High resolution intravital imaging of subcellular structures of mouse abdominal organs using a microstage device

Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated 'microstage' that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in real-time as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox.     

Biomechanical imaging of cell stiffness and prestress with subcellular resolution

Knowledge of cell mechanical properties, such as elastic modulus, is essential to understanding the mechanisms by which cells carry out many integrated functions in health and disease. Cellular stiffness is regulated by the composition, structural organization, and indigenous mechanical stress (or prestress) borne by the cytoskeleton. Current methods for measuring stiffness and cytoskeletal prestress of living cells necessitate either limited spatial resolution but with high speed, or spatial maps of the entire cell at the expense of long imaging times. We have developed a novel technique, called biomechanical imaging, for generating maps of both cellular stiffness and prestress that requires less than 30 s of interrogation time, but which provides subcellular spatial resolution. The technique is based on the ability to measure tractions applied to the cell while simultaneously observing cell deformation, combined with capability to solve an elastic inverse problem to find cell stiffness and prestress distributions. We demonstrated the application of this technique by carrying out detailed mapping of the shear modulus and cytoskeletal prestress distributions of 3T3 fibroblasts, making no assumptions regarding those distributions or the correlation between them. We also showed that on the whole cell level, the average shear modulus is closely associated with the average prestress, which is consistent with the data from the literature. Data collection is a straightforward procedure that lends itself to other biochemical/biomechanical interventions. Biomechanical imaging thus offers a new tool that can be used in studies of cell biomechanics and mechanobiology where fast imaging of cell properties and prestress is desired at subcellular resolution.     

Intracellular dynamics of topoisomerase I inhibitor,CPT-11, by slit-scanning confocal Raman microscopy

Most molecular imaging technologies require exogenous probes and may have some influence on the intracellular dynamics of target molecules. In contrast, Raman scattering light measurement can identify biomolecules in their innate state without application of staining methods. Our aim was to analyze intracellular dynamics of topoisomerase I inhibitor, CPT-11, by using slit-scanning confocal Raman microscopy, which can take Raman images with high temporal and spatial resolution. We could acquire images of the intracellular distribution of CPT-11 and its metabolite SN-38 within several minutes without use of any exogenous tags. Change of subcellular drug localization after treatment could be assessed by Raman imaging. We also showed intracellular conversion from CPT-11 to SN-38 using Raman spectra. The study shows the feasibility of using slit-scanning confocal Raman microscopy for the non-labeling evaluation of the intracellular dynamics of CPT-11 with high temporal and spatial resolution. We conclude that Raman spectromicroscopic imaging is useful for pharmacokinetic studies of anticancer drugs in living cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Joint tagging assisted fluctuation nanoscopy enables fast high‐density super‐resolution imaging

In fluctuation‐based optical nanoscopy, investigating high‐density labeled subcellular structures with high fidelity has been a significant challenge. In this study, based on super‐resolution radial fluctuation (SRRF) microscopy, the joint tagging (JT) strategy is employed to enable fast high‐density nanoscopic imaging and tracking. In fixed cell experiment, multiple types of quantum dots with distinguishable fluorescence spectra are jointly tagged to subcellular microtubules. In each spectral channel, the decrease in labeling density guarantees the high‐fidelity super‐resolution reconstruction using SRRF microscopy. Subsequently, the combination of all spectral channels achieves high‐density super‐resolution imaging of subcellular microtubules with a resolution of ~62 nm using JT assisted SRRF technique. In the live‐cell experiment, 3‐channel JT is utilized to track the dynamic motions of high‐density toxin‐induced lipid clusters for 1 minute, achieving the simultaneous tracking of many individual toxin‐induced lipid clusters spatially distributed significantly below the optical diffraction limit in living cells.      

Multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures: assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale.

