Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging.

Molecular imaging using singlechain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are either expressed in E. coli cytoplasm as insoluble inclusion bodies, that often need cumbersome denaturation and refolding processes, or secreted toward the periplasm as soluble proteins that highly reduce the overall yield. However, production of active scFvs in their native form, without any heterologous fusion, is required for clinical applications. In this study, we expressed an anti-thymocyte differentiation antigen-scFv (Thy1-scFv) as a fusion protein with a N-terminal sequence including 3 × hexa-histidines, as purification tags, together with a Trx-tag and a S-tag for enhanced-solubility. Our strategy allowed to recover ~ 35% of Thy1-scFv in the soluble cytoplasmic fraction. An enterokinase cleavage site in between Thy1-scFv and the upstream tags was used to regenerate the protein with 97.7 ± 2.3% purity without any tags. Thy1-scFv showed functionality towards its target on flow cytometry assays. Finally, in vivo molecular imaging using Thy1-scFv conjugated to an ultrasound contrast agent (MBThy1-scFv) demonstrated signal enhancement on a transgenic pancreatic ductal adenocarcinoma (PDAC) mouse model (3.1 ± 1.2 a.u.) compared to non-targeted control (0.4 ± 0.4 a.u.) suggesting potential for PDAC early diagnosis. Overall, our strategy facilitates the expression and purification of Thy1-scFv while introducing its ability for diagnostic molecular imaging of pancreatic cancer. The presented methodology could be expanded to other important eukaryotic proteins for various applications, including but not limited to molecular imaging.

A simple microscopy setup for visualizing cellular responses to DNA damage at particle accelerator facilities.

Cellular responses to DNA double-strand breaks (DSBs) not only promote genomic integrity in healthy tissues, but also largely determine the efficacy of many DNA-damaging cancer treatments, including X-ray and particle therapies. A growing body of evidence suggests that activation of the mechanisms that detect, signal and repair DSBs may depend on the complexity of the initiating DNA lesions. Studies focusing on this, as well as on many other radiobiological questions, require reliable methods to induce DSBs of varying complexity, and to visualize the ensuing cellular responses. Accelerated particles of different energies and masses are exceptionally well suited for this task, due to the nature of their physical interactions with the intracellular environment, but visualizing cellular responses to particle-induced damage - especially in their early stages - at particle accelerator facilities, remains challenging. Here we describe a straightforward approach for real-time imaging of early response to particle-induced DNA damage. We rely on a transportable setup with an inverted fluorescence confocal microscope, tilted at a small angle relative to the particle beam, such that cells can be irradiated and imaged without any microscope or beamline modifications. Using this setup, we image and analyze the accumulation of fluorescently-tagged MDC1, RNF168 and 53BP1-key factors involved in DSB signalling-at DNA lesions induced by 254 MeV α-particles. Our results provide a demonstration of technical feasibility and reveal asynchronous initiation of accumulation of these proteins at different individual DSBs.

Expanding Molecular Coverage in Mass Spectrometry Imaging of Microbial Systems Using Metal-Assisted Laser Desorption/Ionization.

Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial (∼67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels.

Dielectric nanohole array metasurface for high-resolution near-field sensing and imaging.

Dielectric metasurfaces support resonances that are widely explored both for far-field wavefront shaping and for near-field sensing and imaging. Their design explores the interplay between localised and extended resonances, with a typical trade-off between Q-factor and light localisation; high Q-factors are desirable for refractive index sensing while localisation is desirable for imaging resolution. Here, we show that a dielectric metasurface consisting of a nanohole array in amorphous silicon provides a favourable trade-off between these requirements. We have designed and realised the metasurface to support two optical modes both with sharp Fano resonances that exhibit relatively high Q-factors and strong spatial confinement, thereby concurrently optimizing the device for both imaging and biochemical sensing. For the sensing application, we demonstrate a limit of detection (LOD) as low as 1 pg/ml for Immunoglobulin G (IgG); for resonant imaging, we demonstrate a spatial resolution below 1 µm and clearly resolve individual E. coli bacteria. The combined low LOD and high spatial resolution opens new opportunities for extending cellular studies into the realm of microbiology, e.g. for studying antimicrobial susceptibility.

