Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore

Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique.     

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.     

Estimating intercellular surface tension by laser-induced cell fusion

Intercellular surface tension is a key variable in understanding cellular mechanics. However, conventional methods are not well suited for measuring the absolute magnitude of intercellular surface tension because these methods require determination of the effective viscosity of the whole cell, a quantity that is difficult to measure. In this study, we present a novel method for estimating the intercellular surface tension at single-cell resolution. This method exploits the cytoplasmic flow that accompanies laser-induced cell fusion when the pressure difference between cells is large. Because the cytoplasmic viscosity can be measured using well-established technology, this method can be used to estimate the absolute magnitudes of tension. We applied this method to two-cell-stage embryos of the nematode Caenorhabditis elegans and estimated the intercellular surface tension to be in the 30-90 μN m(-1) range. Our estimate was in close agreement with cell-medium surface tensions measured at single-cell resolution.     

Immobilization Methods for NMR Studies of Cellular Metabolism - A Practical Guide

Nuclear magnetic resonance (NMR) can be used in a nondestructive manner to study cellular metabolism in intact cell samples such as a suspension of cells. However, many different cell types require a well-regulated medium that includes a buffered pH, as well as a continuous supply of oxygen. A series of methods that have been used for the maintenance of the extracellular conditions involves the immobilization of cells, followed by perfusion of the immobilized cell sample. NMR studies can then be performed for extended periods of time, as well as under sterile conditions. We discuss methodology, with perfused erythrocytes and thymocytes as specific examples.     

Immobilized soy-sauce yeasts: development and characterization of a new polyethylene-oxide support

Entrapment of cells in alginate gel is a widely used mild immobilization procedure. However, alginate gel is not very suitable for use in long-term continuous soy-sauce processes because alginate is sensitive to abrasion and chemically unstable towards the high salt content of soy-sauce medium. Therefore, a chemically crosslinked polyethylene-oxide gel was used instead. The disadvantage of this gel was that due to the crosslinking reaction, the viability of the cells after immobilization was poor. For this reason, a new mild procedure for immobilizing soy-sauce yeasts in polyethylene-oxide gel was developed, resulting in high survival percentages of the soy-sauce yeasts Zygosaccharomyces rouxii and Candida versatilis. This newly developed polyethylene-oxide gel, unlike alginate gel, appeared not to be sensitive to abrasion, even in the presence of high salt concentrations. Therefore, we concluded that this newly developed polyethylene-oxide gel is more suitable than alginate gel for use as immobilization material in long-term processes with a high salt content, like soy-sauce processes.     

Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission

Imaging of fluorescence resonance energy transfer (FRET) between suitable fluorophores is increasingly being used to study cellular processes with high spatiotemporal resolution. The genetically encoded Cyan (CFP) and Yellow (YFP) variants of Green Fluorescent Protein have become the most popular donor and acceptor pair in cell biology. FRET between these fluorophores can be imaged by detecting sensitized emission. This technique, for which CFP is excited and transfer is detected as emission of YFP, is sensitive, fast, and straightforward, provided that proper corrections are made. In this study, the detection of sensitized emission between CFP and YFP by confocal microscopy is optimized. It is shown that this FRET pair is best excited at 430 nm. We identify major sources of error and variability in confocal FRET acquisition including chromatic aberrations and instability of the excitation sources. We demonstrate that a novel correction algorithm that employs online corrective measurements yields reliable estimates of FRET efficiency, and it is also shown how the effect of other error sources can be minimized.     

Immobilization of animal cells in fixed bed bioreactors

Immobilization of animal cells has become a highly popular means of achieving high-density animal cell cultures. The advantages of immobilization are that it stabilizes cells in culture and enables long-term culture periods to be achieved. Immobilization increases cell productivity by increasing the usable substrate surface area for anchorage-dependent cells, or by facilitating perfusion of anchorage-independent cells. A method for production of secreted biological products from anchorage-dependent and independent cells is described. The method is based on immobilization of animal cells within the polymeric matrix of polyurethane foam, packed in a fixed bed bioreactor.     

