Next generation single-molecule techniques: Imaging, labeling, and manipulation in vitro and in cellulo.

Owing to their unique abilities to manipulate, label, and image individual molecules in vitro and in cellulo, single-molecule techniques provide previously unattainable access to elementary biological processes. In imaging, single-molecule fluorescence resonance energy transfer (smFRET) and protein-induced fluorescence enhancement in vitro can report on conformational changes and molecular interactions, single-molecule pull-down (SiMPull) can capture and analyze the composition and function of native protein complexes, and single-molecule tracking (SMT) in live cells reveals cellular structures and dynamics. In labeling, the abilities to specifically label genomic loci, mRNA, and nascent polypeptides in cells have uncovered chromosome organization and dynamics, transcription and translation dynamics, and gene expression regulation. In manipulation, optical tweezers, integration of single-molecule fluorescence with force measurements, and single-molecule force probes in live cells have transformed our mechanistic understanding of diverse biological processes, ranging from protein folding, nucleic acids-protein interactions to cell surface receptor function.

Technological advances in super-resolution microscopy to study cellular processes.

Since its initial demonstration in 2000, far-field super-resolution light microscopy has undergone tremendous technological developments. In parallel, these developments have opened a new window into visualizing the inner life of cells at unprecedented levels of detail. Here, we review the technical details behind the most common implementations of super-resolution microscopy and highlight some of the recent, promising advances in this field.

The new era of quantitative cell imaging-challenges and opportunities.

Quantitative optical microscopy-an emerging, transformative approach to single-cell biology-has seen dramatic methodological advancements over the past few years. However, its impact has been hampered by challenges in the areas of data generation, management, and analysis. Here we outline these technical and cultural challenges and provide our perspective on the trajectory of this field, ushering in a new era of quantitative, data-driven microscopy. We also contrast it to the three decades of enormous advances in the field of genomics that have significantly enhanced the reproducibility and wider adoption of a plethora of genomic approaches.

Altered substrate metabolism in neurodegenerative disease: new insights from metabolic imaging.

Neurodegenerative diseases (NDs), such as Alzheimer's disease (AD), Parkinson's disease (PD) and multiple sclerosis (MS), are relatively common and devastating neurological disorders. For example, there are 6 million individuals living with AD in the United States, a number that is projected to grow to 14 million by the year 2030. Importantly, AD, PD and MS are all characterized by the lack of a true disease-modifying therapy that is able to reverse or halt disease progression. In addition, the existing standard of care for most NDs only addresses the symptoms of the disease. Therefore, alternative strategies that target mechanisms underlying the neuropathogenesis of disease are much needed. Recent studies have indicated that metabolic alterations in neurons and glia are commonly observed in AD, PD and MS and lead to changes in cell function that can either precede or protect against disease onset and progression. Specifically, single-cell RNAseq studies have shown that AD progression is tightly linked to the metabolic phenotype of microglia, the key immune effector cells of the brain. However, these analyses involve removing cells from their native environment and performing measurements in vitro, influencing metabolic status. Therefore, technical approaches that can accurately assess cell-specific metabolism in situ have the potential to be transformative to our understanding of the mechanisms driving AD. Here, we review our current understanding of metabolism in both neurons and glia during homeostasis and disease. We also evaluate recent advances in metabolic imaging, and discuss how emerging modalities, such as fluorescence lifetime imaging microscopy (FLIM) have the potential to determine how metabolic perturbations may drive the progression of NDs. Finally, we propose that the temporal, regional, and cell-specific characterization of brain metabolism afforded by FLIM will be a critical first step in the rational design of metabolism-focused interventions that delay or even prevent NDs.

Evolution of photodynamic medicine based on fluorescence image-guided diagnosis using indocyanine green and 5-aminolevulinic acid.

