Live-cell imaging of dendritic spines by STED microscopy

Time lapse fluorescence imaging has become one of the most important approaches in neurobiological research. In particular, both confocal and two-photon microscopy have been used to study activity-dependent changes in synaptic morphology. However, the diffraction-limited resolution of light microscopy is often inadequate, forcing researchers to complement the live cell imaging strategy by EM. Here, we report on the first use of a far-field optical technique with subdiffraction resolution to noninvasively image activity-dependent morphological plasticity of dendritic spines. Specifically we show that time lapse stimulated emission depletion imaging of dendritic spines of YFP-positive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. The technique substantially improves the quantification of morphological parameters, such as the neck width and the curvature of the heads of spines, which are thought to play critical roles for the function and plasticity of synaptic connections.     

Surface Morphology of Textured Transparent Conductive Oxide Thin Film Seen by Various Probes: Visible Light,X-rays,Electron Scattering and Contact Probe

Fluorine-doped tin oxide thin films (SnO₂:F) are widely used as transparent conductive oxide electrodes in thin-film solar cells because of their appropriate electrical and optical properties. The surface morphology of these films influences their optical properties and therefore plays an important role in the overall efficiencies of the solar cells in which they are implemented. At rough surfaces light is diffusely scattered, extending the optical path of light inside the active layer of the solar cell, which in term improves light absorption and solar cell conversion efficiency. In this work, we investigated the surface morphology of undoped and doped SnO₂ thin films and their influence on the optical properties of the films. We have compared and analysed the results obtained by several complementary methods for thin-film surface morphology investigation: atomic force microscopy (AFM), transmission electron microscopy (TEM), and grazing-incidence small-angle X-ray scattering (GISAXS). Based on the AFM and TEM results we propose a theoretical model that reproduces well the GISAXS scattering patterns.     

STED microscopy: increased resolution for medical research?

Optical imaging is crucial for addressing fundamental problems in all areas of life science. With the use of confocal and two‐photon fluorescence microscopy, complex dynamic structures and functions in a plethora of tissue and cell types have been visualized. However, the resolution of ‘classical’ optical imaging methods is poor due to the diffraction limit and does not allow resolution of the cellular microcosmos. On the other hand, the novel stimulated emission depletion (STED) microscopy technique, because of its targeted on/off‐switching of fluorescence, is not hampered by a diffraction‐limited resolution barrier. STED microscopy can therefore provide much sharper images, permitting nanoscale visualization by sequential imaging of individual‐labelled biomolecules, which should allow previous findings to be reinvestigated and provide novel information. The aim of this review is to highlight promising developments in and applications of STED microscopy and their impact on unresolved issues in biomedical science.     

A case study of physicians' use of liver-spleen scans. Are we doing what we think we're doing?

We evaluated physicians' perceptions of the performance capabilities of the liver-spleen scan (LSS) in detecting metastases and the inferences physicians draw from LSS results. Physicians' perceptions of the sensitivity and specificity of the LSS in detection of metastases, as well as their estimates of likelihood ratios for various scan results, varied broadly over a range that could result in clinically important variations in patient treatment. In addition, independent of any variations in estimates of test performance characteristics, physicians had difficulty drawing appropriate probabilistic inferences from LSS results. Our findings suggest that data regarding the performance capabilities of the LSS and other diagnostic tests within a particular hospital should be made available to physicians and that physicians should be given microcomputer assistance in estimating the impact of test results on the probability of disease.     

Advances in colonic imaging: new endoscopic imaging methods

There is a need for better endoscopic visualization in specific circumstances like detection of flat colorectal lesions and dysplasia-screening in ulcerative colitis. Chromoendoscopy is a technique with proven success, but many more, novel endoscopic techniques are currently under investigation. In this article different point measurement and still imaging methods are discussed: Raman spectroscopy, elastic (light) scattering spectroscopy, fluorescence spectroscopy, optical coherence tomography and confocal laser microscopy. Furthermore, real-time endoscopic imaging methods are discussed. These include narrow band imaging, fluorescence imaging and endocytoscopy. The results of fluoroscence imaging might be improved by application of photosensitizers or coupling of fluorescent dyes to tumour-related antigens (immunoscopy). Most of these techniques still have to be developed further and are not yet available for routine use. In our opinion, a combination of a red-flag technique and a microscopic technique carries an enormous potential.     