Immobilization of cultured Catharanthus roseus cells using a fibreglass substratum

Summary Cultured Catharanthus roseus cells were immobilized using geometrically identical needled fibreglass mats prepared with a range of surface coatings. The phenyl (PS), polyglycol (PG), aldehyde (CHO), alkyl (CTMS), and silanol (AW) coatings, along with the untreated glass (HC) surface, produced surfaces with a range of surface tensions. The immobilization efficiency of the substratum, measured as the percentage of cells immobilized, increased with increasing substratum surface tension in the order PS < PG < CHO < CTMS < AW < HC. The dependence of immobilization efficiency on substratum surface tension can be described using a thermodynamic model of adhesion that considers the extent of plant cell adhesion to be a function of the surface tensions of the substratum, the suspending liquid, and the plant cells. In addition, this dependence also demonstrates the fundamental role of adhesion in the immobilization process involving a glass fibre matrix. However, cell entrapment processes are also implicated. The untreated glass fibre substratum (HC), which demonstrated the greatest immobilization efficiency, was used for further characterization of the immobilization strategy. Maximum inoculum biomass was determined to be approximately 1.9 g cells (fresh weight)/g substratum (dry weight) to achieve greater than 90% immobilization efficiency. The growth rate of immobilized cultures was slower than suspension cultures, probably due to mass transfer limitations. Production of the indole alkaloids, tryptamine, catharanthine, and ajmalicine, was also suppressed relative to suspension-cultured cells. These results are considered in relation to other immobilization strategies and their apparent effects on cellular processes. Offprint requests to: F. Dicosmo     

Assessing the significance of conserved genomic aberrations using high resolution genomic microarrays

Genomic aberrations recurrent in a particular cancer type can be important prognostic markers for tumor progression. Typically in early tumorigenesis, cells incur a breakdown of the DNA replication machinery that results in an accumulation of genomic aberrations in the form of duplications, deletions, translocations, and other genomic alterations. Microarray methods allow for finer mapping of these aberrations than has previously been possible; however, data processing and analysis methods have not taken full advantage of this higher resolution. Attention has primarily been given to analysis on the single sample level, where multiple adjacent probes are necessarily used as replicates for the local region containing their target sequences. However, regions of concordant aberration can be short enough to be detected by only one, or very few, array elements. We describe a method called Multiple Sample Analysis for assessing the significance of concordant genomic aberrations across multiple experiments that does not require a-priori definition of aberration calls for each sample. If there are multiple samples, representing a class, then by exploiting the replication across samples our method can detect concordant aberrations at much higher resolution than can be derived from current single sample approaches. Additionally, this method provides a meaningful approach to addressing population-based questions such as determining important regions for a cancer subtype of interest or determining regions of copy number variation in a population. Multiple Sample Analysis also provides single sample aberration calls in the locations of significant concordance, producing high resolution calls per sample, in concordant regions. The approach is demonstrated on a dataset representing a challenging but important resource: breast tumors that have been formalin-fixed, paraffin-embedded, archived, and subsequently UV-laser capture microdissected and hybridized to two-channel BAC arrays using an amplification protocol. We demonstrate the accurate detection on simulated data, and on real datasets involving known regions of aberration within subtypes of breast cancer at a resolution consistent with that of the array. Similarly, we apply our method to previously published datasets, including a 250K SNP array, and verify known results as well as detect novel regions of concordant aberration. The algorithm has been fully implemented and tested and is freely available as a Java application at http://www.cbil.upenn.edu/MSA.     

Comparison of atomic force microscopy interaction forces between bacteria and silicon nitride substrata for three commonly used immobilization methods

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.     

Comparison of Atomic Force Microscopy Interaction Forces between Bacteria and Silicon Nitride Substrata for Three Commonly Used Immobilization Methods

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.     

