Real‐time in vivo imaging of adult Zebrafish brain using optical coherence tomography

We report noninvasive imaging of the brain of adult Zebrafish (Danio rerio) using real time optical coherence tomography (OCT) capable of acquiring cross sectional 2D OCT images @ 8 frames/sec. Anatomic features such as telencephalon, tectum opticum, eminentia Granularis and cerebellum were clearly resolved in the OCT images. A 3D model of Zebrafish brain was reconstructed, for the first time to our knowledge, using these 2D OCT images. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Real‐time video mosaicking to guide handheld in vivo microscopy

Handheld and endoscopic optical‐sectioning microscopes are being developed for noninvasive screening and intraoperative consultation. Imaging a large extent of tissue is often desired, but miniature in vivo microscopes tend to suffer from limited fields of view. To extend the imaging field during clinical use, we have developed a real‐time video mosaicking method, which allows users to efficiently survey larger areas of tissue. Here, we modified a previous post‐processing mosaicking method so that real‐time mosaicking is possible at >30 frames/second when using a device that outputs images that are 400 × 400 pixels in size. Unlike other real‐time mosaicking methods, our strategy can accommodate image rotations and deformations that often occur during clinical use of a handheld microscope. We perform a feasibility study to demonstrate that the use of real‐time mosaicking is necessary to enable efficient sampling of a desired imaging field when using a handheld dual‐axis confocal microscope.     

Real‐time imaging of photodynamic action in bacteria

Fluorescence imaging studies of the processes leading to photodynamic inactivation of bacteria have been limited due to the small size of microorganisms as well as by the faint fluorescence of most photosensitizers. A versatile method based on highly‐sensitive fluorescence microscopy is presented which allows to study, in real time, the incorporation of photosensitizers inside S. aureus upon photodynamic action. The method takes advantage of the fluorescence enhancement of phenothiazine and porphyrin photosensitizers upon entering the bacterial cytosol after the cell wall has been compromised. In combination with typical assays, such as the addition of specific enhancers of reactive oxygen species, it is possible to extract mechanistic information about the pathway of photodynamic damage at the single‐cell level. Imaging experiments in deuterated buffer strongly support a Type‐I mechanism for methylene blue and a very minor role of singlet oxygen.

     

Real‐time mid‐infrared imaging of living microorganisms

The speed and efficiency of quantum cascade laser‐based mid‐infrared microspectroscopy are demonstrated using two different model organisms as examples. For the slowly moving Amoeba proteus, a quantum cascade laser is tuned over the wavelength range of 7.6 µm to 8.6 µm (wavenumbers 1320 cm^–1 and 1160 cm^–1, respectively). The recording of a hyperspectral image takes 11.3 s whereby an average signal‐to‐noise ratio of 29 is achieved. The limits of time resolution are tested by imaging the fast moving Caenorhabditis elegans at a discrete wavenumber of 1265 cm^–1. Mid‐infrared imaging is performed with the 640 × 480 pixel video graphics array (VGA) standard and at a full‐frame time resolution of 0.02 s (i.e. well above the most common frame rate standards). An average signal‐to‐noise ratio of 16 is obtained. To the best of our knowledge, these findings constitute the first mid‐infrared imaging of living organisms at VGA standard and video frame rate.

     

Real‐time in vivo cancer diagnosis using raman spectroscopy

Raman spectroscopy has becoming a practical tool for rapid in vivo tissue diagnosis. This paper provides an overview on the latest development of real‐time in vivo Raman systems for cancer detection. Instrumentation, data handling, as well as oncology applications of Raman techniques were covered. Optic fiber probes designs for Raman spectroscopy were discussed. Spectral data pre‐processing, feature extraction, and classification between normal/benign and malignant tissues were surveyed. Applications of Raman techniques for clinical diagnosis for different types of cancers, including skin cancer, lung cancer, stomach cancer, oesophageal cancer, colorectal cancer, cervical cancer, and breast cancer, were summarized.

Schematic of a real‐time Raman spectrometer for skin cancer detection. Without correction, the image captured on CCD camera for a straight entrance slit has a curvature. By arranging the optic fiber array in reverse orientation, the curvature could be effectively corrected.     

Real‐time imaging of single cancer‐cell dynamics of lung metastasis

We have developed a new in vivo mouse model to image single cancer‐cell dynamics of metastasis to the lung in real‐time. Regulating airflow volume with a novel endotracheal intubation method enabled controlling lung expansion adequate for imaging of the exposed lung surface. Cancer cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected in the tail vein of the mouse. The right chest wall was then opened in order to image metastases on the lung surface directly. After each observation, the chest wall was sutured and the air was suctioned in order to re‐inflate the lung, in order to keep the mice alive. Observations have been carried out for up to 8 h per session and repeated up to six times per mouse thus far. The seeding and arresting of single cancer cells on the lung, accumulation of cancer‐cell emboli, cancer‐cell viability, and metastatic colony formation were imaged in real‐time. This new technology makes it possible to observe real‐time monitoring of cancer‐cell dynamics of metastasis in the lung and to identify potential metastatic stem cells. J. Cell. Biochem. 109: 58–64, 2010. © 2009 Wiley‐Liss, Inc.     

