Multispectral imaging fluorescence microscopy for living cells

Multispectral imaging technologies have been widely used in fields of astronomy and remote sensing. Interdisciplinary approaches developed in, for example, the National Aeronautics and Space Administration (NASA, USA), the Jet Propulsion Laboratory (JPL, USA), or the Communications Research Laboratory (CRL, Japan) have extended the application areas of these technologies from planetary systems to cellular systems. Here we overview multispectral imaging systems that have been devised for microscope applications. We introduce these systems with particular interest in live cell imaging. Finally we demonstrate examples of spectral imaging of living cells using commercially available systems with no need for user engineering.     

The changing point-spread function: single-molecule-based super-resolution imaging

Over the past decade, many techniques for imaging systems at a resolution greater than the diffraction limit have been developed. These methods have allowed systems previously inaccessible to fluorescence microscopy to be studied and biological problems to be solved in the condensed phase. This brief review explains the basic principles of super-resolution imaging in both two and three dimensions, summarizes recent developments, and gives examples of how these techniques have been used to study complex biological systems.     

Targeted molecular imaging of angiogenesis in PET and SPECT: a review

Over the past few decades, there have been significant advancements in the imaging techniques of positron emission tomography (PET) and single photon emission tomography (SPECT). These changes have allowed for the targeted imaging of cellular processes and the development of hybrid imaging systems (e.g., SPECT/CT and PET/CT), which provide both functional and structural images of biological systems. One area that has garnered particular attention is angiogenesis as it relates to ischemic heart disease and limb ischemia. Though the aforementioned techniques have benefits and consequences, they enable scientists and clinicians to identify regions that are vulnerable to or have been exposed to ischemic injury via non-invasive means. This literature review highlights the advancements in molecular imaging techniques and specific probes as they pertain to the process of angiogenesis in cardiovascular disease.     

Stereoscopic Integrated Imaging Goggles for Multimodal Intraoperative Image Guidance

We have developed novel stereoscopic wearable multimodal intraoperative imaging and display systems entitled Integrated Imaging Goggles for guiding surgeries. The prototype systems offer real time stereoscopic fluorescence imaging and color reflectance imaging capacity, along with in vivo handheld microscopy and ultrasound imaging. With the Integrated Imaging Goggle, both wide-field fluorescence imaging and in vivo microscopy are provided. The real time ultrasound images can also be presented in the goggle display. Furthermore, real time goggle-to-goggle stereoscopic video sharing is demonstrated, which can greatly facilitate telemedicine. In this paper, the prototype systems are described, characterized and tested in surgeries in biological tissues ex vivo. We have found that the system can detect fluorescent targets with as low as 60 nM indocyanine green and can resolve structures down to 0.25 mm with large FOV stereoscopic imaging. The system has successfully guided simulated cancer surgeries in chicken. The Integrated Imaging Goggle is novel in 4 aspects: it is (a) the first wearable stereoscopic wide-field intraoperative fluorescence imaging and display system, (b) the first wearable system offering both large FOV and microscopic imaging simultaneously, (c) the first wearable system that offers both ultrasound imaging and fluorescence imaging capacities, and (d) the first demonstration of goggle-to-goggle communication to share stereoscopic views for medical guidance.     

A review of high-resolution X-ray computed tomography and other imaging modalities for small animal research

Dedicated high-resolution small animal systems have recently emerged as important new tools for laboratory animal research. These imaging systems permit researchers to noninvasively screen animal models for mutations or pathologies and to monitor disease progression and response to therapy. The authors survey various small animal imaging modalities, including MRI, PET, SPECT, and microCT, and discuss several representative microCT mouse imaging studies.     

