In vivo clinical molecular imaging of T cell activity.

Tumor immunotherapy is refashioning traditional treatments in the clinic for certain tumors, especially by relying on the activation of T cells. However, the safety and effectiveness of many antitumor immunotherapeutic agents are suboptimal due to difficulties encountered in assessing T cell responses and adjusting treatment regimens accordingly. Here, we review advances in the clinical visualization of T cell activity in vivo, and focus particularly on molecular imaging probes and biomarkers of T cell activation. Current challenges and prospects are also discussed that aim to achieve a better strategy for real-time monitoring of T cell activity, predicting prognoses and responses to tumor immunotherapy, and assessing disease management.

Accelerated MINFLUX Nanoscopy, through Spontaneously Fast-Blinking Fluorophores.

The introduction of MINFLUX nanoscopy allows single molecules to be localized with one nanometer precision in as little as one millisecond. However, current applications have so far focused on increasing this precision by optimizing photon collection, rather than minimizing the localization time. Concurrently, commonly used fluorescent switches are specifically designed for stochastic methods (e.g., STORM), optimized for a high photon yield and rather long on-times (tens of milliseconds). Here, accelerated MINFLUX nanoscopy with up to a 30-fold gain in localization speed is presented. The improvement is attained by designing spontaneously blinking fluorescent markers with remarkably fast on-times, down to 1-3 ms, matching the iterative localization process used in a MINFLUX microscope. This design utilizes a silicon rhodamine amide core, shifting the spirocyclization equilibrium toward an uncharged closed form at physiological conditions and imparting intact live cell permeability, modified with a fused (benzo)thiophene spirolactam fragment. The best candidate for MINFLUX microscopy (also suitable for STORM imaging) is selected through detailed characterization of the blinking behavior of single fluorophores, bound to different protein tags. Finally, optimization of the localization routines, customized to the fast blinking times, renders a significant speed improvement on a commercial MINFLUX microscope.

Non-invasive molecular imaging for precision diagnosis of metastatic lymph nodes: opportunities from preclinical to clinical applications.

Lymph node metastasis is an indicator of the invasiveness and aggressiveness of cancer. It is a vital prognostic factor in clinical staging of the disease and therapeutic decision-making. Patients with positive metastatic lymph nodes are likely to develop recurrent disease, distant metastasis, and succumb to death in the coming few years. Lymph node dissection and histological analysis are needed to detect whether regional lymph nodes have been infiltrated by cancer cells and determine the likely outcome of treatment and the patient's chances of survival. However, these procedures are invasive, and tissue biopsies are prone to sampling error. In recent years, advanced molecular imaging with novel imaging probes has provided new technologies that are contributing to comprehensive management of cancer, including non-invasive investigation of lymphatic drainage from tumors, identifying metastatic lymph nodes, and guiding surgeons to operate efficiently in patients with complex lesions. In this review, first, we outline the current status of different molecular imaging modalities applied for lymph node metastasis management. Second, we summarize the multi-functional imaging probes applied with the different imaging modalities as well as applications of cancer lymph node metastasis from preclinical studies to clinical translations. Third, we describe the limitations that must be considered in the field of molecular imaging for improved detection of lymph node metastasis. Finally, we propose future directions for molecular imaging technology that will allow more personalized treatment plans for patients with lymph node metastasis.

Molecular Imaging for Early-Stage Disease Diagnosis.

With the development of cellular biology, molecular biology, and other subjects, targeted molecular probe was combined with medical imaging technologies to launch a new scientific discipline of molecular imaging that is a research discipline to visualize, characterize, and analyze biological process at the cellular and molecular levels for real-time tracking and precision therapy, also termed as the medical imaging in the twenty-first century. An array of imaging techniques has been developed to image specific targets of living cells or tissues by molecular probes, including optical molecular imaging (OI), magnetic resonance molecular imaging, ultrasound (US) molecular imaging, nuclear medicine molecular imaging, X-ray molecular imaging, and multi-mode molecular imaging. These imaging techniques make the early diagnosis of various diseases possible by means of visualization of gene expression, interactions between proteins, signal transduction, cell metabolism, cell traces, and other physiological or pathological processes in the living system, which bridge the gap between molecular biology and clinical medicine. This chapter will lay the emphasis on the early-stage diagnosis of fatal diseases, such as malignant tumors, cardio- or cerebrovascular diseases, digestive system disease, central nervous system disease, and other diseases employing molecular imaging in a real-time visualized manner.