We have applied multiple site optical recording of transmembrane voltage (MSORTV) to patterned growth cultures of heart cells to analyze the effect of geometry per se on impulse propagation in excitable tissue, with cellular and subcellular resolution. Extensive dye screening led to the choice of di-8-ANEPPS as the most suitable voltage-sensitive dye for this application; it is internalized slowly and permits optical recording with signal-to-noise ratios as high as 40:1 (measured peak-to-peak) and average fractional fluorescence changes of 15% per 100 mV. Using a x 100 objective and a fast data acquisition system, we could resolve impulse propagation on a microscopic scale (15 microns) with high temporal resolution (uncertainty of +/- 5 microseconds). We could observe the decrease in conduction velocity of an impulse propagating along a narrow cell strand as it enters a region of abrupt expansion, and we could explain this phenomenon in terms of the micro-architecture of the tissue. In contrast with the elongated and aligned cells forming the narrow strands, the cells forming the expansions were aligned at random and presented 2.5 times as many cell-to-cell appositions per unit length. If the decrease in conduction velocity results entirely from this increased number of cell-to-cell boundaries per unit length, the mean activation delay introduced by each boundary can be estimated to be 70 microseconds. Using this novel experimental system, we could also demonstrate the electrical coupling of fibroblasts and endotheloid cells to myocytes in culture.     

Glucagon-like peptide 1 and peptide YY are in separate storage organelles in enteroendocrine cells

A sub-group of enteroendocrine cells (L cells) release gastrointestinal hormones, GLP-1 and PYY, which have different but overlapping physiological effects, in response to intraluminal nutrients. Whilst their release profiles are not identical, how the plasma levels of these two hormones are differentially regulated is not well understood. We investigate the possibility that GLP-1 and PYY are in separate storage vesicles. In this study, the subcellular location of GLP-1 and PYY storage organelles is investigated using double-labelling immunohistochemistry, super resolution microscopy and high-resolution confocal microscopy. In all species tested, human, pig, rat and mouse, most cytoplasmic stores that exhibited GLP-1 or PYY immunofluorescence were distinct from each other. The volume occupancy, determined by 3D analysis, overlapped by only about 10～20 %. At the lower resolution achieved by conventional confocal microscopy, there was also evidence of GLP-1 and PYY being in separate storage compartments but, in subcellular regions where there were many storage vesicles, separate storage could not be resolved. The results indicate that different storage vesicles in L cells contain predominantly GLP-1 or predominantly PYY. Whether GLP-1 and PYY storage vesicles are selectively mobilised and their products are selectively released needs to be determined.     

Visualizing the subcellular localization of RHOB-GTP and GTPase-Effector complexes using a split-GFP/nanobody labelling assay

Small GTPases are highly regulated proteins that control essential signaling pathways through the activity of their effector proteins. Among the RHOA subfamily, RHOB regulates peculiar functions that could be associated with the control of the endocytic trafficking of signaling proteins. Here, we used an optimized assay based on tripartite split-GFP complementation to localize GTPase-effector complexes with high-resolution. The detection of RHOB interaction with the Rhotekin Rho binding domain (RBD) that specifically recognizes the active GTP-bound GTPase, is performed in vitro by the concomitant addition of recombinant GFP1–9 and a GFP nanobody. Analysis of RHOB-RBD complexes localization profiles combined with immunostaining and live cell imaging indicated a serum-dependent reorganization of the endosomal and membrane pool of active RHOB. We further applied this technology to the detection of RHO-effector complexes that highlighted their subcellular localization with high resolution among the different cellular compartments.     

Sensitive and High Resolution Localization and Tracking of Membrane Proteins in Live Cells with BRET

Peripheral and integral membrane proteins can be located in several different subcellular compartments, and it is often necessary to determine the location of such proteins or to track their movement in living cells. Image‐based colocalization of labeled membrane proteins and compartment markers is frequently used for this purpose, but this method is limited in terms of throughput and resolution. Here we show that bioluminescence resonance energy transfer (BRET) between membrane proteins of interest and compartment‐targeted BRET partners can report subcellular location and movement of membrane proteins in live cells. The sensitivity of the method is sufficient to localize a few hundred protein copies per cell. The spatial resolution can be sufficient to determine membrane topology, and the temporal resolution is sufficient to track changes that occur in less than 1 second. BRET requires little user intervention, and is thus amenable to large‐scale experimental designs with standard instruments.     