Imaging of chlorophyll a fluorescence: theoretical and practical aspects of an emerging technique for the monitoring of photosynthetic performance

The development of chlorophyll (Chl) a fluorescence imaging systems has greatly increased the versatility of Chl a fluorometry as a non-invasive technique for the investigation of photosynthesis in plants and algae. For example, systems that image at the microscopic level have made it possible to measure PSII photochemical efficiencies from chloroplasts within intact leaves and from individual algal cells within mixed populations, while systems that image over much larger areas have been used to investigate heterogeneous patterns of photosynthetic performance across leaves and in screening programmes that image tens or even hundreds of plants simultaneously. In addition, it is now practical to use fluorescence imaging systems as real-time, multi-channel fluorometers, which can be used to record continuous fluorescence traces from multiple leaves, plants, or algal cells. This paper discusses some of the theoretical and practical issues associated with the imaging of Chl a fluorescence and with Chl a fluorometry in general. This discussion includes a review of the most commonly used Chl a fluorescence parameters.     

Positron emission tomography: measurement of the activity of second messenger systems

Phosphoinositide turnover is closely connected to modulation of synaptic function and is part of an important second messenger-producing system. New radioligands for imaging second messenger systems by positron emission tomography have been developed: carbon-11-labeled 1,2-diacylglycerols. The theoretical background of second messenger imaging is described in detail and the relation between the biologically active compounds and potential tracers for imaging second messenger systems is discussed. We report informative findings on postsynaptic biological responses in the living human brain of healthy normal subjects and with various diseases.     

Magnetic resonance imaging in patients with ICDs and pacemakers

Magnetic resonance (MR) imaging has unparalleled soft-tissue imaging capabilities. The presence of devices such as pacemakers and implantable cardioverter/defibrillators (ICDs), however, is historically considered a contraindication to MR imaging. These devices are now smaller, with less magnetic material and improved electromagnetic interference protection. This review summarizes the potential hazards of the device-MR environment interaction, and presents updated information regarding in-vivo and in-vitro experiments. Recent reports on patients with implantable pacemakers and ICDs who underwent MR scan shows that under certain conditions patients with these implanted systems may benefit from this imaging modality. The data presented suggests that certain modern pacemaker and ICD systems may indeed be MR safe. This may have major clinical implications on current imaging practice.     

Protein microarray biosensors based on imaging ellipsometry techniques and their applications

After years of development, biosensors based on imaging ellipsometry and biosensors based on total internal reflection imaging ellipsometry have been successfully implemented in various engineering systems. Their experimental setups, detection principles, and biological and clinical applications are briefly reviewed.     

Over the rainbow: 25 years of confocal imaging

Confocal microscopes have evolved over the past 25 years from the early stage scanning systems to a collection of sophisticated laser scanning systems designed for a range of biomedical applications. Major improvements to the photon efficiency of the instrumentation coupled with the development of novel fluorescent reporters have enabled multidimensional imaging of living cells and tissues.     

Optical calcium imaging in the nervous system of Drosophila melanogaster

BACKGROUND: Drosophila melanogaster is one of the best-studied model organisms in biology, mainly because of the versatility of methods by which heredity and specific expression of genes can be traced and manipulated. Sophisticated genetic tools have been developed to express transgenes in selected cell types, and these techniques can be utilized to target DNA-encoded fluorescence probes to genetically defined subsets of neurons. Neuroscientists make use of this approach to monitor the activity of restricted types or subsets of neurons in the brain and the peripheral nervous system. Since membrane depolarization is typically accompanied by an increase in intracellular calcium ions, calcium-sensitive fluorescence proteins provide favorable tools to monitor the spatio-temporal activity across groups of neurons. SCOPE OF REVIEW: Here we describe approaches to perform optical calcium imaging in Drosophila in consideration of various calcium sensors and expression systems. In addition, we outline by way of examples for which particular neuronal systems in Drosophila optical calcium imaging have been used. Finally, we exemplify briefly how optical calcium imaging in the brain of Drosophila can be carried out in practice. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: Drosophila provides an excellent model organism to combine genetic expression systems with optical calcium imaging in order to investigate principles of sensory coding, neuronal plasticity, and processing of neuronal information underlying behavior. This article is part of a Special Issue entitled Biochemical, Biophysical and Genetic Approaches to Intracellular Calcium Signaling.     

