Tracing DiO-labelled tumour cells in liver sections by confocal laser scanning microscopy

Investigating rare cellular events is facilitated by studying thick sections with confocal laser scanning microscopy (CLSM). Localization of cells in tissue sections can be done by immunolabelling or by fluorescent labelling of cells prior to intravenous administration. Immunolabelling is technically complicated because of the preservation of antigens during fixation and the problems associated with the penetration of the antibodies. In this study, an alternative and simple approach for the labelling of cells in vitro with the fluorescent probe DiO and its subsequent application in vivo will be outlined. The method was applied to trace DiO‐labelled colon carcinoma cells (CC531s) in 100 µm thick liver sections. In vitro and in vivo experiments revealed that DiO‐labelling of CC531s cells had no cytotoxic or antiproliferative effect and the cells preserved their susceptibility towards hepatic NK cells or Kupffer cells. In addition, DiO remained stable for at least 72 h in the living cell. DiO‐labelled CC531s cells could be traced all over the tissue depth and anti‐metastatic events such as phagocytosis of tumour cells by Kupffer cells could be easily observed. In situ staining with propidium iodide (nucleic acids) or rhodamine‐phalloidin (filamentous actin) resulted in additional tissue information. The data presented improved the understanding of the possible effects of the vital fluorescent probe DiO on cell function and provided a limit of confidence for CLSM imaging of DiO‐labelled cells in tissue sections.     

Monitoring of neuronal and glial calcium activity using a novel direct-contact probe

In vivo neuronal and glial calcium activity was monitored using a novel direct‐contact probe that was designed for fluorescence detection deep within biological tissue. A small diameter fibre bundle direct‐contact probe was employed with a laser scanning confocal microscope to detect evoked neuronal and glial activity in the posteromedial barrel subfield of the rat somatosensory cortex in vivo. Resolution of the probe allowed discrimination of single cells, and calcium dynamics spanning milliseconds to several seconds were observed. Initial results indicate that the probe has useful practical applications in the imaging of individual cells and monitoring rapid calcium fluctuations within their cell body and large processes.     

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two‐photon excitation microscopy including 2‐photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2‐photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in‐tandem combination of 2‐photon fluorescence and second harmonic generated signal microscopy as two‐modality microscopy allows for in situ co‐localization imaging of various microstructural components in the whole‐mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2‐photon‐controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two‐modal 2‐photon microscopy/tomography, acting as an efficient and sensitive non‐injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non‐linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.     

Use of independent component analysis to improve signal‐to‐noise ratio in multi‐probe fluorescence microscopy

In conventional multi‐probe fluorescence microscopy, narrow bandwidth filters on detectors are used to avoid bleed‐through artefacts between probes. The limited bandwidth reduces the signal‐to‐noise ratio of the detection, often severely compromising one or more channels. Herein, we describe a process of using independent component analysis to discriminate the position of different probes using only a dichroic mirror to differentiate the signals directed to the detectors. Independent component analysis was particularly effective in samples where the spatial overlap between the probes is minimal, a very common case in cellular microscopy. This imaging scheme collects nearly all of the emitted light, significantly improving the image signal‐to‐noise ratio. In this study, we focused on the detection of two fluorescence probes used in vivo, NAD(P)H and ANEPPS. The optimal dichroic mirror cutoff frequency was determined with simulations using the probes spectral emissions. A quality factor, defined as the cross‐channel contrast‐to‐noise ratio, was optimized to maximize signals while maintaining spatial discrimination between the probes after independent component analysis post‐processing. Simulations indicate that a ～3 fold increase in signal‐to‐noise ratio using the independent component analysis approach can be achieved over the conventional narrow‐band filtering approach without loss of spatial discrimination. We confirmed this predicted performance from experimental imaging of NAD(P)H and ANEPPS in mouse skeletal muscle, in vivo. For many multi‐probe studies, the increased sensitivity of this ‘full bandwidth’ approach will lead to improved image quality and/or reduced excitation power requirements.     