Molecular Design of Magnetic Resonance Imaging Agents Binding to Amyloid Deposits.

The ability to detect and monitor amyloid deposition in the brain using non-invasive imaging techniques provides valuable insights into the early diagnosis and progression of Alzheimer's disease and helps to evaluate the efficacy of potential treatments. Magnetic resonance imaging (MRI) is a widely available technique offering high-spatial-resolution imaging. It can be used to visualize amyloid deposits with the help of amyloid-binding diagnostic agents injected into the body. In recent years, a number of amyloid-targeted MRI probes have been developed, but none of them has entered clinical practice. We review the advances in the field and deduce the requirements for the molecular structure and properties of a diagnostic probe candidate. These requirements make up the base for the rational design of MRI-active small molecules targeting amyloid deposits. Particular attention is paid to the novel cryo-EM structures of the fibril aggregates and their complexes, with known binders offering the possibility to use computational structure-based design methods. With continued research and development, MRI probes may revolutionize the diagnosis and treatment of neurodegenerative diseases, ultimately improving the lives of millions of people worldwide.

Radiolabeled Liposomes for Nuclear Imaging Probes.

Quantitative nuclear imaging techniques are in high demand for various disease diagnostics and cancer theranostics. The non-invasive imaging modality requires radiotracing through the radioactive decay emission of the radionuclide. Current preclinical and clinical radiotracers, so-called nuclear imaging probes, are radioisotope-labeled small molecules. Liposomal radiotracers have been rapidly developing as novel nuclear imaging probes. The physicochemical properties and structural characteristics of liposomes have been elucidated to address their long circulation and stability as radiopharmaceuticals. Various radiolabeling methods for synthesizing radionuclides onto liposomes and synthesis strategies have been summarized to render them biocompatible and enable specific targeting. Through a variety of radionuclide labeling methods, radiolabeled liposomes for use as nuclear imaging probes can be obtained for in vivo biodistribution and specific targeting studies. The advantages of radiolabeled liposomes including their use as potential clinical nuclear imaging probes have been highlighted. This review is a comprehensive overview of all recently published liposomal SPECT and PET imaging probes.

Fluorinated Human Serum Albumin as Potential 19F Magnetic Resonance Imaging Probe.

Fluorinated human serum albumin conjugates were prepared and tested as potential metal-free probes for 19F magnetic resonance imaging (MRI). Each protein molecule was modified by several fluorine-containing compounds via the N-substituted natural acylating reagent homocysteine thiolactone. Albumin conjugates retain the protein's physical and biological properties, such as its 3D dimensional structure, aggregation ability, good solubility, proteolysis efficiency, biocompatibility, and low cytotoxicity. A dual-labeled with cyanine 7 fluorescence dye and fluorine reporter group albumin were synthesized for simultaneous fluorescence imaging and 19F MRI. The preliminary in vitro studies show the prospects of albumin carriers for multimodal imaging.

Expanding the Range of Bioorthogonal Tags for Multiplex Stimulated Raman Scattering Microscopy.

Multiplex optical detection in live cells is challenging due to overlapping signals and poor signal-to-noise associated with some chemical reporters. To address this, the application of spectral phasor analysis to stimulated Raman scattering (SRS) microscopy for unmixing three bioorthogonal Raman probes within cells is reported. Triplex detection of a metallacarborane using the B-H stretch at 2480-2650 cm-1 , together with a bis-alkyne and deuterated fatty acid can be achieved within the cell-silent region of the Raman spectrum. When coupled to imaging in the high-wavenumber region of the cellular Raman spectrum, nine discrete regions of interest can be spectrally unmixed from the hyperspectral SRS dataset, demonstrating a new capability in the toolkit of multiplexed Raman imaging of live cells.

Molecular imaging of cellular immunotherapies in experimental and therapeutic settings.

