were surrounded by larger Ca-rich clusters. The transition metal Fe, which is part of nitrogenase enzyme, was detected as irregular shaped clusters. The elemental composition and cellular morphology of diazotrophic Anabaena sp. was visualized by multimodal imaging using AFM, SEM, and fluorescence microscopy. This paper discusses the first experimental results obtained with a combined in-line optical and X-ray fluorescence microscope at the Bionanoprobe.

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or "nanodrug") administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Direct measurement of multi-angle evanescent field by whispering gallery mode of the microsphere and fluorescent spots in an optical waveguide fluorescence microscopy

Evanescent field (EF) has been widely applied in various micro-imaging techniques for its ability to excite surfaces. However, measuring the multi-angle EF remains challenging. In this paper, the whispering gallery mode (WGM) is observed in an excited microsphere on an optical waveguide (OWG) with a thickness of 0.6 mm, which is used to determine the diameter of the excited microsphere. A simple and nondestructive measurement method for multi-angle OWG-EF is proposed, which is based on the reliable diameter of the microsphere, determined by WGM and the TIRF spots. The measurement result for multi-angle OWG-EF shows a dynamic range between ∼180 and ∼2400 nm, which shows good agreement with the simulation results. The method synchronization possible for EF measurements and fluorescent experiments.

Tumor spheroid elasticity estimation using mechano-microscopy combined with a conditional generative adversarial network

Background and ObjectivesTechniques for imaging the mechanical properties of cells are needed to study how cell mechanics influence cell function and disease progression. Mechano-microscopy (a high-resolution variant of compression optical coherence elastography) generates elasticity images of a sample undergoing compression from the phase difference between optical coherence microscopy (OCM) B-scans. However, the existing mechano-microscopy signal processing chain (referred to as the algebraic method) assumes the sample stress is uniaxial and axially uniform, such that violation of these assumptions reduces the accuracy and precision of elasticity images. Furthermore, it does not account for prior information regarding the sample geometry or mechanical property distribution. In this study, we investigate the feasibility of training a conditional generative adversarial network (cGAN) to generate elasticity images from phase difference images of samples containing a cell spheroid embedded in a hydrogel. MethodsTo construct the cGAN training and simulated test sets, we generated 30,000 artificial elasticity images using a parametric model and computed the corresponding phase difference images using finite element analysis to simulate compression applied to the artificial samples. We also imaged real MCF7 breast tumor spheroids embedded in hydrogel using mechano-microscopy to construct the experimental test set and evaluated the cGAN using the algebraic elasticity images and co-registered OCM and confocal fluorescence microscopy (CFM) images. ResultsComparison with the simulated test set ground truth elasticity images shows the cGAN produces a lower root mean square error (median: 3.47 kPa, 95 % confidence interval (CI) [3.41, 3.52]) than the algebraic method (median: 4.91 kPa, 95 % CI [4.85, 4.97]). For the experimental test set, the cGAN elasticity images contain features resembling stiff nuclei at locations corresponding to nuclei seen in the algebraic elasticity, OCM, and CFM images. Furthermore, the cGAN elasticity images are higher resolution and more robust to noise than the algebraic elasticity images. ConclusionsThe cGAN elasticity images exhibit better accuracy, spatial resolution, sensitivity, and robustness to noise than the algebraic elasticity images for both simulated and real experimental data.

Observation of Polymerization‐Induced Phase Separation During Polymerization of Methyl Methacrylate in the Presence of Poly(Ethylene Glycol)

AbstractPolymerization‐induced phase separation takes place when a miscible component of the monomer becomes immiscible as the polymer is formed. One example is the polymerization of methyl methacrylate (MMA) in the presence of poly(ethylene glycol) (PEG). Depending on the initial conditions, microscopic structures, including porous structures, co‐continuous monolith structures, and particle aggregation structures, are obtained. In this study, the phase separation process is analyzed using an optical microscope and fluorescence microscope. The nucleation or spinodal decomposition in the initial stage, subsequent growth in the microscopic domains, and eventual fixation of the structure are observed. The observed images are analyzed using image analysis software. The particle number and the particle size as a function of reaction time are evaluated. Conversion and molecular weight distribution as a function of reaction time are also analyzed.