Technical advance: an automated device for cryofixation of specimens of electron microscopy using liquid helium

Metal-contact rapid freezing using liquid helium is theoretically the best method for preserving the fine structure of living cells with high temporal resolution in preparation of tissue samples for electron microscopy. However, this method is not commonly used, because of its technical difficulty and low reproducibility. We have designed and constructed an automatic device which allows simple, rapid and reproducible preparation of high-quality electron microscopic specimens by the non-specialist. We assessed the quality of cryofixation in samples prepared using this device by examining the preservation of cellular ultrastructure in relation to distance from the freezing block, and found that the region within 10 microm of the metal-contact plane was fixed with the highest quality. We applied this device, in combination with freeze-substitution methods and immunocytochemical techniques, to two phenomena involving rapid movement of subcellular components: (1) active movement of subcellular structures in the papillar cells of stigma and (2) light-induced rapid subcellular translocation of phytochrome A. Considering the importance of understanding subcellular processes of living cells for molecular and cell biology, this device will be a useful tool for diverse biological applications in the near future.     

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.     

Depicting the Spatial Distribution of Proteins in Human Tumor Tissue Combining SELDI and MALDI Imaging and Immunohistochemistry

Carcinoma tissue consists of not only tumor cells but also fibroblasts, endothelial cells or vascular structures, and inflammatory cells forming the supportive tumor stroma. Therefore, the spatial distribution of proteins that promote growth and proliferation in these complex functional units is of high interest. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a newly developed technique that generates spatially resolved profiles of protein signals directly from thin tissue sections. Surface-enhanced laser desorption/ionization mass spectrometry (MS)combined with tissue microdissection allows analysis of defined parts of the tissue with a higher sensitivity and a broader mass range. Nevertheless, both MS-based techniques have a limited spatial resolution. IHC is a technique that allows a resolution down to the subcellular level. However, the detection and measurement of a specific protein expression level is possible only by semiquantitative methods. Moreover, prior knowledge about the identity of the proteins of interest is necessary. In this study, we combined all three techniques to gain highest spatial resolution, sensitivity, and quantitative information. We used frozen tissue from head and neck tumors and chose two exemplary proteins (HNP1–3 and S100A8) to highlight the advantages and disadvantages of each technique. It could be shown that the combination of these three techniques results in congruent but also synergetic data. (J Histochem Cytochem 58:929–937, 2010)     

Imaging the subcellular structure of human coronary atherosclerosis using micro-optical coherence tomography

Progress in understanding, diagnosis, and treatment of coronary artery disease (CAD) has been hindered by our inability to observe cells and extracellular components associated with human coronary atherosclerosis in situ. The current standards for microstructural investigation, histology and electron microscopy are destructive and prone to artifacts. The highest-resolution intracoronary imaging modality, optical coherence tomography (OCT), has a resolution of ~10 μm, which is too coarse for visualizing most cells. Here we report a new form of OCT, termed micro-optical coherence tomography (μOCT), whose resolution is improved by an order of magnitude. We show that μOCT images of cadaver coronary arteries provide clear pictures of cellular and subcellular features associated with atherogenesis, thrombosis and responses to interventional therapy. These results suggest that μOCT can complement existing diagnostic techniques for investigating atherosclerotic specimens, and that μOCT may eventually become a useful tool for cellular and subcellular characterization of the human coronary wall in vivo.     

A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images.     

Multi‐ATOM: Ultrahigh‐throughput single‐cell quantitative phase imaging with subcellular resolution

A growing body of evidence has substantiated the significance of quantitative phase imaging (QPI) in enabling cost‐effective and label‐free cellular assays, which provides useful insights into understanding the biophysical properties of cells and their roles in cellular functions. However, available QPI modalities are limited by the loss of imaging resolution at high throughput and thus run short of sufficient statistical power at the single‐cell precision to define cell identities in a large and heterogeneous population of cells—hindering their utility in mainstream biomedicine and biology. Here we present a new QPI modality, coined multiplexed asymmetric‐detection time‐stretch optical microscopy (multi‐ATOM) that captures and processes quantitative label‐free single‐cell images at ultrahigh throughput without compromising subcellular resolution. We show that multi‐ATOM, based upon ultrafast phase‐gradient encoding, outperforms state‐of‐the‐art QPI in permitting robust phase retrieval at a QPI throughput of >10 000 cell/sec, bypassing the need for interferometry which inevitably compromises QPI quality under ultrafast operation. We employ multi‐ATOM for large‐scale, label‐free, multivariate, cell‐type classification (e.g. breast cancer subtypes, and leukemic cells vs peripheral blood mononuclear cells) at high accuracy (>94%). Our results suggest that multi‐ATOM could empower new strategies in large‐scale biophysical single‐cell analysis with applications in biology and enriching disease diagnostics.      