Cell-based cancer immunotherapies are becoming a routine part of the armamentarium against cancer. While remarkable successes have been seen, including durable remissions, not all patients will benefit from these therapies and many can suffer from life-threatening side effects. These differences in efficacy and safety across patients and across tumor types (e.g., blood vs. solid), are thought to be due to differences in how well the immune cells traffic to their target tissue (e.g., tumor, lymph nodes, etc.) whilst avoiding non-target tissues. Across patient variability can also stem from whether the cells interact with (i.e., communicate with) their intended target cells (e.g., cancer cells), as well as if they proliferate and survive long enough to yield potent and long-lasting therapeutic effects. However, many cell-based therapies are monitored by relatively simple blood tests that lack any spatial information and do not reflect how many immune cells have ended up at particular tissues. The ex vivo labeling and imaging of infused therapeutic immune cells can provide a more precise and dynamic understanding of whole-body immune cell biodistribution, expansion, viability, and activation status in individual patients. In recent years numerous cellular imaging technologies have been developed that may provide this much-needed information on immune cell fate. For this review, we summarize various ex vivo labeling and imaging approaches that allow for tracking of cellular immunotherapies for cancer. Our focus is on clinical imaging modalities and summarize the progression from experimental to therapeutic settings. The imaging information provided by these technologies can potentially be used for many purposes including improved real-time understanding of therapeutic efficacy and potential side effects in individual patients after cell infusion; the ability to more readily compare new therapeutic cell designs to current designs for various parameters such as improved trafficking to target tissues and avoidance of non-target tissues; and the long-term ability to identify patient populations that are likely to be positive responders and at low-risk of side effects.

Modulation of Cellular Fate of Vinyl Triarylamines through Structural Fine Tuning: To Stay or Not To Stay in the Mitochondria?

Mitochondria are involved in many cellular pathways and dysfunctional mitochondria are linked to various diseases. Hence efforts have been made to design mitochondria-targeted fluorophores for monitoring the mitochondrial status. However, the factors that govern the mitochondria-targeted potential of dyes are not well-understood. In this context, we synthesized analogues of the TP-2Bzim probe belonging to the vinyltriphenylamine (TPA) class and already described for its capacity to bind nuclear DNA in fixed cells and mitochondria in live cells. These analogues (TP-1Bzim, TPn -2Bzim, TP1+ -2Bzim, TN-2Bzim) differ in the cationic charge, the number of vinylbenzimidazolium branches and the nature of the triaryl core. Using microscopy, we demonstrated that the cationic derivatives accumulate in mitochondria but do not reach mtDNA. Under depolarisation of the mitochondrial membrane, TP-2Bzim and TP1+ -2Bzim translocate to the nucleus in direct correlation with their strong DNA affinity. This reversible phenomenon emphasizes that these probes can be used to monitor ΔΨm variations.

Engineered TALE Repeats for Enhanced Imaging-Based Analysis of Cellular 5-Methylcytosine.

Transcription-activator-like effectors (TALEs) are repeat-based, programmable DNA-binding proteins that can be engineered to recognize sequences of canonical and epigenetically modified nucleobases. Fluorescent TALEs can be used for the imaging-based analysis of cellular 5-methylcytosine (5 mC) in repetitive DNA sequences. This is based on recording fluorescence ratios from cell co-stains with two TALEs: an analytical TALE targeting the cytosine (C) position of interest through a C-selective repeat that is blocked by 5 mC, and a control TALE targeting the position with a universal repeat that binds both C and 5 mC. To enhance this approach, we report herein the development of novel 5 mC-selective repeats and their integration into TALEs that can replace universal TALEs in imaging-based 5 mC analysis, resulting in a methylation-dependent response of both TALEs. We screened a library of size-reduced repeats and identified several 5 mC binders. Compared to the 5 mC-binding repeat of natural TALEs and to the universal repeat, two repeats containing aromatic residues showed enhancement of 5 mC binding and selectivity in cellular transcription activation and electromobility shift assays, respectively. In co-stains of cellular SATIII DNA with a corresponding C-selective TALE, this selectivity results in a positive methylation response of the new TALE, offering perspectives for studying 5 mC functions in chromatin regulation by in situ imaging with increased dynamic range.

Imaging NF-κB activity in a murine model of early stage diabetes.