Real‐time endoscopic Raman spectroscopy for in vivo early lung cancer detection

Currently the most sensitive method for localizing lung cancers in central airways is autofluorescence bronchoscopy (AFB) in combination with white light bronchoscopy (WLB). The diagnostic accuracy of WLB + AFB for high grade dysplasia (HGD) and carcinoma in situ is variable depending on physician's experience. When WLB + AFB are operated at high diagnostic sensitivity, the associated diagnostic specificity is low. Raman spectroscopy probes molecular vibrations and gives highly specific, fingerprint‐like spectral features and has high accuracy for tissue pathology classification. In this study we present the use of a real‐time endoscopy Raman spectroscopy system to improve the specificity. A spectrum is acquired within 1 second and clinical data are obtained from 280 tissue sites (72 HGDs/malignant lesions, 208 benign lesions/normal sites) in 80 patients. Using multivariate analyses and waveband selection methods on the Raman spectra, we have demonstrated that HGD and malignant lung lesions can be detected with high sensitivity (90%) and good specificity (65%).

     

In vivo imaging of the diseased nervous system

In vivo microscopy is an exciting tool for neurological research because it can reveal how single cells respond to damage of the nervous system. This helps us to understand how diseases unfold and how therapies work. Here, we review the optical imaging techniques used to visualize the different parts of the nervous system, and how they have provided fresh insights into the aetiology and therapeutics of neurological diseases. We focus our discussion on five areas of neuropathology (trauma, degeneration, ischaemia, inflammation and seizures) in which in vivo microscopy has had the greatest impact. We discuss the challenging issues in the field, and argue that the convergence of new optical and non-optical methods will be necessary to overcome these challenges.     

Real‐time imaging and genetic dissection of host–microbe interactions in zebrafish

Many aspects of host interactions with microbes can only be studied in the context of a whole organism. The zebrafish as a model organism has shown to be highly successful for studies of infection biology and the interactions of commensal microbiota with their hosts. Zebrafish are transparent during embryo and larval development and these early life stages are optimally suited for high‐resolution imaging of host–microbe interactions in a vertebrate organism. This is facilitated by the development of a variety of fluorescent reporter lines that mark different immune cell types or subcellular compartments where pathogens reside. The zebrafish is an excellent vertebrate model for forward genetic screening and efficient tools for gene knock‐down and targeted mutagenesis add further to the strength of this model organism. The use of zebrafish larvae for studying microbial infections has recently led to important new insights in host defence mechanisms, which are highlighted in this review focused on bacterial pathogens. Considering the highly conserved nature of the processes involved, including innate immune recognition, immunometabolism and autophagy, it is to be expected that these recent findings in zebrafish will have great translational value for biomedical applications.     

In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy

Loss of myelin in the central nervous system (CNS) leads to debilitating neurological deficits. High-resolution optical imaging of myelin in the CNS of animal models is limited by a lack of in vivo myelin labeling strategies. We demonstrated that third harmonic generation (THG) microscopy—a coherent, nonlinear, dye-free imaging modality—provides micrometer resolution imaging of myelin in the mouse CNS. In fixed tissue, we found that THG signals arose from white matter tracts and were colocalized with two-photon excited fluorescence (2PEF) from a myelin-specific dye. In vivo, we used simultaneous THG and 2PEF imaging of the mouse spinal cord to resolve myelin sheaths surrounding individual fluorescently-labeled axons, and followed myelin disruption after spinal cord injury. Finally, we suggest optical mechanisms that underlie the myelin specificity of THG. These results establish THG microscopy as an ideal tool for the study of myelin loss and recovery.     

Presenilin 1 in migration and morphogenesis in the central nervous system

Morphogenesis of the central nervous system relies in large part upon the correct migration of neuronal cells from birthplace to final position. Two general modes of migration govern CNS morphogenesis: radial, which is mostly glia-guided and topologically relatively simple; and tangential, which often involves complex movement of neurons in more than one direction. We describe the consequences of loss of function of presenilin 1 on these fundamental processes. Previous studies of the central nervous system in presenilin 1 homozygote mutant embryos identified a premature neuronal differentiation that is transient and localized, with cortical dysplasia at later stages. We document widespread effects on CNS morphogenesis that appear strongly linked to defective neuronal migration. Loss of presenilin 1 function perturbs both radial and tangential migration in cerebral cortex, and several tangential migratory pathways in the brainstem. The inability of cells to execute their migratory trajectories affects cortical lamination, formation of the facial branchiomotor nucleus, the spread of cerebellar granule cell precursors to form the external granule layer and development of the pontine nuclei. Finally, overall morphogenesis of the mid-hindbrain region is abnormal, resulting in incomplete midline fusion of the cerebellum and overgrowth of the caudal midbrain. These observations indicate that in the absence of presenilin 1 function, the ability of a cell to move can be severely impaired regardless of its mode of migration, and, at a grosser level, brain morphogenesis is perturbed. Our results demonstrate that presenilin 1 plays a much more important role in brain development than has been assumed, consistent with a pleiotropic involvement of this molecule in cellular signaling.     