Enzyme immobilization on electrospun polymer nanofibers: An overview

Enzyme immobilization has attracted continuous attention in the fields of fine chemistry, biomedicine, and biosensor. The performance of immobilized enzyme largely depends on the structure of supports. Nanostructured supports are believed to be able to retain the catalytic activity as well as ensure the immobilization efficiency of enzyme to a high extent. Electrospinning provides a simple and versatile method to fabricate nanofibrous supports. Compared with other nanostructured supports (e.g. mesoporous silica, nanoparticles), nanofibrous supports show many advantages for their high porosity and interconnectivity. This review mainly discusses the recent advances in using nanofibers as hosts for enzyme immobilization by two different methods, surface attachment and encapsulation. Surface attachment refers to physical adsorption or covalent attachment of enzymes on pristine or modified nanofibrous supports, and encapsulation means electrospinning a mixture of enzyme and polymer. We make a detailed comparison between these two immobilization approaches and highlight their distinct characteristics. The prospective applications of enzyme immobilized electrospun nanofibers in the development of biosensors, biofuel cells and biocatalysts are also discussed.     

Hydrodynamic deposition: a novel method of cell immobilization.

A novel method of cell immobilization is described. The cell support consists of ceramic microspheres of approximately 50-75 microns diameter. The spheres are hollow, having a wall thickness of 10-15 microns and one entrance (ca. 20 microns diameter). The walls are porous with a mean pore size of approximately 90 nm. When a cell suspension (of S. cerevisiae) is passed through a column of such particles, cells are immobilized. Conditions are devised such that the overwhelming majority of cells are held in the central cavity of the support and not between the particles. Provided turbulence is avoided, the distribution of cells along the column length in the steady state is rather homogeneous. The facts that (a) essentially all particles, regardless of orientation, entrap cells, and (b) nonporous particles also entrap cells with high efficiency, indicate that filtration effects are irrelevant and that heretofore unrecognized hydrodynamic forces are alone responsible for the cell immobilization. Cells can be immobilized to high biomass densities, while the hydrodynamic properties of columns containing such immobilized cells are excellent. We describe an on-line electronic method for the real-time measurement of immobilized cellular biomass. Cell growth (so recorded) and metabolism continue to occur in such particles at high rates. Using the glycolytic production of ethanol by S. cerevisiae as a model reaction, volumetric productivities as great as any published are obtained. Thus the "lobster-pot effect" or "hydrodynamic deposition" represents a novel, promising, and generally applicable method of cell immobilization.     

Protein immobilization on plasma-polymerized ethylenediamine-coated glass slides

For protein chip construction, protein immobilization on the surface of the glass slide is essential. It was previously reported that glass slides are embedded with chemicals that contain primary amines and aldehydes for protein immobilization. We fabricated a plasma-polymerized ethylenediamine (PPEDA)-coated slide that exposed primary amines. For the plasma polymer deposition on the glass slide, the inductively coupled plasma (ICP) power was found to be a critical factor in sustaining a high density of amine on the surface of the PPEDA films. We prepared PPEDA-coated slides at three different ICP powers (3, 30, or 70 W). In the slide that was prepared at a low ICP power (3 W), we detected a high density of primary amine. Therefore, the fluorescein isothiocyanate-conjugated immunoglobulin G (IgG) was highly immobilized to the PPEDA-coated slide that was prepared at the low ICP power. For protein immobilization, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used as a cross-linker. The immobilization of the protein to the PPEDA-coated slide was carried out by consecutive incubations with 1 mg/ml EDC for 5 min and 0.1 mg/ml IgG for 1 h. This efficiently produced the functionally active protein-immobilized slide. Therefore, this work shows that the plasma technique can be applied to produce a high-quality glass surface for the immobilization of proteins and other materials.     

Whole-cell biochips for bio-sensing: integration of live cells and inanimate surfaces

Recent advances in the convergence of the biological, chemical, physical, and engineering sciences have opened new avenues of research into the interfacing of diverse biological moieties with inanimate platforms. A main aspect of this field, the integration of live cells with micro-machined platforms for high throughput and bio-sensing applications, is the subject of the present review. These unique hybrid systems are configured in a manner that ensures positioning of the cells in designated patterns, and enables cellular viability maintenance, and monitoring of cellular functionality. Here we review both animate and inanimate surface properties and how they affect cellular attachment, describe relevant modifications of both types of surfaces, list technologies for platform engineering and for cell deposition in the desired configurations, and discuss the influence of various deposition and immobilization methods on the viability and performance of the immobilized cells.     