Applications of mass spectrometry imaging in virus research.

Mass spectrometry imaging (MSI) is a label-free molecular imaging technique allowing an untargeted detection of a broad range of biomolecules and xenobiotics. MSI enables imaging of the spatial distribution of proteins, peptides, lipids and metabolites from a wide range of samples. To date, this technique is commonly applied to tissue sections in cancer diagnostics and biomarker development, but also molecular histology in general. Advances in the methodology and bioinformatics improved the resolution of MS images below the single cell level and increased the flexibility of the workflow. However, MSI-based research in virology is just starting to gain momentum and its full potential has not been exploited yet. In this review, we discuss the main applications of MSI in virology. We review important aspects of matrix-assisted laser desorption/ionization (MALDI) MSI, the most widely used MSI technique in virology. In addition, we summarize relevant literature on MSI studies that aim to unravel virus-host interactions and virus pathogenesis, to elucidate antiviral drug kinetics and to improve current viral disease diagnostics. Collectively, these studies strongly improve our general understanding of virus-induced changes in the proteome, metabolome and metabolite distribution in host tissues of humans, animals and plants upon infection. Furthermore, latest MSI research provided important insights into the drug distribution and distribution kinetics, especially in antiretroviral research. Finally, MSI-based investigations of oncogenic viruses greatly increased our knowledge on tumor mass signatures and facilitated the identification of cancer biomarkers.

Toward molecular imaging using spectral photon-counting computed tomography?

Molecular imaging is a valuable tool in drug discovery and development, early screening and diagnosis of diseases, and therapy assessment among others. Although many different imaging modalities are in use today, molecular imaging with computed tomography (CT) is still challenging owing to its low sensitivity and soft tissue contrast compared with other modalities. Recent technical advances, particularly the introduction of spectral photon-counting detectors, might allow overcoming these challenges. Herein, the fundamentals and recent advances in CT relevant to molecular imaging are reviewed and potential future preclinical and clinical applications are highlighted. The review concludes with a discussion of potential future advancements of CT for molecular imaging.

Ultra-high-frequency radio-frequency acoustic molecular imaging with saline nanodroplets in living subjects.

Molecular imaging is a crucial technique in clinical diagnostics but it relies on radioactive tracers or strong magnetic fields that are unsuitable for many patients, particularly infants and pregnant women. Ultra-high-frequency radio-frequency acoustic (UHF-RF-acoustic) imaging using non-ionizing RF pulses allows deep-tissue imaging with sub-millimetre spatial resolution. However, lack of biocompatible and targetable contrast agents has prevented the successful in vivo application of UHF-RF-acoustic imaging. Here we report our development of targetable nanodroplets for UHF-RF-acoustic molecular imaging of cancers. We synthesize all-liquid nanodroplets containing hypertonic saline that are stable for at least 2 weeks and can produce high-intensity UHF-RF-acoustic signals. Compared with concentration-matched iron oxide nanoparticles, our nanodroplets produce at least 1,600 times higher UHF-RF-acoustic signals at the same imaging depth. We demonstrate in vivo imaging using the targeted nanodroplets in a prostate cancer xenograft mouse model expressing gastrin release protein receptor (GRPR), and show that targeting specificity is increased by more than 2-fold compared with untargeted nanodroplets or prostate cancer cells not expressing this receptor.

Complementary performances of convolutional and capsule neural networks on classifying microfluidic images of dividing yeast cells.

Microfluidic-based assays have become effective high-throughput approaches to examining replicative aging of budding yeast cells. Deep learning may offer an efficient way to analyze a large number of images collected from microfluidic experiments. Here, we compare three deep learning architectures to classify microfluidic time-lapse images of dividing yeast cells into categories that represent different stages in the yeast replicative aging process. We found that convolutional neural networks outperformed capsule networks in terms of accuracy, precision, and recall. The capsule networks had the most robust performance in detecting one specific category of cell images. An ensemble of three best-fitted single-architecture models achieves the highest overall accuracy, precision, and recall due to complementary performances. In addition, extending classification classes and data augmentation of the training dataset can improve the predictions of the biological categories in our study. This work lays a useful framework for sophisticated deep-learning processing of microfluidic-based assays of yeast replicative aging.