Innovations technologiques récentes en détection pour la Tomographie par Emission de Positons

Since the recognition of the clinical value of Positron Emission Tomography (PET) for the diagnosis and staging of several cancers, the PET systems have evolved to systems associating PET and Computed Tomography (CT). The main constraint for clinical imaging is to reduce the acquisition duration. As a consequence, PET detectors are faster and emit more light than the BGO crystal used previously. These detectors allow an improvement of the count rate performance of the PET systems, reducing the scattered and the random events while increasing the true events at high activity concentration. Among the new crystals, some allow measuring the time of flight of the annihilation photons. This measurement further improves the performance of the systems. The spatial resolution of clinical PET systems is still equal to 5 mm at best. High spatial resolution PET systems dedicated to small animal imaging have been developed. These systems use similar crystal materials as the clinical systems. However, in order to permit spatial resolution close to 1 mm, the crystal elements have much smaller transverse dimensions than that of clinical systems. The detectors are compact using position sensitive photomultipliers or photodiodes. In order to preserve the uniformity of the spatial resolution over the transverse field of view of the tomographs, solutions allowing the measurement of the depth of interaction of the photons in the crystal have been designed. New compact detectors based on semi conductors are currently investigated.     

Cancer imaging: Gene transcription-based imaging and therapeutic systems

Molecular-genetic imaging of cancer is in its infancy. Over the past decade gene reporter systems have been optimized in preclinical models and some have found their way into the clinic. The search is on to find the best combination of gene delivery vehicle and reporter imaging system that can be translated safely and quickly. The goal is to have a combination that can detect a wide variety of cancers with high sensitivity and specificity in a way that rivals the current clinical standard, positron emission tomography with [(18)F]fluorodeoxyglucose. To do so will require systemic delivery of reporter genes for the detection of micrometastases, and a nontoxic vector, whether viral or based on nanotechnology, to gain widespread acceptance by the oncology community. Merger of molecular-genetic imaging with gene therapy, a strategy that has been employed in the past, will likely be necessary for such imaging to reach widespread clinical use.     

Novel Immunohistochemical Techniques Using Discrete Signal Amplification Systems for Human Cutaneous Peripheral Nerve Fiber Imaging

Confocal imaging uses immunohistochemical binding of specific antibodies to visualize tissues, but technical obstacles limit more widespread use of this technique in the imaging of peripheral nerve tissue. These obstacles include same-species antibody cross-reactivity and weak fluorescent signals of individual and co-localized antigens. The aims of this study were to develop new immunohistochemical techniques for imaging of peripheral nerve fibers. Three-millimeter punch skin biopsies of healthy individuals were fixed, frozen, and cut into 50-µm sections. Tissues were stained with a variety of antibody combinations with two signal amplification systems, streptavidin-biotin-fluorochrome (sABC) and tyramide-horseradish peroxidase-fluorochrome (TSA), used simultaneously to augment immunohistochemical signals. The combination of the TSA and sABC amplification systems provided the first successful co-localization of sympathetic adrenergic and sympathetic cholinergic nerve fibers in cutaneous human sweat glands and vasomotor and pilomotor systems. Primary antibodies from the same species were amplified individually without cross-reactivity or elevated background interference. The confocal fluorescent signal-to-noise ratio increased, and image clarity improved. These modifications to signal amplification systems have the potential for widespread use in the study of human neural tissues.     

Inorganic nanoparticles for multimodal molecular imaging

Multimodal molecular imaging can offer a synergistic improvement of diagnostic ability over a single imaging modality. Recent development of hybrid imaging systems has profoundly impacted the pool of available multimodal imaging probes. In particular, much interest has been focused on biocompatible, inorganic nanoparticle-based multimodal probes. Inorganic nanoparticles offer exceptional advantages to the field of multimodal imaging owing to their unique characteristics, such as nanometer dimensions, tunable imaging properties, and multifunctionality. Nanoparticles mainly based on iron oxide, quantum dots, gold, and silica have been applied to various imaging modalities to characterize and image specific biologic processes on a molecular level. A combination of nanoparticles and other materials such as biomolecules, polymers, and radiometals continue to increase functionality for in vivo multimodal imaging and therapeutic agents. In this review, we discuss the unique concepts, characteristics, and applications of the various multimodal imaging probes based on inorganic nanoparticles.     