Monitoring of three-dimensional live-cell cultures using a multimode, multiscale imaging system combining confocal fluorescence microscopy and optical coherence microscopy

Live-cell monitoring involves long-term observation and analysis of living cells in tissue cultures, which develop cells or tissues in an artificial environment and are essential for tissue engineering. Fluorescence microscopy (FM) is widely used as a monitoring tool owing to its powerful ability to visualize protein distribution within cells and tissues, thereby providing information on their functions in biological processes. To reduce the photobleaching effect, which is a major limiting factor in FM, FM is generally combined with other imaging modalities, such as phase contrast and differential interference contrast microscopy, which are nondestructive to fluorochromes, to selectively use fluorescence signals only in specific areas of interest within a sample. However, these methods are restricted to thin samples and cannot produce depth-resolved images. Here, we propose a method to determine three-dimensional (3D) positions in volumetric samples for application in high-resolution 3D fluorescence imaging using a multimode and multiscale imaging system that combines optical coherence microscopy (OCM) and line confocal FM (LC–FM). We also demonstrate the benefits of multimodal imaging in 3D cell culture monitoring. To evaluate the performance of the proposed system and method, we rapidly and accurately located the regions of interest in 3D tissue culture models, such as tumor spheroids and microvascular bed, and instantaneously acquired volumetric high-resolution fluorescence images. We also used an integrated system to monitor tumor spheroids mixed with extracellular matrix (ECM) over five days of the culture process, and the FM–OCM integrated technique successfully imaged the overall 3D structures, such as the distribution and boundaries of the spheroids and ECM as well as stained tumor cells. This complementary information is useful for understanding the relationship between cellular behaviors, such as the proliferation and migration of tumor cells, and microenvironments, such as the ECM.

The supramolecular processing of liposomal doxorubicin hinders its therapeutic efficacy in cells

The successful trajectory of liposome-encapsulated Doxorubicin (e.g. FDA-approved Doxil®) as an anticancer nanodrug in clinical applications is contradicted by in-vitro cell-viability data which highlight its reduced efficacy in promoting cell death compared to non-encapsulated Doxorubicin. No reports to date have provided a mechanistic explanation for this apparently discordant evidence. Taking advantage of Doxorubicin intrinsic fluorescence and time-resolved optical microscopy, we analyze the uptake and intracellular processing of liposome-encapsulated Doxorubicin (hereafter ‘L-DOX’) in several in-vitro cellular models. Cell-entry of L-DOX was found to lead to a rapid (seconds to minutes), energy- and temperature-independent release of crystallized Doxorubicin nanorods into the cell cytoplasm, which then disassemble into a pool of fibril-shaped derivatives capable of crossing the cellular membrane while simultaneously releasing active drug monomers. Thus, a steady state is rapidly established in which the continuous supply of crystal nanorods from incoming liposomes is counteracted by a concentration-guided efflux in the extracellular medium of fibril-shaped derivatives and active drug monomers. These results demonstrate that liposome-mediated delivery is constitutively less efficient than isolated drug in establishing favorable conditions for drug retention in the cell. In addition to explaining previous contradictory evidence, present results impose careful rethinking of the synthetic identity of encapsulated anticancer drugs.

Acupotomy alleviates knee osteoarthritis in rabbit by regulating chondrocyte mitophagy Pink1-Parkin pathway.