Friction and lubrication of human skin

The in vivo friction of human skin has been measured in the dry, wet and damp states using smooth glass and polypropylene spherically tipped probes. They were selected to be representative of hydrophilic and hydrophobic countersurfaces. The data are interpreted using the adhesion model of friction, which provides an explanation for the influence of the normal load and the surface free energies of the probe materials on the frictional characteristics of the skin. In particular, explanations based on this model are given for the tendency of wet skin to exhibit stick-slip motion and of damp skin to exhibit a peak frictional force against a glass probe.     

Utilization of 3D printing for an intravital microscopy platform to study the intestinal microcirculation

Intravital microscopy of the intestine is a sophisticated technique that allows qualitative and quantitative in vivo observation of dynamic cellular interactions and blood flow at a high resolution. Physiological conditions of the animal and in particular of the observed organ, such as temperature and moisture are crucial for intravital imaging. Often, the microscopy stage with the animal or the organ of interest imposes limitations on how well the animal can be maintained. In addition, the access for additional oxygen supply or drug administration during the procedure is rather restricted. To address these limitations, we developed a novel intravital microscopy platform, allowing us to have improved access to the animal during the intravital microscopy procedure, as well as improved microenvironmental maintenance. The production process of this prototype platform is based on 3D printing of device parts in a single‐step process. The simplicity of production and the advantages of this versatile and customizable design are shown and discussed in this paper. Our design potentially represents a major step forward in facilitating intestinal intravital imaging using fluorescent microscopy.     

A Nuclear Magnetic Resonance Probe for Testing Substances with Large Values of T1

An autodyne nuclear magnetic resonance (NMR) probe for testing solids with a long spin–lattice relaxation time T ₁ is described. The main advantage of the probe circuit suggested in this work over conventional NMR probes is an increase in the signal-to-noise ratio, whereas the induction of the radio-frequency field at the site of the sample remains the same and the voltage across the sample coil varies from 2 to 20 mV. The probe can be used at frequencies of 5 to 25 MHz.     

In vivo monitoring of the transfer kinetics of trace elements in animal brains with hyphenated inductively coupled plasma mass spectrometry techniques

The roles of metal ions to sustain normal function and to cause dysfunction of neurological systems have been confirmed by various studies. However, because of the lack of adequate analytical method to monitor the transfer kinetics of metal ions in the brain of a living animal, research on the physiopathological roles of metal ions in the CNS remains in its early stages and more analytical efforts are still needed. To explicitly model the possible links between metal ions and physiopathological alterations, it is essential to develop in vivo monitoring techniques that can bridge the gap between metalloneurochemistry and neurophysiopathology. Although inductively coupled plasma mass spectrometry (ICP‐MS) is a very powerful technique for multiple trace element analyses, when dealing with chemically complex microdialysis samples, the detection capability is largely limited by instrumental sensitivity, selectivity, and contamination that arise from the experimental procedure. As a result, in recent years several high efficient and clean on‐line sample pretreatment systems have been developed and combined with microdialysis and ICP‐MS for the continuous and in vivo determination of the concentration‐time profiles of metal ions in the extracellular space of rat brain. This article reviews the research relevant to the development of analytical techniques for the in vivo determination of dynamic variation in the concentration levels of metal ions in a living animal. © 2009 Wiley Periodicals, Inc. Mass Spec Rev 29:392‐424, 2010     

KIF18A is a potential prognostic factor and promotes tumor progression in oral tongue squamous cell carcinoma