Applications of stable isotopes in MALDI imaging: current approaches and an eye on the future.

Matrix-assisted laser desorption/ionisation-imaging mass spectrometry (MALDI-IMS) is now an established imaging modality with particular utility in the study of biological, biomedical and pathological processes. In the first instance, the use of stable isotopically labelled (SIL) compounds in MALDI-IMS has addressed technical barriers to increase the accuracy and versatility of this technique. This has undoubtedly enhanced our ability to interpret the two-dimensional ion intensity distributions produced from biological tissue sections. Furthermore, studies using delivery of SIL compounds to live tissues have begun to decipher cell, tissue and inter-tissue metabolism while maintaining spatial resolution. Here, we review both the technical and biological applications of SIL compounds in MALDI-IMS, before using the uptake and metabolism of glucose in bovine ocular lens tissue to illustrate the current limitations of SIL compound use in MALDI-IMS. Finally, we highlight recent instrumentation advances that may further enhance our ability to use SIL compounds in MALDI-IMS to understand biological and pathological processes. Graphical Abstract.

Real-time interleaved spectroscopic photoacoustic and ultrasound (PAUS) scanning with simultaneous fluence compensation and motion correction.

For over two decades photoacoustic imaging has been tested clinically, but successful human trials have been limited. To enable quantitative clinical spectroscopy, the fundamental issues of wavelength-dependent fluence variations and inter-wavelength motion must be overcome. Here we propose a real-time, spectroscopic photoacoustic/ultrasound (PAUS) imaging approach using a compact, 1-kHz rate wavelength-tunable laser. Instead of illuminating tissue over a large area, the fiber-optic delivery system surrounding an US array sequentially scans a narrow laser beam, with partial PA image reconstruction for each laser pulse. The final image is then formed by coherently summing partial images. This scheme enables (i) automatic compensation for wavelength-dependent fluence variations in spectroscopic PA imaging and (ii) motion correction of spectroscopic PA frames using US speckle tracking in real-time systems. The 50-Hz video rate PAUS system is demonstrated in vivo using a murine model of labelled drug delivery.

Localization of Major Ephedra Alkaloids in Whole Aerial Parts of Ephedrae Herba Using Direct Analysis in Real Time-Time of Flight-Mass Spectrometry.

Mass spectrometry-based molecular imaging has been utilized to map the spatial distribution of target metabolites in various matrixes. Among the diverse mass spectrometry techniques, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is the most popular for molecular imaging due to its powerful spatial resolution. This unparalleled high resolution, however, can paradoxically act as a bottleneck when the bio-imaging of large areas, such as a whole plant, is required. To address this issue and provide a more versatile tool for large scale bio-imaging, direct analysis in real-time-time of flight-mass spectrometry (DART-TOF-MS), an ambient ionization MS, was applied to whole plant bio-imaging of a medicinal plant, Ephedrae Herba. The whole aerial part of the plant was cut into 10-20 cm long pieces, and each part was further cut longitudinally to compare the contents of major ephedra alkaloids between the outer surface and inner part of the stem. Using optimized DART-TOF-MS conditions, molecular imaging of major ephedra alkaloids of the whole aerial part of a single plant was successfully achieved. The concentration of alkaloids analyzed in this study was found to be higher on the inner section than the outer surface of stems. Moreover, side branches, which are used in traditional medicine, represented a far higher concentration of alkaloids than the main stem. In terms of the spatial metabolic distribution, the contents of alkaloids gradually decreased towards the end of branch tips. In this study, a fast and simple macro-scale MS imaging of the whole plant was successfully developed using DART-TOF-MS. This application on the localization of secondary metabolites in whole plants can provide an area of new research using ambient ionization mass spectroscopy and an unprecedented macro-scale view of the biosynthesis and distribution of active components in medicinal plants.

Biomedical Micro-/Nanomotors: From Overcoming Biological Barriers to In Vivo Imaging.