To investigate the effect of acupotomy, on mitophagy and the Pink1-Parkin pathway in chondrocytes from rabbits with knee osteoarthritis (KOA). A KOA model was established via the modified Videman method. Rabbits were randomly divided into a control group (CON), KOA group and KOA + acupotomy group (Acu). Rabbits in the acupotomy group were subjected to acupotomy for 4 weeks after model establishment. The behavior of the rabbits before and after intervention was recorded. Cartilage degeneration was evaluated by optical microscopy and fluorescence microscopy. The level of mitophagy was evaluated by transmission electron microscopy, immunofluorescence and enzyme-linked immunosorbent assay (ELISA). The expression of phosphatase and tensin homolog (PTEN)-induced kinase 1 (Pink1)-Parkin mitophagy pathway components was evaluated by immunofluorescence, Western blotting and real-time polymerase chain reaction. In rabbits with KOA, joint pain, mobility disorders and cartilage degeneration were observed, the Mankin score was increased, collagen type Ⅱ (Col-Ⅱ) expression was significantly decreased, mitophagy was inhibited, mitochondrial function was impaired, and factors associated with the Pink1-Parkin pathway were inhibited. Acupotomy regulated the expression of Pink1-Parkin pathway-related proteins, the mitophagy-related protein microtubule-associated protein-1 light chain-3, the translocase of the outer membrane, and the inner mitochondrial membrane 23; increased the colocalization of mitochondria and autophagosomes; promoted the removal of damaged mitochondria; restored mitochondrial adenosine-triphosphate (ATP) production; and alleviated cartilage degeneration in rabbits with KOA. Acupotomy played a role in alleviating KOA in rabbits by activating mitophagy in chondrocytes via the regulation of proteins that are related to the Pink1-Parkin pathway.

High-Moisture Extrusion of a Dietary Protein Blend Impairs In Vitro Digestion and Delays In Vivo Postprandial Plasma Amino Acid Availability in Humans

BackgroundIndustrial processing can alter the structural complexity of dietary proteins and, potentially, their digestion and absorption upon ingestion. High-moisture extrusion (HME), a common processing method used to produce meat alternative products, affects in vitro digestion, but human data are lacking. We hypothesized that HME of a mycoprotein/pea protein blend would impair in vitro digestion and in vivo postprandial plasma amino acid availability. MethodsIn Study A, 9 healthy volunteers completed 2 experimental trials in a randomized, double-blind, crossover design. Participants consumed a beverage containing 25 g protein from a “dry” blend (CON) of mycoprotein/pea protein (39%/61%) or an HME content-matched blend (EXT). Arterialized venous blood samples were collected in the postabsorptive state and regularly over a 5-h postprandial period to assess plasma amino acid concentrations. In Study B, in vitro digestibility of the 2 beverages were assessed using bicinchoninic acid assay and optical fluorescence microscopy at baseline and during and following gastric and intestinal digestion using the INFOGEST model of digestion. ResultsProtein ingestion increased plasma total, essential (EAA), and branched-chain amino acid (BCAA) concentrations (time effect, P < 0.0001) but more rapidly and to a greater magnitude in the CON compared with the EXT condition (condition × time interaction, P < 0.0001). This resulted in greater plasma availability of EAA and BCAA concentrations during the early postprandial period (0–150 min). These data were corroborated by the in vitro approach, which showed greater protein availability in the CON (2150 ± 129 mg/mL) compared with the EXT (590 ± 41 mg/mL) condition during the gastric phase. Fluorescence microscopy revealed clear structural differences between the 2 conditions. ConclusionsThese data demonstrate that HME delays in vivo plasma amino acid availability following ingestion of a mycoprotein/pea protein blend. This is likely due to impaired gastric phase digestion as a result of HME-induced aggregate formation in the pea protein.This trial was registered at clinicaltrials.gov as NCT05584358.

Nonlinear viscoelasticity of Melaleuca alternifolia essential oil Pickering emulsion stabilized with cellulose nanofibrils

Pickering emulsions are suitable technological approaches to stabilize the bioactive compounds of essential oils (EOs) with economic interests for the pharmaceutical, cosmetic and food industries using atoxic and renewable stabilizers, such as cellulose nanostructures. The Melaleuca alternifolia essential oil (MaEO) is recognized for its pronounced antimicrobial performance, which has attracted your interest in developing eco-friendly antiseptic products. In this contribution, we report the nonlinear viscoelasticity behavior of MaEO-in-water Pickering emulsions stabilized by cellulose nanofibrils (CNF) using Large Amplitude Oscillatory Shear (LAOS) tests. The rheological characterizations by Fourier Transform (FT) rheology spectroscopy and Lissajous plots enabled the correlation of the viscous and elastic behaviors with the physical processes involving the deformation of the emulsion. Also, the internal structure of the emulsion was elucidated by fluorescence optical microscopy. Several findings relative to the effects of the shear deformation amplitude over the nonlinear viscoelasticity of the emulsified system are reported here, which are essential to the industrial processes used to develop technological products.