The kinesin family member 18A protein was dysregulated in several human cancers and involved in cancer progression. However, the significance in oral tongue squamous cell carcinoma (OTSCC) has not been studied. The present study was intended to explore the functions of KIF18A in oral tongue squamous cell carcinoma. The immunohistochemistry (IHC) assay was performed to assess the relationships between the KIF18A protein expression level and clinical-pathological features of the patients. The biological functions of KIF18A in OTSCC cells were investigated by the experiments in vitro and in vivo. Based on immunohistochemistry, we found that KIF18A was correlated with the clinical-pathological features of OTSCC patients. High expression of KIF18A was associated with the lymph node metastasis, and clinical stages. In vitro experiments revealed that silencing of KIF18A significantly inhibited the expression of the proliferation and migration related proteins such as Ki67, proliferating cell nuclear antigen, matrix metalloproteinase-7 and matrix metalloproteinase-9, and thereby inhibiting the proliferation, migration and invasion of tumor cells. In vivo, knocking-down of KIF18A could inhibit the tumor growth in nude mice. In conclusion, we found KIF18A promoted tumor progression in vivo and in vitro and might become an effective target for the treatment of OTSCC.     

Cell viability in the cadmium-stressed cell suspension cultures of tobacco is regulated by extracellular ATP,possibly by a reactive oxygen species-associated mechanism

Cadmium (Cd) is one of the most widespread and toxic heavy metals to plants. Extracellular ATP (exATP) is thought to be an extracellular effector in regulating the physiological responses of plant cells to environmental stresses. However, the function of exATP in Cd-stressed plant cells is much unknown. The present work showed that treating tobacco (Nicotiana tabacum L. cv. Bright Yellow-2) cell-suspension cultures with exogenous CdCl₂ reduced the cell viability, exATP level, and Mg content. However, the production of reactive oxygen species (ROS), Cd content, and electrolyte leakage of the cells were enhanced by exogenous CdCl₂. When the Cd-induced accumulation of ROS was decreased by the supplement with DMTU (dimethylthiourea, a scavenger of ROS), the Cd-induced increases of the electrolyte leakage and Cd content were alleviated, and the Cd-induced reductions of cell viability were partly rescued, suggesting that Cd-induced reduction of cell viability could be related to the ROS accumulation. Under the condition of Cd stress, when the reduction of exATP level was partly rescued by exogenous ATP (20 μM), the increases of ROS production, electrolyte leakage, and Cd content were attenuated, and the reduction of cell viability was also alleviated. These observations indicate that exATP can regulate the cell viability in the Cd–stressed plant cells possibly by an ROSassociated mechanism.     

Endocytosis: The different energy requirements for the uptake of particles by small and large vesicles into peritoneal macrophages

The uptake of various, different-sized particles by macrophages was studied using the electron microscope. In addition to observing normal cells, cells were examined which had been inhibited by exposing them to low temperature (4°C), and to a number of metabolic poisons. It was found that large particles (> 01 μm) enter the cells and are contained in large vesicles (0·1–5 μm). Small particles (< 50 nm) may also enter the cells by this process. They enter most frequently, however, by passing into small (～ 70 nm) vesicles. These may later coalesce and their contents adhere to give a second kind of large vesicle. The various inhibitors prevented the ingestion of the large particles (and of the small particles en masse) into large vesicles, but did not prevent their initial adsorption onto the plasma membranes. They did not prevent nearly normal numbers of small particles from entering the pre-existing small vesicles, nor their subsequent fusion into the second class of large vesicles.     

Near-field scanning optical microscopy probes: a comparison of pulled and double-etched bent NSOM probes for fluorescence imaging of biological samples

Bent near‐field optical probes for biological applications have been fabricated using a combination of a two‐step chemical etching method and focused ion beam milling to create a well‐defined aperture. The transmission efficiencies have been evaluated as a function of laser wavelength (λ) and aperture size (D) for both large and small core fibres. The probe transmission behaviour follows a (D/λ)³ relationship. The double‐etched probes are compared to pulled probes fabricated from highly GeO₂‐doped dispersion compensating fibre and a standard single‐mode optical fibre. The transmission efficiencies of both types of pulled probes are approximately two orders of magnitude lower than double‐etched probes with similar aperture sizes. To demonstrate the utility of the various probes, their imaging performance has been evaluated for samples of polymer beads and phase‐separated phospholipid monolayers of dipalmitoylphosphatidylcholine or cholesterol/phosphatidylcholine/sphingomyelin mixtures. Both pulled and double‐etched probes are suitable for fluorescence imaging of polymer spheres. However, pulled probes are rapidly damaged at the higher input laser intensities required for fluorescence imaging of monolayer samples doped with < 1% of a fluorescent dye‐labelled lipid. The images obtained with the double‐etched probes show excellent spatial resolution and signal/noise, illustrating the potential of such probes for imaging of biological samples.     

Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy

Using the tandem scanning microscope, in vivo confocal microscopic images of living eyes were compared to images obtained from ex vivo, freshly enucleated or fixed tissue in the rabbit. In the normal cornea, microscopic details of the superficial epithelium, basal lamina, stromal fibrocyte nuclei, nerves and endothelial cell borders were easily discernible. Removal of the eye from the intact animal resulted in loss of detail with distortion of the normal structural interrelationships within the corneal stroma whilst enhancing details of the corneal epithelium. Formalin fixation further enhanced details of the basal and suprabasal corneal epithelial cell nuclei and the stromal fibrocyte cell borders whilst inducing prominent brightly reflecting folds in the thickened stroma with concomitant enhancement of the edge contrast of the collagen lamellae. These changes appeared to be related, in part, to hydration of the cornea and artefactual pooling of water between structures that may enhance reflectivity by increasing the difference between the refractive index of the cellular and extracellular elements. We conclude that microscopic examination of ex vivo preparations of corneal tissue, although providing increased resolution similar to conventional light microscopic techniques, significantly altered the normal structural relationships and could lead to erroneous measurements of the physiological properties of the tissue as compared to in vivo microscopy of undisturbed, intact tissue.     

Harmonic demodulation and minimum enhancement factors in field‐enhanced near‐field optical microscopy

Field‐enhanced scanning optical microscopy relies on the design and fabrication of plasmonic probes which had to provide optical and chemical contrast at the nanoscale. In order to do so, the scattering containing the near‐field information recorded in a field‐enhanced scanning optical microscopy experiment, has to surpass the background light, always present due to multiple interferences between the macroscopic probe and sample. In this work, we show that when the probe–sample distance is modulated with very low amplitude, the higher the harmonic demodulation is, the better the ratio between the near‐field signal and the interferometric background results. The choice of working at a given n harmonic is dictated by the experiment when the signal at the n + 1 harmonic goes below the experimental noise. We demonstrate that the optical contrast comes from the nth derivative of the near‐field scattering, amplified by the interferometric background. By modelling the far and near field we calculate the probe–sample approach curves, which fit very well the experimental ones. After taking a great amount of experimental data for different probes and samples, we conclude with a table of the minimum enhancement factors needed to have optical contrast with field‐enhanced scanning optical microscopy.     

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1. 77. p1–79 and the plasmid probe α-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.     

Measurement of probe displacement to the thermal resolution limit in photonic force microscopy using a miniature quadrant photodetector

A photonic force microscope comprises of an optically trapped micro-probe and a position detection system to track the motion of the probe. Signal collection for motion detection is often carried out using the backscattered light off the probe-however, this mode has problems of low S/N due to the small backscattering cross sections of the micro-probes typically used. The position sensors often used in these cases are quadrant photodetectors. To ensure maximum sensitivity of such detectors, it would help if the detector size matched with the detection beam radius after the condenser lens (which for backscattered detection would be the trapping objective itself). To suit this condition, we have used a miniature displacement sensor whose dimensions makes it ideal to work with 1:1 images of micrometer-sized trapped probes in the backscattering detection mode. The detector is based on the quadrant photo-integrated chip in the optical pick-up head of a compact disc player. Using this detector, we measured absolute displacements of an optically trapped 1.1 μm probe with a resolution of ～10 nm for a bandwidth of 10 Hz at 95% significance without any sample or laser stabilization. We characterized our optical trap for different sized probes by measuring the power spectrum for each probe to 1% accuracy, and found that for 1.1 μm diameter probes, the noise in our position measurement matched the thermal resolution limit for averaging times up to 10 ms. We also achieved a linear response range of around 385 nm with cross talk between axes ?4% for 1.1 μm diameter probes. The detector has extremely high bandwidth (few MHz) and low optical power threshold-other factors that can lead to its widespread use in photonic force microscopy.     