Self-propelled micro- and nanomotors (MNMs) have shown great potential for applications in the biomedical field, such as active targeted delivery, detoxification, minimally invasive diagnostics, and nanosurgery, owing to their tiny size, autonomous motion, and navigation capacities. To enter the clinic, biomedical MNMs request the biodegradability of their manufacturing materials, the biocompatibility of chemical fuels or externally physical fields, the capability of overcoming various biological barriers (e.g., biofouling, blood flow, blood-brain barrier, cell membrane), and the in vivo visual positioning for autonomous navigation. Herein, the recent advances of synthetic MNMs in overcoming biological barriers and in vivo motion-tracking imaging techniques are highlighted. The challenges and future research priorities are also addressed. With continued attention and innovation, it is believed that, in the future, biomedical MNMs will pave the way to improve the targeted drug delivery efficiency.

An overview of biological applications and fundamentals of new inlet and vacuum ionization technologies.

RATIONALE: The developments of new ionization technologies based on processes previously unknown to mass spectrometry (MS) have gained significant momentum. Herein we address the importance of understanding these unique ionization processes, demonstrate the new capabilities currently unmet by other methods, and outline their considerable analytical potential. METHODS: The inlet and vacuum ionization methods of solvent-assisted ionization (SAI), matrix-assisted ionization (MAI), and laserspray ionization can be used with commercial and dedicated ion sources producing ions from atmospheric or vacuum conditions for analyses of a variety of materials including drugs, lipids, and proteins introduced from well plates, pipet tips and plate surfaces with and without a laser using solid or solvent matrices. Mass spectrometers from various vendors are employed. RESULTS: Results are presented highlighting strengths relative to ionization methods of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization. We demonstrate the utility of multi-ionization platforms encompassing MAI, SAI, and ESI and enabling detection of what otherwise is missed, especially when directly analyzing mixtures. Unmatched robustness is achieved with dedicated vacuum MAI sources with mechanical introduction of the sample to the sub-atmospheric pressure (vacuum MAI). Simplicity and use of a wide array of matrices are attained using a conduit (inlet ionization), preferably heated, with sample introduction from atmospheric pressure. Tissue, whole blood, urine (including mouse, chicken, and human origin), bacteria strains and chemical on-probe reactions are analyzed directly and, especially in the case of vacuum ionization, without concern of carryover or instrument contamination. CONCLUSIONS: Examples are provided highlighting the exceptional analytical capabilities associated with the novel ionization processes in MS that reduce operational complexity while increasing speed and robustness, achieving mass spectra with low background for improved sensitivity, suggesting the potential of this simple ionization technology to drive MS into areas currently underserved, such as clinical and medical applications.

Neuropeptidomics: Improvements in Mass Spectrometry Imaging Analysis and Recent Advancements.

Neuropeptides are an important class of endogenous peptides in the nervous system that regulate physiological functions such as feeding, glucose homeostasis, pain, memory, reproduction, and many others. In order to understand the functional role of neuropeptides in diseases or disorders, studies investigating their dysregulation in terms of changes in abundance and localization must be carried out. As multiple neuropeptides are believed to play a functional role in each physiological process, techniques capable of global profiling multiple neuropeptides simultaneously are desired. Mass spectrometry is well-suited for this goal due to its ability to perform untargeted measurements without prior comprehensive knowledge of the analytes of interest. Mass spectrometry imaging (MSI) is particularly useful because it has the capability to image a large variety of peptides in a single experiment without labeling. Like all analytical techniques, careful sample preparation is critical to successful MSI analysis. The first half of this review focuses on recent developments in MSI sample preparation and instrumentation for analyzing neuropeptides and other biomolecules in which the sample preparation technique may be directly applicable for neuropeptide analysis. The benefit offered by incorporating these techniques is shown as improvement in a number of observable neuropeptides, enhanced signal to noise, increased spatial resolution, or a combination of these aspects. The second half of this review focuses on recent biological discoveries about neuropeptides resulting from these improvements in MSI analysis. The recent progress in neuropeptide detection and analysis methods, including the incorporation of various tissue washes, matrices, instruments, ionization sources, and computation approaches combined with the advancements in understanding neuropeptide function in a variety of model organisms, indicates the potential for the utilization of MSI analysis of neuropeptides in clinical settings.