Zero-shot learning enables instant denoising and super-resolution in optical fluorescence microscopy.

Computational super-resolution methods, including conventional analytical algorithms and deep learning models, have substantially improved optical microscopy. Among them, supervised deep neural networks have demonstrated outstanding performance, however, demanding abundant high-quality training data, which are laborious and even impractical to acquire due to the high dynamics of living cells. Here, we develop zero-shot deconvolution networks (ZS-DeconvNet) that instantly enhance the resolution of microscope images by more than 1.5-fold over the diffraction limit with 10-fold lower fluorescence than ordinary super-resolution imaging conditions, in an unsupervised manner without the need for either ground truths or additional data acquisition. We demonstrate the versatile applicability of ZS-DeconvNet on multiple imaging modalities, including total internal reflection fluorescence microscopy, three-dimensional wide-field microscopy, confocal microscopy, two-photon microscopy, lattice light-sheet microscopy, and multimodal structured illumination microscopy, which enables multi-color, long-term, super-resolution 2D/3D imaging of subcellular bioprocesses from mitotic single cells to multicellular embryos of mouse and C. elegans.

Optical coherence tomography-guided Brillouin microscopy highlights regional tissue stiffness differences during anterior neural tube closure in the Mthfd1l murine mutant.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

Biocompatibility assessment of organic semiconductor pigments epindolidione and quinacridone

Organic colorants epindolidione (EPI) and quinacridone (QUI) are commercially available hydrogen-bonded semiconductor pigments. Despite their suitable properties for bioelectronic applications, their biocompatibility has not been thoroughly examined yet. In this study, thin EPI and QUI layers were applied on well plates by vacuum deposition technique followed by short and long-term in vitro biocompatibility study. In vitro testing represents a relatively fast and cheap approach, especially suitable for screening testing and prioritization of materials for further research. LIVE/DEAD assay, cell cycle analysis, adhesion and morphology of NIH 3T3 mouse fibroblasts have been investigated up to 7 days using flow cytometry and fluorescent optical microscopy. In summary, no significant differences were observed in viability, cell morphology, or attachment capabilities between control cells and cells grown on the EPI or QUI surfaces or cells cultivated in medium harvested from EPI- or QUI-coated wells. Our results suggest that both pigments are highly biocompatible. The absence of adverse biological effects of EPI and QUI together with their low cost and availability indicate their high application potential in next-generation bioelectronic devices.

A beginner’s guide to immunohistochemistry

Using the affinity between antibodies and antigens, immunohistochemistry (IHC) is a helpful method for analysing the cellular components within tissue sections placed on glass slides. The chemistry of antibodies was not discovered until 1941, and since then, their use has increased due to the discovery of enzymes and fluorochromes conjugated to antibodies, enabling observations through optical or fluorescence microscopy (FM). Hence, immunofluorescence (IF), or IHC with fluorescent antibodies, has been associated with the advancement of FM and fluorochromes. Specifically, the discovery of three fluorochromes that emit blue, red and green led to a rise in IF application in the context of visualizing numerous components in a single cell. The proximity ligation assay (PLA) method, so termed because the antibody is conjugated with short DNA strands called PLA probes, is the most recent development in the field of antibody conjugation. Protein–protein interactions and sub-localization of proteins can both be studied using this technique. The improvement of conjugation techniques has occurred in parallel with the development of sophisticated microscopies, including confocal and slide scanners. Confocal microscopy allows for the reconstruction of three-dimensional structures while a slide scanner can digitally convert up to 100 slides' worth of high-resolution pictures. The first step in the IHC process is the collection and fixation of tissues from animal models and humans. Paraformaldehyde is the most commonly used fixative. Fixation prevents antigen blocking and creates cross-links between proteins to preserve tissue structures. Fixation is followed by cutting tissues using microtome or cryostat, then slides are treated for antigen exposition (antigen retrieval) and then included in Triton X solution, which increases membrane permeability. Subsequently, incubations with enzyme- or fluorochrome-conjugated antibodies enable observations via optical microscopy or FM, respectively. In clinical practice and research, IHC and IF are still helpful methods for identifying the antigen-proteins linked to specific diseases.

