Fluorescent Dye Imaging Research Articles

Current state of sentinel lymph nodes for women with endometrial cancer

Sentinel lymph node (SLN) biopsy has been investigated as an alternative to conventional pelvic and para-aorticlymphadenectomy for the surgical staging of endometrial cancer. Clinical trials have established the accuracy of sentinel nodes in the detecting metastatic disease. Novel advancements in tracers from the historically favored blue dyes and radio labeled colloids to near infrared imaging of fluorescent dyes has improved the ability to detect sentinel nodes and increased options for surgeons. The uterine cervix has been shown to be a feasible and accurate injection site for tracer, though the potential for under-evaluation of the para-aortic nodes remains a controversy, particularly for high-risk cancers. Additionally, sentinel node evaluation provides qualitatively different information than traditional staging techniques by identifying lymph nodes outside of traditional sampling locations and through the identification of very low volume meta static disease implants, such as isolated tumor cells. It is unclear how this altered staging information should be interpreted, guide the prescription of adjuvant therapy and its impact on long term clinical outcomes such as recurrence and survival. In this review we will discuss the evidence that has supported the use of the SLN technique in the staging of endometrial cancer, the options for surgical technique and the implications of managing the results of staging pathology.

Histone Deacetylases Enhance Ca2+-Activated K⁺ Channel KCa3.1 Expression in Murine Inflammatory CD4⁺ T Cells.

The up-regulated expression of the Ca2+-activated K+ channel KCa3.1 in inflammatory CD4+ T cells has been implicated in the pathogenesis of inflammatory bowel disease (IBD) through the enhanced production of inflammatory cytokines, such as interferon-γ (IFN-γ). However, the underlying mechanisms have not yet been elucidated. The objective of the present study is to clarify the involvement of histone deacetylases (HDACs) in the up-regulation of KCa3.1 in the CD4+ T cells of IBD model mice. The expression levels of KCa3.1 and its regulators, such as function-modifying molecules and transcription factors, were quantitated using a real-time polymerase chain reaction (PCR) assay, Western blotting, and depolarization responses, which were induced by the selective KCa3.1 blocker TRAM-34 (1 μM) and were measured using a voltage-sensitive fluorescent dye imaging system. The treatment with 1 μM vorinostat, a pan-HDAC inhibitor, for 24 h repressed the transcriptional expression of KCa3.1 in the splenic CD4+ T cells of IBD model mice. Accordingly, TRAM-34-induced depolarization responses were significantly reduced. HDAC2 and HDAC3 were significantly up-regulated in the CD4+ T cells of IBD model mice. The down-regulated expression of KCa3.1 was observed following treatments with the selective inhibitors of HDAC2 and HDAC3. The KCa3.1 K+ channel regulates inflammatory cytokine production in CD4+ T cells, mediating epigenetic modifications by HDAC2 and HDAC3.

Impaired Bioenergetics in Mutant Mitochondrial DNA Determines Cell Fate During Seizure-Like Activity

Mutations in genes affecting mitochondrial proteins are increasingly recognised in patients with epilepsy, but the factors determining cell fate during seizure activity in these mutations remain unknown. Fluorescent dye imaging techniques were applied to fibroblast cell lines from patients suffering from common mitochondrial mutations and to age-matched controls. Using live cell imaging techniques in fibroblasts, we show that fibroblasts with mutations in the mitochondrial genome had reduced mitochondrial membrane potential and NADH pools and higher redox indices, indicative of respiratory chain dysfunction. Increasing concentrations of ferutinin, a Ca2+ ionophore, led to oscillatory Ca2+ signals in fibroblasts resembling dynamic Ca2+ changes that occur during seizure-like activity. Co-monitoring of mitochondrial membrane potential (ΔΨm) changes induced by ferutinin showed accelerated membrane depolarisation and cell collapse in fibroblasts with mutations in the mitochondrial genome when compared to controls. Ca2+ flash photolysis using caged Ca2+ confirmed impaired Ca2+ handling in fibroblasts with mitochondrial mutations. Findings indicate that intracellular Ca2+ levels cannot be compensated during periods of hyperexcitability, leading to Ca2+ overload and subsequent cell death in mitochondrial diseases.