Early pro-inflammatory signaling in the endocrine pancreas involves activation of NF-κB, which is believed to be important for determining the ultimate fate of ß-cells and hence progression of type 1 diabetes (T1D). Thus, early non-invasive detection of NF-κB in pancreatic islets may serve as a potential strategy for monitoring early changes in pancreatic endocrine cells eventually leading to T1D. We investigated the feasibility of optical imaging of NF-κB transcription factor activation induced by low-dose streptozocin (LD-STZ) treatment in the immunocompetent SKH1 mouse model of early stage diabetes. In this model, we showed that the levels of NF-κB may be visualized and measured by fluorescence intensity of specific near-infrared (NIR) fluorophore-labeled oligodeoxyribonucleotide duplex (ODND) probes. In addition, NF-κB activation following LD-STZ treatment was validated using immunofluorescence and transgenic animals expressing NF-κB inducible imaging reporter. We showed that LD-STZ-treated SKH1 mice had significantly higher (2-3 times, P < .01) specific NIR FI in the nuclei and cytoplasm of islets cells than in non-treated control mice and this finding was corroborated by immunoblotting and electrophoretic mobility shift assays. Finally, using semi-quantitative confocal analysis of non-fixed pancreatic islet microscopy we demonstrated that ODND probes may be used to distinguish between the islets with high levels of NF-κB transcription factor and control islet cells.

Microwave Non-Destructive Testing for Delamination Detection in Layered Composite Pipelines.

Microwave imaging and defectoscopy are promising techniques for dielectric composite evaluation. Their most significant advantage is their relatively high penetration depth. Another feature worth noting is that traditional methods could not acquire an internal content with such a low impact on both the sample and surrounding environment, including the test operator, compared to other techniques. This paper presents microwave non-destructive and noninvasive methods for quality evaluation of layered composite materials using an open-ended waveguide probe. Pure |S11| parameters only exceptionally give a clear answer about the location of material cracks. Therefore, this makes it necessary to analyze these parameters simultaneously along with several other factors, such as stand-off distance, probe type or wave polarization. The purpose of the work was to find the dependency between the physical state of a layered composite powerplant pipeline and the S-matrix parameters response (reflection and transmission parameters) in a Ku frequency band that has not yet been extensively researched. Lower-frequency measurements broaden the application possibility for thicker composites, mainly because of a higher penetration depth and measurement setup availability. Different methods have been shown, including reflection and transmission/reflection methods, both in close proximity and in stand-off distance. The measurements are based on a low-complexity experimental setup.

Fluorination Triggers Fluoroalkylation: Nucleophilic Perfluoro-tert-butylation with 1,1-Dibromo-2,2-bis(trifluoromethyl)ethylene (DBBF) and CsF.

Perfluoro-tert-butylation reaction has long remained a challenging task. We now report the use of 1,1-dibromo-2,2-bis(trifluoromethyl)ethylene (DBBF) as a practical reagent for perfluoro-tert-butylation reactions for the first time. Through a consecutive triple-fluorination process with DBBF and CsF, the (CF3 )3 C- species can be liberated and observed, which is able to serve as a robust nucleophilic perfluoro-tert-butylating agent for various electrophiles. The power of this synthetic protocol is evidenced by the efficient synthesis of structurally diverse perfluoro-tert-butylated molecules. Multiple applications demonstrate the practicability of this method, as well as the superiority of perfluoro-tert-butylated compounds as sensitive probes. The perfluoro-tert-butylated product was successfully applied in 1 H- and 19 F-magnetic resonance imaging (MRI) experiment with an ultra-low field (ULF) MRI system.

Multifunctional Magnetic Resonance Imaging Probes.

Magnetic resonance imaging is characterized by high spatial resolution and unsurpassed soft tissue discrimination. Development and characterization of both intrinsic and extrinsic magnetic resonance (MR) imaging probes in the last decade has further strengthened the pivotal role MR imaging holds in the assessment of cancer in preclinical and translational settings. Sophisticated chemical modifications of a variety of nanoparticulate probes hold the potential to deliver valuable multifunctional tools applicable in diagnostics and/or treatment in human oncology. MR imaging suffers from a lack of sensitivity achievable by, e.g., nuclear medicine imaging methods. Advantages of including additional functionality/functionalities in a probe suitable for MR imaging are thus numerous, comprising the addition of fundamentally different imaging information (diagnostics), drug delivery (therapy), or the combination of both (theranostics). In recent years, we have witnessed a plethora of preclinical multimodal or multifunctional imaging probes being published mainly as proof-of-principle studies, yet only a handful are readily applicable in clinical settings. This chapter summarizes recent innovations in the development of multifunctional MR imaging probes and discusses the suitability of these probes for clinical transfer.

Advances in the development of imaging probes and aggregation inhibitors for alpha-synuclein.