Illuminating Polysulfide Distribution in Lithium Sulfur Batteries; Tracking Polysulfide Shuttle Using Operando Optical Fluorescence Microscopy.

High-energy-density lithium sulfur (Li-S) batteries suffer heavily from the polysulfide shuttle effect, a result of the dissolution and transport of intermediate polysulfides from the cathode, into the electrolyte, and onto the anode, leading to rapid cell degradation. If this primary mechanism of cell failure is to be overcome, the distribution, dynamics, and degree of polysulfide transport must first be understood in depth. In this work, operando optical fluorescence microscope imaging of optically accessible Li-S cells is shown to enable real-time qualitative visualization of the spatial distribution of lithium polysulfides, both within the electrolyte and porous cathode. Quantitative determinations of spatial concentration are also possible at a low enough concentration. The distribution throughout cycling is monitored, including direct observation of polysulfide shuttling to the anode and consequent dendrite formation. This was enabled through the optimization of a selective fluorescent dye, verified to fluoresce proportionally with concentration of polysulfides within Li-S cells. This ability to directly and conveniently track the spatial distribution of soluble polysulfide intermediates in Li-S battery electrolytes, while the cell operates, has the potential to have a widespread impact across the field, for example, by enabling the influence of a variety of polysulfide mitigation strategies to be assessed and optimized, including in this work the LiNO3 additive.

An MR-based brain template and atlas for optical projection tomography and light sheet fluorescence microscopy in neuroscience.

Optical Projection Tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high resolution optical imaging techniques, ideally suited for ex vivo 3D whole mouse brain imaging. Although they exhibit high specificity for their targets, the anatomical detail provided by tissue autofluorescence remains limited. T1-weighted images were acquired from 19 BABB or DBE cleared brains to create an MR template using serial longitudinal registration. Afterwards, fluorescent OPT and LSFM images were coregistered/normalized to the MR template to create fusion images. Volumetric calculations revealed a significant difference between BABB and DBE cleared brains, leading to develop two optimized templates, with associated tissue priors and brain atlas, for BABB (OCUM) and DBE (iOCUM). By creating fusion images, we identified virus infected brain regions, mapped dopamine transporter and translocator protein expression, and traced innervation from the eye along the optic tract to the thalamus and superior colliculus using cholera toxin B. Fusion images allowed for precise anatomical identification of fluorescent signal in the detailed anatomical context provided by MR. The possibility to anatomically map fluorescent signals on magnetic resonance (MR) images, widely used in clinical and preclinical neuroscience, would greatly benefit applications of optical imaging of mouse brain. These specific MR templates for cleared brains enable a broad range of neuroscientific applications integrating 3D optical brain imaging.

Microstructuring of Thermoresponsive Biofunctional Hydrogels by Multiphoton Photocrosslinking

AbstractA pioneering method is reported for creating thermoresponsive biofunctional hydrogel microstructures using maskless multiphoton lithography. Departing from conventional multiphoton‐triggered polymerization‐based techniques, this approach relies on simultaneous photocrosslinking and attachment of already pre‐synthesized polymer chains onto solid substrates. The method allows improving control over polymer network characteristics and enables facile integration of additional functionalities through postmodification with biomolecules at specific sites. Exploring two distinct benzophenone‐ and anthraquinone‐based photocrosslinkers incorporated into specially designed poly(N‐isopropyl acrylamide)‐based co‐ and terpolymers, the photocrosslinking efficacy is scrutinized with the use of a custom femtosecond near‐infrared laser lithographer. Comprehensive characterization via surface plasmon resonance imaging, atomic force microscopy, and optical fluorescence microscopy reveals swelling behavior and demonstrates postmodification feasibility. Notably, within a specific range of multiphoton photocrosslinking parameters, the surface‐attached microstructures exhibit a quasiperiodic topography akin to wrinkle‐pattern formation. Leveraging the capabilities of established multiphoton lithographer systems that offer fast pattern writing with high resolution, this approach holds great promise for the versatile fabrication of multifunctional 3D micro‐ and nanostructures. Such tailored responsive biofunctional materials with spatial control over composition, swelling behavior, and postmodification are particularly attractive in the areas of bioanalytical and biomedical technologies.