Advancing biomedical imaging

Imaging reveals complex structures and dynamic interactive processes, located deep inside the body, that are otherwise difficult to decipher. Numerous imaging modalities harness every last inch of the energy spectrum. Clinical modalities include magnetic resonance imaging (MRI), X-ray computed tomography (CT), ultrasound, and light-based methods [endoscopy and optical coherence tomography (OCT)]. Research modalities include various light microscopy techniques (confocal, multiphoton, total internal reflection, superresolution fluorescence microscopy), electron microscopy, mass spectrometry imaging, fluorescence tomography, bioluminescence, variations of OCT, and optoacoustic imaging, among a few others. Although clinical imaging and research microscopy are often isolated from one another, we argue that their combination and integration is not only informative but also essential to discovering new biology and interpreting clinical datasets in which signals invariably originate from hundreds to thousands of cells per voxel.     

Temporal Imaging of Live Cells by High-Speed Confocal Raman Microscopy

Label-free live cell imaging was performed using a custom-built high-speed confocal Raman microscopy system. For various cell types, cell-intrinsic Raman bands were monitored. The high-resolution temporal Raman images clearly delineated the intracellular distribution of biologically important molecules such as protein, lipid, and DNA. Furthermore, optical phase delay measured using quantitative phase microscopy shows similarity with the image reconstructed from the protein Raman peak. This reported work demonstrates that Raman imaging is a powerful label-free technique for studying various biomedical problems in vitro with minimal sample preparation and external perturbation to the cellular system.     

Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction

Biological systems are known to be highly transparent to 700- to 1,100-nm near-infrared (NIR) light. It is shown here that the strong optical absorbance of single-walled carbon nanotubes (SWNTs) in this special spectral window, an intrinsic property of SWNTs, can be used for optical stimulation of nanotubes inside living cells to afford multifunctional nanotube biological transporters. For oligonucleotides transported inside living cells by nanotubes, the oligos can translocate into cell nucleus upon endosomal rupture triggered by NIR laser pulses. Continuous NIR radiation can cause cell death because of excessive local heating of SWNT in vitro. Selective cancer cell destruction can be achieved by functionalization of SWNT with a folate moiety, selective internalization of SWNTs inside cells labeled with folate receptor tumor markers, and NIR-triggered cell death, without harming receptor-free normal cells. Thus, the transporting capabilities of carbon nanotubes combined with suitable functionalization chemistry and their intrinsic optical properties can lead to new classes of novel nanomaterials for drug delivery and cancer therapy.     

Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell

We demonstrate far-field optical imaging with subdiffraction resolution of the endoplasmic reticulum (ER) in the interior of a living mammalian cell. The diffraction barrier is overcome by applying stimulated emission depletion (STED) on a yellow fluorescent protein tag. Imaging individual structural elements of the ER revealed a focal plane (x, y) resolution of <50 nm inside the living cell, corresponding to a 4-fold improvement over that of a confocal microscope and a 16-fold reduction in the focal-spot cross-sectional area. A similar gain in resolution is realized with both pulsed- and continuous-wave laser illumination. Images of highly convoluted parts of the ER reveal a similar resolution improvement in 3D optical sectioning by a factor of 3 along the optic axis (z). Time-lapse STED recordings document morphological changes of the ER over time. Thus, nanoscale 3D imaging of organelles in the interior of living cells greatly expands the scope of light microscopy in cell biology.     

Multimodal Gold Nanostars as SERS Tags for Optically-Driven Doxorubicin Release Study in Cancer Cells

Surface Enhanced Raman Scattering (SERS) active gold nanostars represent an opportunity in the field of bioimaging and drug delivery. The combination of gold surface chemical versatility with the possibility to tune the optical properties changing the nanoparticles shape constitutes a multimodal approach for the investigation of the behavior of these carriers inside living cells. In this work, SERS active star-shaped nanoparticles were functionalized with doxorubicin molecules and covered with immuno-mimetic thiolated polyethylene glycol (PEG). Doxorubicin-conjugate gold nanoparticles show an intense Raman enhancement, a good stability in physiological conditions, and a low cytotoxicity. The strong adsorption of the anticancer drug doxorubicin in close contact with the gold nanostars surface enables their use as SERS tag imaging probes in vivo. Upon laser irradiation of the nanoparticles, a strong SERS signal is generated by the doxorubicin molecules close to the nanostars surface, enabling the localization of the nanoparticles inside the cells. After long time irradiation, the SERS signal drops, indicating the thermally driven delivery of the drug inside the cell. Therefore, the combination of SERS and laser scanning confocal microscopy is a powerful technique for the real-time analysis of drug release in living cells.     

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to “pinhole cross-talk,” which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.     