Abnormal protein aggregation has been linked to many neurodegenerative diseases, including Parkinson's disease (PD). The main pathological hallmark of PD is the formation of Lewy bodies (LBs) and Lewy neurites, both of which contain the presynaptic protein alpha-synuclein (α-syn). Under normal conditions, native α-syn exists in a soluble unfolded state but undergoes misfolding and aggregation into toxic aggregates under pathological conditions. Toxic α-syn species, especially oligomers, can cause oxidative stress, membrane penetration, synaptic and mitochondrial dysfunction, as well as other damage, leading to neuronal death and eventually neurodegeneration. Early diagnosis and treatments targeting PD pathogenesis are urgently needed. Given its critical role in PD, α-syn is an attractive target for the development of both diagnostic tools and effective therapeutics. This review summarizes the progress toward discovering imaging probes and aggregation inhibitors for α-syn. Relevant strategies and techniques in the discovery of α-syn-targeted drugs are also discussed.

Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers for In Situ Live-Cell Molecular Imaging of Dengue Virus Replication.

Current methods to detect and monitor pathogens in biological systems are largely limited by the tradeoffs between spatial context and temporal detail. A new generation of molecular tracking that provides both information simultaneously involves in situ detection coupled with non-invasive imaging. An example is antisense imaging that uses antisense oligonucleotide probes complementary to a target nucleotide sequence. In this study, we explored the potential of repurposing antisense oligonucleotides initially developed as antiviral therapeutics as molecular probes for imaging of viral infections in vitro and in vivo. We employed nuclease-resistant phosphorodiamidate synthetic oligonucleotides conjugated with cell-penetrating peptides (i.e., PPMOs) previously established as antivirals for dengue virus serotype-2 (DENV2). As proof of concept, and before further development for preclinical testing, we evaluated its validity as in situ molecular imaging probe for tracking cellular DENV2 infection using live-cell fluorescence imaging. Although the PPMO was designed to specifically target the DENV2 genome, it was unsuitable as in situ molecular imaging probe. This study details our evaluation of the PPMOs to assess specific and sensitive molecular imaging of DENV2 infection and tells a cautionary tale for those exploring antisense oligonucleotides as probes for non-invasive imaging and monitoring of pathogen infections in experimental animal models.

PET Radiopharmaceuticals for Alzheimer's Disease and Parkinson's Disease Diagnosis, the Current and Future Landscape.

Ironically, population aging which is considered a public health success has been accompanied by a myriad of new health challenges, which include neurodegenerative disorders (NDDs), the incidence of which increases proportionally to age. Among them, Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common, with the misfolding and the aggregation of proteins being common and causal in the pathogenesis of both diseases. AD is characterized by the presence of hyperphosphorylated τ protein (tau), which is the main component of neurofibrillary tangles (NFTs), and senile plaques the main component of which is ß-amyloid peptide aggregates (Aß). The neuropathological hallmark of PD is α-synuclein aggregates (α-syn), which are present as insoluble fibrils, the primary structural component of Lewy body (LB) and neurites (LN). An increasing number of non-invasive PET examinations have been used for AD, to monitor the pathological progress (hallmarks) of disease. Notwithstanding, still the need for the development of novel detection tools for other proteinopathies still remains. This review, although not exhaustively, looks at the timeline of the development of existing tracers used in the imaging of Aß and important moments that led to the development of these tracers.

Designer Receptors for Nucleotide-Resolution Analysis of Genomic 5-Methylcytosine by Cellular Imaging.

We report programmable receptors for the imaging-based analysis of 5-methylcytosine (5mC) in user-defined DNA sequences of single cells. Using fluorescent transcription-activator-like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co-stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation in situ with nucleotide, locus, and cell resolution.

A Multi-responsive Fluorescent Probe Reveals Mitochondrial Nucleoprotein Dynamics with Reactive Oxygen Species Regulation through Super-resolution Imaging.

Understanding the biomolecular interactions in a specific organelle has been a long-standing challenge because it requires super-resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties are the scarcity of suitable probes for super-resolution nanoscopy and the complications that arise from the use of multiple probes. Herein, we report a quinolinium derivative probe that is selectively enriched in mitochondria and switches on in three different fluorescence modes in response to hydrogen peroxide (H2 O2 ), proteins, and nucleic acids, enabling the visualization of mitochondrial nucleoprotein dynamics. STED nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H2 O2 level leads to disassociation of nucleic acid-protein complexes.