Understanding the Role of Biofilms in Acute Recurrent Tonsillitis through 3D Bioprinting of a Novel Gelatin-PEGDA Hydrogel.

Acute recurrent tonsillitis is a chronic, biofilm-related infection that is a significant burden to patients and healthcare systems. It is often treated with repeated courses of antibiotics, which contributes to antimicrobial resistance. Studying biofilms is key to understanding this disease. In vitro modelling using 3D bioprinted hydrogels is a promising approach to achieve this. A novel gelatin-PEGDA pseudomonas fluorescens-laden bioink was developed and bioprinted in a 3D hydrogel construct fabricated using computer-aided design to mimic the tonsillar biofilm environment. The bioprinted constructs were cultured at 37 °C in lysogeny broth for 12 days. Bacterial growth was assessed by spectrophotometry. Cellular viability analysis was conducted using optical fluorescence microscopy (FDA/PI staining). A biocompatible 3D-printed bacteria-laden hydrogel construct was successfully fabricated. Bacterial growth was observed using optical fluorescence microscopy. A live/dead cellular-staining protocol demonstrated bacterial viability. Results obtained after the 12-day culture period showed higher bacterial growth in the 1% gelatin concentration construct compared to the 0% control. This study demonstrates the first use of a bacteria-laden gelatin-PEGDA hydrogel for biofabrication of a 3D-printed construct designed to model acute recurrent tonsillitis. Initiating a study with clinically relevant ex vivo tonsil bacteria will be an important next step in improving treatment of this impactful but understudied disease.

Single-molecule analysis of purified proteins and nuclear extracts: Insights from 8-oxoguanine glycosylase 1

By observing one molecule at a time, single-molecule studies can offer detailed insights about biomolecular processes including on rates, off rates, and diffusivity of molecules on strands of DNA. A recent technological advance (Single-molecule Analysis of DNA-binding proteins from Nuclear Extracts, SMADNE) has lowered the barrier to entry for single-molecule studies, and single-molecule dynamics can now be determined directly out of nuclear extracts, providing information in an intermediate environment between purified proteins in isolation and the heterogeneity of a nucleus. To compare and contrast the single-molecule DNA binding dynamics in nuclear extracts versus purified proteins, combined optical tweezers and fluorescence microscopy experiments were performed with purified GFP-tagged 8-oxoguanine glycosylase 1 (OGG1), purified GFP-OGG1 spiked into nuclear extracts, and nuclear extracts from human cells overexpressing GFP-OGG1. We observed differences in undamaged DNA binding during DNA damage search in each of the three conditions. Purified GFP-OGG1 engaged undamaged DNA for a weighted average lifetime of 5.7 s and 21% of these events underwent DNA diffusion after binding. However, unlike other glycosylases studied by SMADNE, OGG1 does not bind non-damaged DNA efficiently in nuclear extracts. In contrast, GFP-OGG1 binding dynamics on DNA substrates containing oxidative damage were relatively similar in all three conditions, with the weighted average binding lifetimes varying from 2.2 s in nuclear extracts to 7.8 s with purified GFP-OGG1 in isolation. Finally, we compared the purified protein and nuclear extract approaches for a catalytically dead OGG1 variant (GFP-OGG1-K249Q). This variant greatly increased the binding lifetime for oxidative DNA damage, with the weighted average lifetime for GFP-OGG1–249Q in nuclear extracts at 15.4 s vs 10.7 s for the purified protein. SMADNE will provide a new window of observation into the behavior of nucleic acid binding proteins only accessible by biophysicists trained in protein purification and protein labeling.

Correlative multimodal optical and X-ray fluorescence imaging of brominated fluorophores.

Imaging with multiple modalities can maximise the information gained from the analysis of a single sample. probes for optical fluorescence and X-ray fluorescence microscopy based on brominated 4-amino-1,8-naphthalimide and BODIPY scaffolds have been successfully designed and synthesised. Herein we show that these prototype probes, based on each of these scaffolds, can be imaged in two different cancer cell lines, and that the respective optical fluorescence and X-ray fluorescence signals are well correlated in these images.