Optical measurement of cycle-dependent cell growth

Determining the growth patterns of single cells offers answers to some of the most elusive questions in contemporary cell biology: how cell growth is regulated and how cell size distributions are maintained. For example, a linear growth in time implies that there is no regulation required to maintain homeostasis; an exponential pattern indicates the opposite. Recently, there has been great effort to measure single cells using microelectromechanical systems technology, and several important questions have been explored. However, a unified, easy-to-use methodology to measure the growth rate of individual adherent cells of various sizes has been lacking. Here we demonstrate that a newly developed optical interferometric technique, known as spatial light interference microscopy, can measure the cell dry mass of many individual adherent cells in various conditions, over spatial scales from micrometers to millimeters, temporal scales ranging from seconds to days, and cell types ranging from bacteria to mammalian cells. We found evidence of exponential growth in Escherichia coli, which agrees very well with other recent reports. Perhaps most importantly, combining spatial light interference microscopy with fluorescence imaging provides a unique method for studying cell cycle-dependent growth. Thus, by using a fluorescent reporter for the S phase, we measured single cell growth over each phase of the cell cycle in human osteosarcoma U2OS cells and found that the G2 phase exhibits the highest growth rate, which is mass-dependent and can be approximated by an exponential.     

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.Fluorescence microscopy is an extremely powerful research tool in the life sciences. It combines highest sensitivity with molecular specificity and exceptional image contrast. However, as with all light-based microscopy techniques, its resolution is limited by the diffraction of light to a typical lateral resolution of ∼200 nm and an axial resolution of ∼500 nm (for 500-nm wavelength light). Only recently, this diffraction limit was broken by using the quantum, or nonlinear, character of fluorescence excitation and emission. The first of these superresolution methods was stimulated emission depletion (STED) microscopy (1). Later, methods based on single-molecule localization, such as photoactivated localization microscopy (PALM) (2) and stochastic optical reconstruction microscopy (STORM) (3), joined the field. These methods “break” the diffraction limit because they all use principles that operate beyond the diffraction of light.Although still bound to light diffraction, increased spatial resolution can be achieved in a class of advanced resolution methods that exploit a clever combination of excitation and detection modalities (4–7). Although these methods do not reach the resolution achievable with STED, PALM, STORM, and related techniques, they do not require any specialized labels or high excitation intensities, and they may be applied to any fluorescent sample at any excitation/emission wavelength. The most prominent example of this class is structured illumination microscopy (SIM) (5), in which one scans a sample with a structured illumination pattern while taking images with a wide-field imaging system. Meanwhile, several commercial instruments for SIM have become available. The disadvantages of SIM are its technical complexity, reflected in the rather large cost of the commercially available systems, and its sensitivity to optical imperfections and aberrations, which are unavoidable in biological samples.In a theoretical study in 1988, Sheppard (8) pointed out that it is possible to double the resolution of a scanning confocal microscope in a manner closely related to SIM. In SIM, one starts with a conventional wide-field imaging microscope, and by implementing a scanning structured illumination, one subsequently obtains, after appropriate deconvolution of the recorded images, an image with increased resolution. In image-scanning microscopy (ISM), as proposed by Sheppard, one starts with a conventional confocal microscope that uses a diffraction-limited laser focus for scanning a sample but replaces the point detector typically used for recording the excited fluorescence signal with an imaging detector. Also here, an image with enhanced resolution is obtained after applying an appropriate algorithm to the recorded images.We experimentally realized this idea first in 2010 (4), indeed demonstrating a substantial increase in resolution. The major drawback of this implementation was the slowness of the imaging. At each scan position of the laser focus, an image of the excited region had to be recorded, limiting the scan speed by the frame rate of the imaging camera used. For the small scan area of 2 µm × 2 µm shown with the original ISM setup, data acquisition took 25 s. In 2012, York et al. (6) demonstrated that this limitation may be overcome by using a multifocal excitation scheme. They generated an array of multiple excitation foci by implementing a digital micro-mirror device (DMD) into the excitation path of a wide-field microscope. Using this system, ISM images can be obtained with excellent speed, in two excitation/emission wavelengths (two-color imaging) and in three dimensions. However, this approach requires the incorporation of a DMD with all the necessity of perfect optical alignment.Here, we demonstrate that existing imaging detector-based confocal systems can be converted easily into a doubly resolving ISM system. This mainly includes two kinds of microscopes that are widely available in research laboratories: confocal spinning-disk (CSD) microscopes and rapid laser scanning confocal microscopes with an imaging camera as the detector. We present the results obtained with a CSD system.     