Alpharadiography: A Simple Method for Determination of Mass Concentration in Cells and Tissues

A simple apparatus for the production of contact microradiographs with the help of a polonium alpha source and nuclear emulsion plates is described. This apparatus best adapted for soft tissue and low grade mineralization studies offers advantages as to resolution, geometry of specimen as well as ease of operation and cost.     

Uptake of taurine into subcellular fractions of C-6 glioma cells.

Subcellular fractions from cultured C-6 glioma cells prepared by methods similar to those for crude synaptosomal fractions of rat cerebral cortex accumulated [³⁵S]taurine as did intact glioma cells. Thus, the accumulation of taurine was dependent on temperature and sodium concentration and sensitive to osmotic shock. The kinetic properties of this uptake are characterized by an apparent K_m, of about 25 μm, The properties of taurine uptake into subcellular fractions from C-6 glioma cells were compared with those of crude synaptosomal fractions and differences could be observed in temperature sensitivity and with metabolic inhibitors, which were less potent in the glioma preparation. Equilibrium density gradient centrifugation of subcellular fractions from glioma cells revealed that particles containing [³⁵S]taurine sediment to a lower buoyant density than mitochondria. But on co-sedimentation of subcellular fractions from glioma cells with synaptosomal fractions derived from cerebral cortex, differences in the buoyant density between these two preparations could be found. The findings support the possibility of a contamination of synaptosomal fractions with subcellular fractions derived from glial origin.     

Digital evaluation of compacta cross-section preparations

In order to be able to evaluate compacta cross-section preparations morphometrically, we produced contrasting microradiographs of this material. We recorded the compacta structures in the microradiographs as digital images by means of a TAS image analysis system (Leitz/Bosch) and stored them on discs. The digital images thus restored can be evaluated immediately or at a later date by means of suitable image analysis programs.     

Probing the Subcellular Localization of Hopanoid Lipids in Bacteria Using NanoSIMS

The organization of lipids within biological membranes is poorly understood. Some studies have suggested lipids group into microdomains within cells, but the evidence remains controversial due to non-native imaging techniques. A recently developed NanoSIMS technique indicated that sphingolipids group into microdomains within membranes of human fibroblast cells. We extended this NanoSIMS approach to study the localization of hopanoid lipids in bacterial cells by developing a stable isotope labeling method to directly detect subcellular localization of specific lipids in bacteria with ca. 60 nm resolution. Because of the relatively small size of bacterial cells and the relative abundance of hopanoid lipids in membranes, we employed a primary ²H-label to maximize our limit of detection. This approach permitted the analysis of multiple stable isotope labels within the same sample, enabling visualization of subcellular lipid microdomains within different cell types using a secondary label to mark the growing end of the cell. Using this technique, we demonstrate subcellular localization of hopanoid lipids within alpha-proteobacterial and cyanobacterial cells. Further, we provide evidence of hopanoid lipid domains in between cells of the filamentous cyanobacterium Nostoc punctiforme. More broadly, our method provides a means to image lipid microdomains in a wide range of cell types and test hypotheses for their functions in membranes.     

Demonstration of aluminum in amyloid fibers in the cores of senile plaques in the brains of patients with Alzheimer’s disease

Aluminum (Al) exposure has been reported to be a risk factor for Alzheimer’s disease (senile dementia of Alzheimer type), although the role of Al in the etiology of Alzheimer’s disease remains controversial. We examined the presence of Al in the Alzheimer’s brain using energy-dispersive X-ray spectroscopy combined with transmission electron microscopy (TEM-EDX). TEM-EDX analysis allows simultaneous imaging of subcellular structures with high spatial resolution and analysis of small quantities of elements contained in the same subcellular structures. We identified senile plaques by observation using TEM and detected Al in amyloid fibers in the cores of senile plaques located in the hippocampus and the temporal lobe by EDX. Phosphorus and calcium were also present in the amyloid fibers. No Al could be detected in the extracellular space in senile plaques or in the cytoplasm of nerve cells. In this study, we demonstrated colocalization of Al and beta-amyloid (Abeta) peptides in amyloid fibers in the cores of senile plaques. The results support the following possibilities in the brains of patients with Alzheimer’s disease: Al could be involved in the aggregation of Abeta peptides to form toxic fibrils; Al might induce Abeta peptides into the beta-sheet structure; and Al might facilitate iron-mediated oxidative reactions, which cause severe damage to brain tissues.