New diagnostic techniques based on photodynamic medicine, such as near-infrared fluorescence using indocyanine green (NIR-ICG) and 5-aminolevulinic acid-mediated photodynamic diagnosis (ALA-PDD), are aiding navigation tasks across various fields of surgery. Specifically, NIR-ICG is being used for the intraoperative identification of sentinel lymph nodes or blood vessels in organ resection and for blood flow evaluation in surgery. These ICG-fluorescent imaging techniques could provide an additional and potentially valuable way to identify vascular and lymphatic structures in surrounding tissue. 5-Aminolevulinic acid is a precursor of a photosensitizing substance with affinity for tumors; thus, diagnostic laparoscopy using ALA-PDD in combination should improve the accuracy of detecting peritoneal dissemination in patients with advanced gastric cancer. The ability to overlay fluorescent imaging with conventional color images in real time using ALA-PDD and NIR with ICG would be of immense benefit to surgeons, providing good visualization and detection of target lesions not seen with the naked eye. A multi-center clinical study examining the safety and efficacy of ALA-PDD during laparoscopic examination for patients with advanced gastric cancer is currently underway in the form of doctor-initiated trials, and further verification studies will be conducted. Such imaging capability could have broad potential across cancer and vascular surgery.

Recent Advances and the Potential for Clinical Use of Autofluorescence Detection of Extra-Ophthalmic Tissues.

The autofluorescence (AF) characteristics of endogenous fluorophores allow the label-free assessment and visualization of cells and tissues of the human body. While AF imaging (AFI) is well-established in ophthalmology, its clinical applications are steadily expanding to other disciplines. This review summarizes clinical advances of AF techniques published during the past decade. A systematic search of the MEDLINE database and Cochrane Library databases was performed to identify clinical AF studies in extra-ophthalmic tissues. In total, 1097 articles were identified, of which 113 from internal medicine, surgery, oral medicine, and dermatology were reviewed. While comparable technological standards exist in diabetology and cardiology, in all other disciplines, comparability between studies is limited due to the number of differing AF techniques and non-standardized imaging and data analysis. Clear evidence was found for skin AF as a surrogate for blood glucose homeostasis or cardiovascular risk grading. In thyroid surgery, foremost, less experienced surgeons may benefit from the AF-guided intraoperative separation of parathyroid from thyroid tissue. There is a growing interest in AF techniques in clinical disciplines, and promising advances have been made during the past decade. However, further research and development are mandatory to overcome the existing limitations and to maximize the clinical benefits.

New observations in neuroscience using superresolution microscopy.

Superresolution microscopy (SM) techniques are among the revolutionary methods for molecular and cellular observations in the 21st century. SM techniques overcome optical limitations, and several new observations using SM lead us to expect these techniques to have a large impact on neuroscience in the near future. Several types of SM have been developed, including structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), and photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM), each with special features. In this Minisymposium, experts in these different types of SM discuss the new structural and functional information about specific important molecules in neuroscience that has been gained with SM. Using these techniques, we have revealed novel mechanisms of endocytosis in nerve growth, fusion pore dynamics, and described quantitative new properties of excitatory and inhibitory synapses. Additional powerful techniques, including single molecule-guided Bayesian localization SM (SIMBA) and expansion microscopy (ExM), alone or combined with super-resolution observation, are also introduced in this session.

WorMachine: machine learning-based phenotypic analysis tool for worms.

BACKGROUND: Caenorhabditis elegans nematodes are powerful model organisms, yet quantification of visible phenotypes is still often labor-intensive, biased, and error-prone. We developed WorMachine, a three-step MATLAB-based image analysis software that allows (1) automated identification of C. elegans worms, (2) extraction of morphological features and quantification of fluorescent signals, and (3) machine learning techniques for high-level analysis. RESULTS: We examined the power of WorMachine using five separate representative assays: supervised classification of binary-sex phenotype, scoring continuous-sexual phenotypes, quantifying the effects of two different RNA interference treatments, and measuring intracellular protein aggregation. CONCLUSIONS: WorMachine is suitable for analysis of a variety of biological questions and provides an accurate and reproducible analysis tool for measuring diverse phenotypes. It serves as a "quick and easy," convenient, high-throughput, and automated solution for nematode research.

Measuring embryo metabolism to predict embryo quality.