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy

ObjectivesWe produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining.MethodsWe improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods.ResultsAn image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging.ConclusionsThe colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis.

Comparison of glomerular activity patterns by fMRI and wide-field calcium imaging: Implications for principles underlying odor mapping

Functional imaging signals arise from distinct metabolic and hemodynamic events at the neuropil, but how these processes are influenced by pre- and post-synaptic activities need to be understood for quantitative interpretation of stimulus-evoked mapping data. The olfactory bulb (OB) glomeruli, spherical neuropil regions with well-defined neuronal circuitry, can provide insights into this issue. Optical calcium-sensitive fluorescent dye imaging (OICa2+) reflects dynamics of pre-synaptic input to glomeruli, whereas high-resolution functional magnetic resonance imaging (fMRI) using deoxyhemoglobin contrast reveals neuropil function within the glomerular layer where both pre- and post-synaptic activities contribute. We imaged odor-specific activity patterns of the dorsal OB in the same anesthetized rats with fMRI and OICa2+ and then co-registered the respective maps to compare patterns in the same space. Maps by each modality were very reproducible as trial-to-trial patterns for a given odor, overlapping by ~80%. Maps evoked by ethyl butyrate and methyl valerate for a given modality overlapped by ~80%, suggesting activation of similar dorsal glomerular networks by these odors. Comparison of maps generated by both methods for a given odor showed ~70% overlap, indicating similar odor-specific maps by each method. These results suggest that odor-specific glomerular patterns by high-resolution fMRI primarily tracks pre-synaptic input to the OB. Thus combining OICa2+ and fMRI lays the framework for studies of OB processing over a range of spatiotemporal scales, where OICa2+ can feature the fast dynamics of dorsal glomerular clusters and fMRI can map the entire glomerular sheet in the OB.

Linking salinity stress tolerance with tissue-specific Na(+) sequestration in wheat roots.

Salinity stress tolerance is a physiologically complex trait that is conferred by the large array of interacting mechanisms. Among these, vacuolar Na+ sequestration has always been considered as one of the key components differentiating between sensitive and tolerant species and genotypes. However, vacuolar Na+ sequestration has been rarely considered in the context of the tissue-specific expression and regulation of appropriate transporters contributing to Na+ removal from the cytosol. In this work, six bread wheat varieties contrasting in their salinity tolerance (three tolerant and three sensitive) were used to understand the essentiality of vacuolar Na+ sequestration between functionally different root tissues, and link it with the overall salinity stress tolerance in this species. Roots of 4-day old wheat seedlings were treated with 100 mM NaCl for 3 days, and then Na+ distribution between cytosol and vacuole was quantified by CoroNa Green fluorescent dye imaging. Our major observations were as follows: (1) salinity stress tolerance correlated positively with vacuolar Na+ sequestration ability in the mature root zone but not in the root apex; (2) contrary to expectations, cytosolic Na+ levels in root meristem were significantly higher in salt tolerant than sensitive group, while vacuolar Na+ levels showed an opposite trend. These results are interpreted as meristem cells playing a role of the “salt sensor;” (3) no significant difference in the vacuolar Na+ sequestration ability was found between sensitive and tolerant groups in either transition or elongation zones; (4) the overall Na+ accumulation was highest in the elongation zone, suggesting its role in osmotic adjustment and turgor maintenance required to drive root expansion growth. Overall, the reported results suggest high tissue-specificity of Na+ uptake, signaling, and sequestration in wheat roots. The implications of these findings for plant breeding for salinity stress tolerance are discussed.