In vivo electroporation in the embryonic mouse central nervous system

This protocol describes a basic method for in vivo electroporation in the nervous system of embryonic mice. Delivery of electric pulses following microinjection of DNA into the brain ventricle or the spinal cord central canal enables efficient transfection of genes into the nervous system. Transfection is facilitated by forceps-type electrodes, which hold the uterus and/or the yolk sac containing the embryo. More than ten embryos in a single pregnant mouse can be operated on within 30 min. More than 90% of operated embryos survive and more than 90% of these survivors express the transfected genes appropriately. Gene expression in neurons persists for a long time, even at postnatal stages, after electroporation. Thus, this method could be used to analyze roles of genes not only in embryonic development but also in higher order function of the nervous system, such as learning.     

Multimodal magnetic resonance imaging of the central nervous system

The physiological and biochemical properties of the diseased brain that can be explored with magnetic resonance imaging (MRI) are increasing. Progress in MR-based technology affords a large panel of MRI sequences that explore different phenomena and, thus, provide complementary informations. The diagnostic accuracy of MRI is improved by the combination of all MR modalities. However, this abundance of data requires an efficient multiparametric analysis to fully achieve the goal of the multimodal strategy. We will discuss the potential impact of this advanced MRI analysis in the clinical management and the therapeutical strategies of the most common brain pathologies (intracranial tumors, multiple sclerosis, stroke, epilepsy and dementia). This non-invasive approach is of utmost importance since it already improves the diagnosis and the therapeutic choice in the management of several central nervous system diseases.     

Fiber optic in vivo imaging in the mammalian nervous system

The compact size, mechanical flexibility, and growing functionality of optical fiber and fiber optic devices are enabling several new modalities for imaging the mammalian nervous system in vivo. Fluorescence microendoscopy is a minimally invasive fiber modality that provides cellular resolution in deep brain areas. Diffuse optical tomography is a non-invasive modality that uses assemblies of fiber optic emitters and detectors on the cranium for volumetric imaging of brain activation. Optical coherence tomography is a sensitive interferometric imaging technique that can be implemented in a variety of fiber based formats and that might allow intrinsic optical detection of brain activity at a high resolution. Miniaturized fiber optic microscopy permits cellular level imaging in the brains of behaving animals. Together, these modalities will enable new uses of imaging in the intact nervous system for both research and clinical applications.     

Real‐time microbial adaptive diversification in soil

Bacteria undergo adaptive diversification over a matter of days in test tubes, but the relevance to natural populations remains unclear. Here, we report real‐time adaptive diversification of the bacterium Pseudomonas fluorescens in its natural environment, soil. Crucially, adaptive diversification was much greater in the absence of the established natural microbial community, suggesting that resident diversity is likely to inhibit, rather than promote, adaptive radiations in natural environments. Rapid diversification is therefore likely to play an important role in the population and community dynamics of microbes in environments where resident communities are perturbed, such as by agriculture, pollution and antibiotics.     

A Real‐time PCR Assay to Identify Meloidogyne minor

Meloidogyne minor is a small root‐knot nematode that causes yellow patch disease in golf courses and severe quality damage in potatoes. It was described in 2004 and has been detected in The Netherlands, England, Wales, Northern Ireland, Ireland and Belgium. The nematode often appears together with M. naasi on grasses. It causes similar symptoms on potato tubers as M. chitwoodi and M. fallax, which are both quarantine organisms in Europe. An accurate identification method therefore is required. This study describes a real‐time PCR assay that enables the identification of M. minor after extraction of nematodes from soil or plant samples. Alignments of sequences of rDNA‐ITS fragments of M. minor and five other Meloidogyne species were used to design a forward primer Mminor_f299, a specific primer Mminor_r362 and the specific MGB TaqMan probe P_Mm_MGB321. PCR with this primers and probe results in an amplicon of 64 bp. The analytical specificity of the real‐time PCR assay was assessed by assaying it on six populations of M. minor and on 10 populations of six other Meloidogyne species. Only DNA from M. minor gave positive results in this assay. The assay was able to identify M. minor using DNA from a single juvenile independent from the DNA extraction method used.