Whole-cell biochips for bio-sensing: integration of live cells and inanimate surfaces

Recent advances in the convergence of the biological, chemical, physical, and engineering sciences have opened new avenues of research into the interfacing of diverse biological moieties with inanimate platforms. A main aspect of this field, the integration of live cells with micro-machined platforms for high throughput and bio-sensing applications, is the subject of the present review. These unique hybrid systems are configured in a manner that ensures positioning of the cells in designated patterns, and enables cellular viability maintenance, and monitoring of cellular functionality. Here we review both animate and inanimate surface properties and how they affect cellular attachment, describe relevant modifications of both types of surfaces, list technologies for platform engineering and for cell deposition in the desired configurations, and discuss the influence of various deposition and immobilization methods on the viability and performance of the immobilized cells.     

Cell immobilization: Application to alcohol production

The immobilization of whole cells is a technique that can be used in several production processes, among them alcoholic fermentation. By concentrating an active cell biomass in a bioreactor, the efficiency of bioconversion increases, as does the reactor productivity which, in turn, results in the reduction of the reactor size for a given production rate. Immobilization can be carried out in different ways; adsorption and entrapment in gelatinous matrices are the methods most commonly used. These techniques can be applied to practically all of the viable and non-viable whole cells systems of interest. Several reactor configurations are used sucessfully; many more have been proposed. Fermentor design imposes limitations on immobilization processes, requiring study of such aspects as mass-transfer, growth of cells in the reactor, aeration and backmixing.     

抗体固定化方法研究进展

在免疫分析和生物芯片中,抗原-抗体特异性结合被广泛应用,其中抗体的固定化是研发高效诊断和分离工具的关键环节。生物分子工程、材料化学与交联剂化学的进步极大地促进了抗体固定化技术的发展。 抗体可以通过物理吸附、共价偶联和亲和相互作用固定到不同类型的固相表面。 抗体固定化的目标是以一种正确的空间取向将抗体固定到固相表面,在完全保留抗体构象和活性的同时最大化抗原的结合能力,这对固相化抗体的分析性能至关重要。 对固定抗体到固相载体表面的各种最新方法进行了阐述,包括物理吸附法,通过羧基、氨基、巯基、糖基和点击化学的共价结合法以及基于生物亲和作用的固定法,并对固定化抗体的表征方法进行了归纳,最后对抗体固定化方法的发展方向进行了展望。     

Ultrathin functional star PEG coatings for DNA microarrays

In this study, star PEG coatings on glass substrates have been used as support material for oligonucleotide microarrays. These coatings are prepared from solutions of six armed star shaped prepolymers that carry reactive isocyanate endgroups. As described earlier, such films prevent the adsorption of proteins and the adhesion of cells but can easily be functionalized for specific biological recognition. Here we used the high functionality of these coatings for the covalent immobilization of amino terminated 20mer oligonucleotides, both by microcontact printing and spotting techniques. The permanent immobilization of fluorescently labeled DNA as well as hybridization of 20mer oligonucleotides have been monitored by fluorescence microscopy. The hybridization efficiency as determined by fluorescence intensity varied from 30% to 80% depending on the way of layer preparation. The direct spotting without additional activation and blocking steps of the surface demonstrates the potential of star PEG coatings as ultrathin surface modification for microarrays.     

Automated classification and quantification of F-actin-containing ruffles in confocal micrographs

Membrane ruffles are actin-rich protrusions of the plasma membrane that can be observed on the surface of many cell types. Phase contrast and fluorescent microscopy are widely used in the analysis of ruffles, which are commonly identified in cells stained with fluorescently labeled phalloidin. Currently, comparison of cellular ruffle formation under different experimental conditions is generally qualitative or semiquantitative. Ruffle structures are often defined using manual tracing and thresholding methods. Here, we report the rapid and accurate segmentation of ruffles from two-dimensional confocal projections of cells using automated method based on well-established image processing and analysis methods. Line-shaped ruffles were detected using line detectors and were then separated from the filtered images. Automated categorizing of the segmented line structures enabled accurate quantification of the ruffles. This automated approach is efficient and reliable and hence can serve as a powerful tool in studies of the mechanism of ruffle formation.