Identification of small-scale low and high permeability layers using single well forced-gradient tracer tests: Fluorescent dye imaging and modelling at the laboratory-scale

Heterogeneity in aquifer permeability, which creates paths of varying mass flux and spatially complex contaminant plumes, can complicate the interpretation of contaminant fate and transport in groundwater. Identifying the location of high mass flux paths is critical for the reliable estimation of solute transport parameters and design of groundwater remediation schemes. Dipole flow tracer tests (DFTTs) and push-pull tests (PPTs) are single well forced-gradient tests which have been used at field-scale to estimate aquifer hydraulic and transport properties. In this study, the potential for PPTs and DFTTs to resolve the location of layered high- and low-permeability layers in granular porous media was investigated with a pseudo 2-D bench-scale aquifer model. Finite element fate and transport modelling was also undertaken to identify appropriate set-ups for in situ tests to determine the type, magnitude, location and extent of such layered permeability contrasts at the field-scale. The characteristics of flow patterns created during experiments were evaluated using fluorescent dye imaging and compared with the breakthrough behaviour of an inorganic conservative tracer. The experimental results show that tracer breakthrough during PPTs is not sensitive to minor permeability contrasts for conditions where there is no hydraulic gradient. In contrast, DFTTs are sensitive to the type and location of permeability contrasts in the host media and could potentially be used to establish the presence and location of high or low mass flux paths. Numerical modelling shows that the tracer peak breakthrough time and concentration in a DFTT is sensitive to the magnitude of the permeability contrast (defined as the permeability of the layer over the permeability of the bulk media) between values of 0.01–20. DFTTs are shown to be more sensitive to deducing variations in the contrast, location and size of aquifer layered permeability contrasts when a shorter central packer is used. However, larger packer sizes are more likely to be practical for field-scale applications, with fewer tests required to characterise a given aquifer section. The sensitivity of DFTTs to identify layered permeability contrasts was not affected by test flow rate.

Anesthetic Protection of Neurons Injured by Hypothermia and Rewarming

Mild hypothermia is neuroprotective after cerebral ischemia but surgery involving profound hypothermia (PH, temperature less than 18°C) is associated with neurologic complications. Rewarming (RW) from PH injures hippocampal neurons by glutamate excitotoxicity, N-methyl-D-aspartate receptors, and intracellular calcium. Because neurons are protected from hypoxia-ischemia by anesthetic agents that inhibit N-methyl-D-aspartic acid receptors, we tested whether anesthetics protect neurons from damage caused by PH/RW. Organotypic cultures of rat hippocampus were used to model PH/RW injury, with hypothermia at 4°C followed by RW to 37°C and assessment of cell death 1 or 24 h later. Cell death and intracellular Ca were assessed with fluorescent dye imaging and histology. Anesthetic agents were present in the culture media during PH and RW or only RW. Injury to hippocampal CA1, CA3, and dentate neurons after PH and RW involved cell swelling, cell rupture, and adenosine triphosphate (ATP) loss; this injury was similar for 4 through 10 h of PH. Isoflurane (1% and 2%), sevoflurane (3%) and xenon (60%) reduced cell loss but propofol (3 μM) and pentobarbital (100 μM) did not. Isoflurane protection involved reduction in N-methyl-D-aspartate receptor-mediated Ca influx during RW but did not involve γ-amino butyric acid receptors or KATP channels. However, cell death increased over the next day. Anesthetic protection of neurons rewarmed from 4°C involves suppression of N-methyl-D-aspartate receptor-mediated Ca overload in neurons undergoing ATP loss and excitotoxicity. Unlike during hypoxia/ischemia, anesthetic agents acting predominantly on γ-aminobutyric acid receptors do not protect against PH/RW. The durability of anesthetic protection against cold injury may be limited.