Measuring the metabolism of early embryos has the potential to be used as a prospective marker for post-transfer development, either alone or in conjunction with other embryo quality assessment tools. This is necessary to maximise the opportunity of couples to have a healthy child from assisted reproduction technology (ART) and for livestock breeders to efficiently improve the genetics of their animals. Nevertheless, although many promising candidate substrates (e.g. glucose uptake) and methods (e.g. metabolomics using different spectroscopic techniques) have been promoted as viability markers, none has yet been widely used clinically or in livestock production. Herein we review the major techniques that have been reported; these are divided into indirect techniques, where measurements are made from the embryo's immediate microenvironment, or direct techniques that measure intracellular metabolic activity. Both have strengths and weaknesses, the latter ruling out some from contention for use in human ART, but not necessarily for use in livestock embryo assessment. We also introduce a new method, namely multi- (or hyper-) spectral analysis, which measures naturally occurring autofluorescence. Several metabolically important molecules have fluorescent properties, which we are pursuing in conjunction with improved image analysis as a viable embryo quality assessment methodology.

White Paper AGA: Advanced Imaging in Barrett's Esophagus.

Enhanced imaging technologies such as narrow band imaging, flexible spectral imaging color enhancement, i-Scan, confocal laser endomicroscopy, and optical coherence tomography are readily available for use by endoscopists in routine clinical practice. In November 2014, the American Gastroenterological Association's Center for GI Innovation and Technology conducted a 2-day workshop to discuss endoscopic image enhancement technologies, focusing on their role in 2 specific clinical conditions (colon polyps and Barrett's esophagus) and on issues relating to training and implementation of these technologies (white papers). Although the majority of the studies that use enhanced imaging technologies have been positive, these techniques ideally need to be validated in larger cohorts and in community centers. As it stands today, detailed endoscopic examination with high-definition white-light endoscopy and random 4-quadrant biopsy remains the standard of care. However, the workshop panelists agreed that in the hands of endoscopists who have met the preservation and incorporation of valuable endoscopic innovation thresholds (diagnostic accuracy) with enhanced imaging techniques (specific technologies), use of the technique in Barrett's esophagus patients is appropriate.

Optical innovations in surgery.

BACKGROUND: In the past decade, there has been a major drive towards clinical translation of optical and, in particular, fluorescence imaging in surgery. In surgical oncology, radical surgery is characterized by the absence of positive resection margins, a critical factor in improving prognosis. Fluorescence imaging provides the surgeon with reliable and real-time intraoperative feedback to identify surgical targets, including positive tumour margins. It also may enable decisions on the possibility of intraoperative adjuvant treatment, such as brachytherapy, chemotherapy or emerging targeted photodynamic therapy (photoimmunotherapy). METHODS: This article reviews the use of optical imaging for intraoperative guidance and decision-making. RESULTS: Image-guided cancer surgery has the potential to be a powerful tool in guiding future surgical care. Photoimmunotherapy is a theranostic concept (simultaneous diagnosis and treatment) on the verge of clinical translation, and is highlighted as an effective combination of image-guided surgery and intraoperative treatment of residual disease. Multispectral optoacoustic tomography, a technique complementary to optical image-guided surgery, is currently being tested in humans and is anticipated to have great potential for perioperative and postoperative application in surgery. CONCLUSION: Significant advances have been achieved in real-time optical imaging strategies for intraoperative tumour detection and margin assessment. Optical imaging holds promise in achieving the highest percentage of negative surgical margins and in early detection of micrometastastic disease over the next decade.

Advanced endoscopic imaging: a review of commercially available technologies.

The rapid strides made in innovative endoscopic technology to improve mucosal visualization have revolutionized endoscopy. Improved lesion detection has allowed the modern endoscopist to provide real-time optical diagnosis. Improvements in image resolution, software processing, and optical filter technology have resulted in the commercial availability of high-definition endoscopy as well as optical contrast techniques such as narrow-band imaging, flexible spectral imaging color enhancement, and i-scan. Along with autofluorescence imaging and confocal laser endomicroscopy, these techniques have complemented and enhanced traditional white light endoscopy. They have the potential to serve as red-flag techniques to improve detection of mucosal abnormalities as well as allow optical diagnosis and virtual histology of detected lesions. This review will focus on these emerging commercially available technologies and aims to provide an overview of the technologies, their clinical applicability, and current status.

Ophthalmic imaging.