IV and IP administration of rhodamine in visualization of WBC‐BBB interactions in cerebral vessels

Epi‐illuminescence intravital fluorescence microscopy has been employed to study leukocyte‐endothelial interactions in a number of brain pathologies. Historically, dyes such as Rhodamine 6G have been injected intravenously. However, intravenous injections can predispose experimental animals to a multitude of complications and requires a high degree of technical skill. Here, we study the efficacy of injecting Rhodamine 6G into the peritoneum (IP) for the purpose of analyzing leukocyte‐endothelial interactions through a cranial window during real time intravital microscopy. After examining the number of rolling and adherent leukocytes through a cranial window, we found no advantage to the intravenous injection (IV). Additionally, we tested blood from both routes of injection by flow cytometry to gain a very precise picture of the two methods. The two routes of administration failed to show any difference in the ability to detect cells. The study supports the notion that IP Rhodamine 6G works as efficaciously as IV and should be considered a viable alternative in experimental design for investigations employing intravital microscopy. Facilitated intravital studies will allow for more exploration into cerebral pathologies and allow for more rapid translation from the laboratory to the patient with less chance of experimental error from failed IV access. Microsc. Res. Tech. 78:894–899, 2015. © 2015 Wiley Periodicals, Inc.     

Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy

Endothelial glycocalyx (GCX) is located on the apical surface of vascular endothelial cells and is composed of a negatively‐charged network of proteoglycans and glycoproteins. The GCX plays an important role in maintaining the integrity of vascular walls and preventing leakage of plasma. Therefore, degradation of the GCX is believed to lead to pathological leakage of plasma. Because the GCX is a very thin layer, its ultrastructural image has been demonstrated on electron microscope. To explore the function of the GCX, it should be visualized by a microscope in vivo. Thus, we developed in vivo visualization technique of the GCX under fluorescence microscopy using a mouse dorsal skinfold chamber (DSC) model. To label and visualize the GCX, we used fluorescein isothiocyanate (FITC)‐labeled lectin, which has a high specificity for sugar moieties. We examined the affinity of the different lectins to epivascular regions under an intravital fluorescent microscope. Among seven different lectins we examined, FITC labeled Triticum vulgaris (wheat germ) agglutinin (WGA) delineated the GCX most clearly. Binding of WGA to the GCX was inhibited by chitin hydrolysate, which contained WGA‐binding polysaccharide chains. Furthermore, the septic condition attenuated this structure, suggesting structural degradation of endothelial GCX layer. In conclusion, FITC‐labeled WGA lectin enabled visualization of endothelial GCX under in vivo fluorescence microscopy. Microsc. Res. Tech. 79:31–37, 2016. © 2015 Wiley Periodicals, Inc.     

Influence of the microenvironment on gene and protein expression of odontogenic-like and osteogenic-like cells

Progenitor cells play an important biological role in tooth and bone formation, and previous analyses during bone and dentine induction have indicated that they may be a good alternative for tissue engineering. Thus, to clarify the influence of the microenvironment on protein and gene expression, MDPC23 cells (mouse dental papilla cell line) and KUSA/A1 cells (bone marrow stromal cell line) were used, both in vitro cell culture and in intra-abdominal diffusion chambers implanted in 4-week-old male immunodefficient mice (SCID mice). Our results indicate that KUSA/A1 cells differentiated into osteoblast-like cells and induced bone tissue inside the chamber, whereas, MDPC-23 showed odontoblast-like characteristics but with a low ability to induce dentin formation. This study shows that MDPC-23 cells are especial cells, which possess morphological and functional characteristics of odontoblast-like cells expressing dentin sialophosphoprotein in vivo. In contrast, dentin sialophosphoprotein gene and protein expression was not detected in both cell lines in vitro. The intra-abdominal diffusion chamber appears as an interesting experimental model for studying phenotypic expression of dental pulp cells in vivo.