Fluorescent dye imaging of the volume sampled by single well forced-gradient tracer tests evaluated in a laboratory-scale aquifer physical model

This study presents a new method to visualise forced-gradient tracer tests in 2-D using a laboratory-scale aquifer physical model. Experiments were designed to investigate the volume of aquifer sampled in vertical dipole flow tracer tests (DFTT) and push–pull tests (PPT), using a miniature monitoring well and straddle packer arrangement equipped with solute injection and recovery chambers. These tests have previously been used to estimate bulk aquifer hydraulic and transport properties for the evaluation of natural attenuation and other remediation approaches. Experiments were performed in a silica glass bead-filled box, using a fluorescent tracer (fluorescein) to deduce conservative solute transport paths. Digital images of fluorescein transport were captured under ultraviolet light and processed to analyse tracer plume geometry and obtain point-concentration breakthrough histories. Inorganic anion mixtures were also used to obtain conventional tracer breakthrough histories. Concentration data from the conservative tracer breakthrough curves was compared with the digital images and a well characterised numerical model. The results show that the peak tracer breakthrough response in dipole flow tracer tests samples a zone of aquifer close to the well screen, while the sampling volume of push–pull tests is limited by the length of the straddle packers used. The effective sampling volume of these single well forced-gradient tests in isotropic conditions can be estimated with simple equations. The experimental approach offers the opportunity to evaluate under controlled conditions the theoretical basis, design and performance of DFTTs and PPTs in porous media in relation to measured flow and transport properties. 2 2 DFTT = Dipole Flow Tracer Test, PPT = Push Pull Tracer Test.

Motmot, an open-source toolkit for realtime video acquisition and analysis

BackgroundVideo cameras sense passively from a distance, offer a rich information stream, and provide intuitively meaningful raw data. Camera-based imaging has thus proven critical for many advances in neuroscience and biology, with applications ranging from cellular imaging of fluorescent dyes to tracking of whole-animal behavior at ecologically relevant spatial scales.ResultsHere we present 'Motmot': an open-source software suite for acquiring, displaying, saving, and analyzing digital video in real-time. At the highest level, Motmot is written in the Python computer language. The large amounts of data produced by digital cameras are handled by low-level, optimized functions, usually written in C. This high-level/low-level partitioning and use of select external libraries allow Motmot, with only modest complexity, to perform well as a core technology for many high-performance imaging tasks. In its current form, Motmot allows for: (1) image acquisition from a variety of camera interfaces (package motmot.cam_iface), (2) the display of these images with minimal latency and computer resources using wxPython and OpenGL (package motmot.wxglvideo), (3) saving images with no compression in a single-pass, low-CPU-use format (package motmot.FlyMovieFormat), (4) a pluggable framework for custom analysis of images in realtime and (5) firmware for an inexpensive USB device to synchronize image acquisition across multiple cameras, with analog input, or with other hardware devices (package motmot.fview_ext_trig). These capabilities are brought together in a graphical user interface, called 'FView', allowing an end user to easily view and save digital video without writing any code. One plugin for FView, 'FlyTrax', which tracks the movement of fruit flies in real-time, is included with Motmot, and is described to illustrate the capabilities of FView.ConclusionMotmot enables realtime image processing and display using the Python computer language. In addition to the provided complete applications, the architecture allows the user to write relatively simple plugins, which can accomplish a variety of computer vision tasks and be integrated within larger software systems. The software is available at

Spatial/temporal correlation of BOLD and optical intrinsic signals in humans.

Comparing the BOLD signal with electrophysiological maps and other perfusion-dependent signals, such as the optical intrinsic signal (OIS), within subjects should provide insight into the etiology of the BOLD signal. Tongue activations were compared in five human subjects using BOLD fMRI, 610-nm OIS, and the electrocortical stimulation map (ESM). Robust fMRI activations centered on the lateral inferior aspect of the central sulcus and extended into pre- and post-central gyri, adjacent to ESM tongue loci. OIS and fMRI maps colocalized, although optical responses were spatially larger (P <.001 across multiple thresholds) and contained more gyral components. The timecourses of the fMRI and OIS signals were similar, appearing within 2.5 s and peaking 6-8 s after task onset. Although many processes contribute to increased 610-nm reflectance, optical spectroscopy and fluorescent dye imaging suggest that a significant part of this signal is due to a concomitant decrease in deoxyhemoglobin and increase in oxyhemoglobin concentrations. The spatial/temporal correlation of BOLD and the positive 610-nm response within subjects suggests that the two signals may share similar etiologies. The OIS/fMRI inconsistencies may be due to cell swelling and light-scattering contributions to OIS and fMRI sensitivity. This study also demonstrates that fMRI maps do not precisely colocalize with ESM, rather they emphasize changes in adjacent venous/sulcal structures.