Chemiluminescence microscopy as a tool in biomedical research.

Chemiluminescence has become a standard tool in biomedical research. Chemiluminescent probes are used for immunoassays, nucleic acid identification, reporter gene assays, measuring enzyme activity, and the detection of ions and small molecules such as Ca2+, ATP, NO, O2- and H2O2. Along with the development of new chemiluminescent probes, significant progress has been made in techniques to measure chemiluminescence. Ultra-sensitive photometers or luminometers have become widely available and can be obtained with automatic injectors and microplate readers. In addition, imaging photon detectors have been developed that allow the imaging of chemiluminescence from gels, blots, and microplates. Imaging photon detectors have also been attached to microscopes and allow imaging of chemiluminescent probes and reporter genes in cells and tissues. Specific methods of photon collection, storage, and analysis have been developed for microscopic imaging of chemiluminescence. Two of these methods are discussed in detail. The first is a method of data storage that allows days of continuous imaging without creating oversized files. The second is a method for calibrating photon imaging microscopes using a low-light standard. Such calibration will be helpful for comparing the performance of various photon imaging systems and for comparing data obtained in different laboratories.     

Live cell imaging using wide-field microscopy and deconvolution

The use of fluorescence imaging methods, most recently based on fluorescent protein technology, and the availability of high quality fluorescence imaging systems have driven a revolution in cell and molecular biology. Live cell imaging, especially using fluorescence, is now used in a wide variety of assays in academic and commercial laboratories. The use of this technology requires particular attention to be paid to cell engineering, the design of the image acquisition system, the imaging protocol, and subsequent processing and analytic methods. In this review, we discuss each of these steps, highlighting practical techniques developed by us and others.     

Multi-scale imaging techniques to investigate solute transport across articular cartilage

As articular cartilage is an avascular tissue, the transport of nutrients and cytokines through the tissue is essential for the health of cells, i.e. chondrocytes. Transport of specific contrast agents through cartilage has been investigated to elucidate cartilage quality. In laboratory, pre-clinical and clinical studies, imaging techniques such as magnetic imaging resonance (MRI), computed tomography (CT) and fluorescent microscopy have been widely employed to visualize and quantify solute transport in cartilage. Many parameters related to the physico-chemical properties of the solute, such as molecular weight, net charge and chemical structure, have a profound effect on the transport characteristics. Information on the interplay of the solute parameters with the imaging-dependent parameters (e.g. resolution, scan and acquisition time) could assist in selecting the most optimal imaging systems and data analysis tools in a specific experimental set up. Here, we provide a comprehensive review of various imaging systems to investigate solute transport properties in articular cartilage, by discussing their potentials and limitations. The presented information can serve as a guideline for applications in cartilage imaging and therapeutics delivery and to improve understanding of the set-up of solute transport experiments in articular cartilage.     

Diagnostic imaging at its centennial: the past,the present and the future.

Since the discovery of x-rays by Wilhelm Conrad Röntgen 100 years ago, diagnostic imaging has profoundly influenced the practice of medicine. As a result of discoveries during this period, ultrasonography, nuclear imaging, computed tomography and magnetic resonance imaging, as well as conventional radiography, have assumed a major role in diagnostic medicine. In addition to their traditional role in diagnosis, imaging techniques are becoming an increasingly important factor in innovative treatment methods, and this role is likely to expand. In the current climate of rising health care costs, radiologists and other health care providers who use imaging must increasingly account to health care funders for the cost-effectiveness of imaging in relation to other diagnostic and interventional techniques. They must also assure minimum standards of quality and training, and determine the appropriate role for diagnostic imaging in health care systems of the future.