Guidelines on Setting Up Stations for Remote Viewing of Nuclear Medicine and Molecular Imaging Studies During COVID-19.

The current pandemic has created a situation where nuclear medicine practitioners and medical physicists read or process nuclear medicine images remotely from their home office. This article presents recommendations on the components and specifications when setting up a remote viewing station for nuclear medicine imaging.

Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a fast-growing technique for visualization of the spatial distribution of the small molecular and macromolecular biomolecules in tissue sections. Challenges in MALDI-MSI, such as poor sensitivity for some classes of molecules or limited specificity, for instance resulting from the presence of isobaric molecules or limited resolving power of the instrument, have encouraged the MSI scientific community to improve MALDI-MSI sample preparation workflows with innovations in chemistry. Recent developments of novel small organic MALDI matrices play a part in the improvement of image quality and the expansion of the application areas of MALDI-MSI. This includes rationally designed/synthesized as well as commercially available small organic molecules whose superior matrix properties in comparison with common matrices have only recently been discovered. Furthermore, on-tissue chemical derivatization (OTCD) processes get more focused attention, because of their advantages for localization of poorly ionizable metabolites and their' in several cases' more specific imaging of metabolites in tissue sections. This review will provide an overview about the latest developments of novel small organic matrices and on-tissue chemical derivatization reagents for MALDI-MSI. Graphical abstract.

Spatially resolved absolute quantitation in thin tissue by mass spectrometry.

Mass spectrometry (MS) has become the de facto tool for routine quantitative analysis of biomolecules. MS is increasingly being used to reveal the spatial distribution of proteins, metabolites, and pharmaceuticals in tissue and interest in this area has led to a number of novel spatially resolved MS technologies. Most spatially resolved MS measurements are qualitative in nature due to a myriad of potential biases, such as sample heterogeneity, sampling artifacts, and ionization effects. As applications of spatially resolved MS in the pharmacological and clinical fields increase, demand has become high for quantitative MS imaging and profiling data. As a result, several varied technologies now exist that provide differing levels of spatial and quantitative information. This review provides an overview of MS profiling and imaging technologies that have demonstrated quantitative analysis from tissue. Focus is given on the fundamental processes affecting quantitative analysis in an array of MS imaging and profiling technologies and methods to address these biases.Graphical abstract.

In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy.

Zebrafish embryos provide a unique opportunity to visualize complex biological processes, yet conventional imaging modalities are unable to access intricate biomolecular information without compromising the integrity of the embryos. Here, we report the use of confocal Raman spectroscopic imaging for the visualization and multivariate analysis of biomolecular information extracted from unlabeled zebrafish embryos. We outline broad applications of this method in: (i) visualizing the biomolecular distribution of whole embryos in three dimensions, (ii) resolving anatomical features at subcellular spatial resolution, (iii) biomolecular profiling and discrimination of wild type and ΔRD1 mutant Mycobacterium marinum strains in a zebrafish embryo model of tuberculosis and (iv) in vivo temporal monitoring of the wound response in living zebrafish embryos. Overall, this study demonstrates the application of confocal Raman spectroscopic imaging for the comparative bimolecular analysis of fully intact and living zebrafish embryos.

Nile-Red-Based Fluorescence Probe for Selective Detection of Biothiols, Computational Study, and Application in Cell Imaging.

A new colorimetric and fluorescence probe NRSH based on Nile-red chromophore for the detection of biothiols has been developed, exhibiting high selectivity towards biothiols over other interfering species. NRSH shows a blue shift in absorption peak upon reacting with biothiols, from 587 nm to 567 nm, which induces an obvious color change from blue to pink and exhibits a 35-fold fluorescence enhancement at 645 nm in red emission range. NRSH displays rapid (<1 min) response for H2S, which is faster than other biothiols (>5 min). The detection limits of probe NRSH towards biothiols are very low (22.05 nM for H2S, 34.04 nM for Cys, 107.28 nM for GSH and 113.65 nM for Hcy). Furthermore, NRSH is low cytotoxic and can be successfully applied as a bioimaging tool for real-time monitoring biothiols in HeLa cells. In addition, fluorescence mechanism of probe NRSH is further understood by theoretical calculations.