INTRODUCTION OR BACKGROUND: The last two decades have seen a revolution in ophthalmic imaging. In this review we present an overview of the breadth of ophthalmic imaging modalities in use today and describe how the role of ophthalmic imaging has changed from documenting abnormalities visible on clinical examination to the detection of clinically silent abnormalities which can lead to an earlier and more precise diagnosis. SOURCES OF DATA: This review is based on published literature in the fields of ophthalmic imaging and with focus on most commonly used imaging modalities. AREAS OF AGREEMENT: New imaging techniques enable non-invasive evaluation of ocular structures at a resolution of a few micrometres. This has led to a re-evaluation of diagnostic criteria for ocular disease, which were previously defined by clinical findings without significant reference to imaging. AREAS OF CONTROVERSY: Lack of formal training and clinical guidelines regarding use of new imaging techniques in diagnosing and monitoring various ocular conditions. Lack of large normative databases and interchangeability issues between different commercial machines can hinder the detection of disease progression. GROWING POINTS: Imaging devices are being constantly refined with improved image capture and image analysis tools. AREAS TIMELY FOR DEVELOPING RESEARCH: Clinical applications of new techniques and devices have yet to be determined using systematic scientific research methods.

Optical methods for single molecule detection and analysis.

As analytical chemists, the highest resolution measurement one can make is at the single molecule level; it just does not get any better than that. To determine the concentration of a molecule in solution, the best way is to count the number of molecules in a given volume. As long as the volume contains a statistically large enough number of molecules and is above the Poisson noise limit, molecular counting is the most accurate way to make a measurement. Molecular counting is the method of the future and is beginning to be performed today.

Imaging methods in mechanosensing: a historical perspective and visions for the future.

Over the past three decades, as mechanobiology has become a distinct area of study, researchers have developed novel imaging tools to discover the pathways of biomechanical signaling. Early work with substrate engineering and particle tracking demonstrated the importance of cell-extracellular matrix interactions on the cell cycle as well as the mechanical flux of the intracellular environment. Most recently, tension sensor approaches allowed directly measuring tension in cell-cell and cell-substrate interactions. We retrospectively analyze how these various optical techniques progressed the field and suggest our vision forward for a unified theory of cell mechanics, mapping cellular mechanosensing, and novel biomedical applications for mechanobiology.

Cardiac optogenetics: a decade of enlightenment.

The electromechanical function of the heart involves complex, coordinated activity over time and space. Life-threatening cardiac arrhythmias arise from asynchrony in these space-time events; therefore, therapies for prevention and treatment require fundamental understanding and the ability to visualize, perturb and control cardiac activity. Optogenetics combines optical and molecular biology (genetic) approaches for light-enabled sensing and actuation of electrical activity with unprecedented spatiotemporal resolution and parallelism. The year 2020 marks a decade of developments in cardiac optogenetics since this technology was adopted from neuroscience and applied to the heart. In this Review, we appraise a decade of advances that define near-term (immediate) translation based on all-optical electrophysiology, including high-throughput screening, cardiotoxicity testing and personalized medicine assays, and long-term (aspirational) prospects for clinical translation of cardiac optogenetics, including new optical therapies for rhythm control. The main translational opportunities and challenges for optogenetics to be fully embraced in cardiology are also discussed.

Recent progress in development and applications of second near-infrared (NIR-II) nanoprobes.

Optical probes for near-infrared (NIR) light have clear advantages over UV/VIS-based optical probes, such as their low levels of interfering auto-fluorescence and high tissue penetration. The second NIR (NIR-II) window (1000-1350 nm) offers better light penetration, lower background signal, higher safety limit, and higher maximum permitted exposure than the first NIR (NIR-I) window (650-950 nm). Therefore, NIR-II laser-based photoacoustic (PA) and fluorescence (FL) imaging can offer higher sensitivity and penetration depth than was previously available, and deeper lesions can be treated in vivo by photothermal therapy (PTT) and photodynamic therapy (PDT) with an NIR-II laser than with an NIR-I laser. Advances in creation of novel nanomaterials have increased options for improving light-induced bioimaging and treatment. Nanotechnology can provide advantages such as good disease targeting ability and relatively long circulation times to supplement the advantages of optical technologies. In this review, we present recent progress in development and applications of NIR-II light-based nanoplatforms for FL, PA, image-guided surgery, PDT, and PTT. We also discuss recent advances in smart NIR-II nanoprobes that can respond to stimuli in the tumor microenvironment and inflamed sites. Finally, we consider the challenges involved in using NIR-II nanomedicine for effective diagnosis and treatment.