Covalent Binding of Heparin to Functionalized PET Materials for Improved Haemocompatibility

The hemocompatibility of vascular grafts made from poly(ethylene terephthalate) (PET) is insufficient due to the rapid adhesion and activation of blood platelets that occur upon incubation with whole blood. PET polymer was treated with NH_x radicals created by passing ammonia through gaseous plasma formed by a microwave discharge, which allowed for functionalization with amino groups. X-ray photoelectron spectroscopy characterization using derivatization with 4-chlorobenzaldehyde indicated that approximately 4% of the –NH₂ groups were associated with the PET surface after treatment with the gaseous radicals. The functionalized polymers were coated with an ultra-thin layer of heparin and incubated with fresh blood. The free-hemoglobin technique, which is based on the haemolysis of erythrocytes, indicated improved hemocompatibility, which was confirmed by imaging the samples using confocal optical microscopy. A significant decrease in number of adhered platelets was observed on such samples. Proliferation of both human umbilical vein endothelial cells and human microvascular endothelial cells was enhanced on treated polymers, especially after a few hours of cell seeding. Thus, the technique represents a promising substitute for wet-chemical modification of PET materials prior to coating with heparin.     

Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo.Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry,super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., realtime quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers,drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and highspeed imaging of cell transient deformability in flow.In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cell's functional state (e.g., live, apoptotic, or necrotic),and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed.     

Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo.Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry,super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., realtime quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers,drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and highspeed imaging of cell transient deformability in flow.In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cell's functional state (e.g., live, apoptotic, or necrotic),and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed.     

Optical clearing facilitates integrated 3D visualization of mouse ileal microstructure and vascular network with high definition

Intestinal microvasculature plays a central role in nutrient absorption and immune response against infections. Microscopic visualization of intestinal microvasculature under normal and pathological conditions such as inflammatory bowel disease is essential for understanding the pathophysiology of the disease. Despite the intensive need to characterize the intestinal microstructure and vasculature in an integrated fashion, 3-dimensional (3D) visualization of the gastrointestinal tissue is often limited by the spatial resolution of the imaging tools. In this research, we aimed to apply optical clearing to minimize the random light scattering in the mouse ileum, thereby facilitating photon penetration for high-resolution, 3D optical microscopy of the tissue network without microtome sectioning. We applied cardiac perfusion of lipophilic dialkylcarbocyanine dye DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) to label the mouse blood vessels, including the intestinal microvasculature. The labeled and paraformaldehyde-fixed ileum was immersed in the aqueous optical-clearing solution to improve photon penetration. Optical clearing revealed the interior domain of the mouse ileal mucosa and submucosa, where random light scattering was suppressed and the size of the microstructure in the fixed specimen remained the same. Using fluorescent labeling, the intestinal microstructure and vasculature were simultaneously imaged by 3D confocal microscopy to allow for an integrated visualization of the tissue network with high definition. This new optical approach provides a useful tool for 3D presentation and analysis of the microvasculature for better understanding the intestinal physiology.     

VEGF165 antisense RNA suppresses oncogenic properties of human esophageal squamous cell carcinoma

AIM: To investigate the effect of antisense RNA to vascularendothelial growth factor165 (VEGF165) on human esophagealsquamous cell carcinoma call line EC109 and the feasibilityof gene therapy for esophageal carcinoma.METHODS: Using subclone technique, the full length ofVEGF165 amino acid cDNA, which was cut from pGEM-3Zf( + ), was cloned inversely into the eukaryotic expressionvector pCEP4 . The recombinant plasmid pCEP-AVEGF165was transfected into EC109 cell with Iipofectamine. After astable transfection, dot Blot, enzyme-linked immunosorbentassay (ELISA), laser confocal imaging system analysis,transmission electron microscopy and flow cytometry wereperformed to determine the biological characteristics ofEC109 cell line before and after transfection in vitro andwhether there was a reversion in the tumorigenic propertiesof the EC109 cell in vivo.RESULTS: The eukaryotic expression vector pCEP-AVEGF165was successfully constructed and transfected into EC109cells. The expression of VEGF165 was significantly decreasedin the transfected cells while the biological characteristics ofthe cells were not influenced by the expression of antisensegene. The tumorigenic and angiogenic capabilities weregreatly reduced in nude mice, as demonstrated by reducedtumorend volume (820 ± 112.5)mm3 vs (7930 ± 1035) mn3and (7850 ± 950) mm3, P ＜ 0.01) and microvessel density(8.5 ± 1.2)mm-2 vs (44.3 ± 9.4)mm-2 and (46.4 ± 12.6)mm-2, P ＜ 0.01) in comparison between experimentalgroup, empty vector transfected group and control group.CONCLUSION: The angiogenesis and tumorigenicity ofhuman esophageal squamous cell carcinoma were effectivelyinhibited by VEGF165 antisense RNA. Antisense RNA toVEGF1, can potentially be used as an adjuvant therapy forsolid tumors.