Kinetics of defibrillation shock-induced response: design implications for the optimal defibrillation waveform.

Implantable cardioverter defibrillator (ICD) therapy is a well-established therapy for treating patients at high risk for sudden cardiac death. Recently formulated virtual electrode polarization theory is a promising foundation for the theory of defibrillation. Yet, continuing optimization of defibrillation therapy is limited to primarily empirical methods due to difficulties in assessing kinetics of cellular response in whole heart models of defibrillation. The aim of this study was to evaluate the response of the myocardium in the context of virtual electrode polarization. We used a Langendorff-perfused rabbit heart model of ICD therapy and voltage-sensitive fluorescent dye imaging in order to map kinetics of trans membrane potential during both mono- and biphasic shocks applied at various phases of the QT-interval. Cellular response was fitted to a single exponential function using the Levenberg-Marquardt method. Time constants (tau) were measured in 45 288 optical records from 17 hearts. We found that cellular response depends upon both QT-phase of application, intensity, polarity, and phase of the biphasic waveform. Shocks of larger strengths produce a faster response. The tau of the first-phase negatively polarizing response was significantly larger compared with the positively polarizing response at intensities below 200 V, but smaller at 200 V and above. The tau of the second phase negatively polarizing response was always slower than the positively polarizing response, regardless of amplitude, and timing. Overall, tau ranged from 1.6 ms to 14.2 ms. The time constant of the membrane depends on the field, action potential phase and the shock polarity, but exceeds 1 msec. Therefore, we suggest using a slower shock leading edge, since the membrane cannot follow potentially damaging faster waveforms.

Reactive Oxidizing Species Produced near the Plasma Membrane Induce Apoptosis in Bovine Aorta Endothelial Cells

Many cytotoxic agents initiate apoptosis by generating reactive oxidizing species (ROS). The goal of this study was to determine whether apoptosis could be induced by initial reactions of ROS near the plasma membrane. Bovine aorta endothelial cells (BAEC) were illuminated with evanescent wave visible radiation, which has limited penetration into the basal surface of cells, or by trans-radiation. Imaging of fluorescent dyes localizing in the plasma membrane, mitochondria, or nucleus confirmed that evanescent wave radiation excited only dyes in and near the plasma membrane. Singlet oxygen, an ROS generated by photosensitization, has a very short lifetime, ensuring that it oxidizes molecules residing in or very close to the plasma membrane when evanescent wave radiation is used. Cells with condensed nuclei were considered apoptotic and were quantified after treatment with varying doses of light. Annexin V staining without propidium iodide staining confirmed that these cells were apoptotic. The doses required to induce apoptosis using evanescent wave radiation were 10-fold greater than those needed for trans-irradiation. Quantitative analysis of the evanescent wave penetration into cells supports a mechanism in which the singlet oxygen created near the plasma membrane, rather than at intracellular sites, was responsible for initiation of apoptosis.

Refractory periods observed by intrinsic signal and fluorescent dye imaging.

All perfusion-based imaging modalities depend on the relationship between neuronal and vascular activity. However, the relationship between stimulus and response was never fully characterized. With the use of optical imaging (intrinsic signals and intravascular fluorescent dyes) during repetitive stimulation paradigms, we observed reduced responses with temporally close stimuli. Cortical evoked potentials, however, did not produce the same reduced responsiveness. We therefore termed these intervals of reduced responsiveness "refractory periods." During these refractory periods an ability to respond was retained, but at a near 60% reduction in the initial magnitude. Although increasing the initial stimulus duration lengthened the observed refractory periods, significantly novel or temporally spaced stimuli overcame them. We observed this phenomenon in both rodent and human subjects in somatosensory and auditory cortices. These results have significant implications for understanding the capacities, mechanisms, and distributions of neurovascular coupling and thereby possess relevance to all perfusion-dependent functional imaging techniques.