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Microscopic imaging is one of the most common techniques for investigating biological systems. In recent years there has been a tremendous growth in the volume of biological imaging data owing to rapid advances in optical instrumentation, high-speed cameras and fluorescent probes. Powerful semantic analysis tools are required to exploit the full potential of the information content of these data. Semantic analysis of multi-modality imaging data, however, poses unique challenges. In this paper we outline the state-of-the-art in this area along with the challenges facing this domain. Information extraction from biological imaging data requires modeling at multiple levels of detail. While some applications require only quantitative analysis at the level of cells and subcellular objects, others require modeling of spatial and temporal changes associated with dynamic biological processes. Modeling of biological data at different levels of detail allows not only quantitative analysis but also the extraction of high-level semantics. Development of powerful image interpretation and semantic analysis tools has the potential to significantly help in understanding biological processes, which in turn will result in improvements in drug development and healthcare.

Recursive procedures used for sequential calculations of polynomial basis coefficients in discrete orthogonal moments produce unreliable results for high moment orders as a result of error accumulation. This paper demonstrates accurate reconstruction of arbitrary-size images using full-order (orders as large as the image size) Tchebichef and Krawtchouk moments by calculating polynomial coefficients directly from their definition formulas in hypergeometric functions and by creating lookup tables of these coefficients off-line. An arbitrary precision calculator is used to achieve greater numerical range and precision than is possible with software using standard 64-bit IEEE floating-point arithmetic. This reconstruction scheme is content and noise independent.

Background: Polychromatic flow cytometry (PFC) allows the simultaneous determination of multiple antigens in the same cell, resulting in the generation of a high number of subsets. As a consequence, data analysis is the main difficulty with this technology. Here we show the use of cluster analysis (CA) and principal component analyses (PCA) to simplify multicolor data visualization and to allow subjects' classification. Methods: By eight-colour cytofluorimetric analysis, we investigated the T cell compartment in donors of different age (young, middle-aged, and centenarians). T cell subsets were identified by combining positive and negative expression of antigens. The resulting data set was organized into a matrix and subjected to CA and PCA. Results: CA clustered people of different ages on the basis of cytofluorimetric profile. PCA of the cellular subsets identified centenarians within a different cluster from young donors, while middle-aged donors were scattered between these groups. These approaches identified T cell phenotypes that changed with increasing age. In young donors, memory T cell subsets tended to be CD127+ and CD95− whereas CD127−, CD95+ phenotypes were found at higher frequencies in people with advanced age. Conclusions: Our data suggest the use of bioinformatic approaches to analyze large data-sets generated by PFC and to obtain the rapid identification of key populations that best characterize a group of subjects. © 2007 International Society for Analytical Cytology

Lymphocyte origin hybridoma Ped-2E9 cell-based cytotoxicity assay can detect virulent Listeria or Bacillus species, and its application in a cell-based biosensor for onsite use would be very attractive. However, maintaining enough viable cells on a sensor platform for a prolonged duration is a challenging task. In this study, key factors affecting the survival and growth of Ped-2E9 cells under modified conditions were investigated. When the Ped-2E9 cells were grown in media containing 5% fetal bovine serum in sealed tubes without any replenishment of nutrients or exogenous CO(2) supply, a large portion of the cells remained viable for 6 to 7 days and cells entered into G0/G1 resting phase. The media pH change was negligible and no cell death was observed in the first 4 days, then cells sequentially underwent apoptotic (fourth day onward) phase until day 7 after which a majority was dead. Subsequent cytotoxicity testing of 3- to 7-day stored Ped-2E9 cells sensitively detected virulent Listeria and Bacillus species. These data strongly suggest that Ped-2E9 cells can be maintained in viable state for 6 days in a sealed tube mimicking the environment in a potential sensor device for onsite use without the need for expensive cell culture facilities.

Three wavelength-merged scatter pattern from E. coli. We report a multispectral elastic-light-scatter instrument that can simultaneously detect three-wavelength scatter patterns and associated optical densities from individual bacterial colonies, overcoming the limits of the single-wavelength predecessor. Absorption measurements on liquid bacterial samples revealed that the spectroscopic information can indeed contribute to sample differentiability. New optical components, including a pellicle beam splitter and an optical cage system, were utilized for robust acquisition of multispectral images. Four different genera and seven shiga toxin producing E. coli serovars were analyzed; the acquired images showed differences in scattering characteristics among the tested organisms. In addition, colony-based spectral optical-density information was also collected. The optical model, which was developed using diffraction theory, correctly predicted wavelength-related differences in scatter patterns, and was matched with the experimental results. Scatter-pattern classification was performed using pseudo-Zernike (GPZ) polynomials/moments by combining the features collected at all three wavelengths and selecting the best features via a random-forest method. The data demonstrate that the selected features provide better classification rates than the same number of features from any single wavelength.

Despite the advancement of recent molecular technologies, culturing is still considered the gold standard for microbial sample analysis. Here we review three different bacterial colony-based screening modalities that provide significant information beyond the simple shape and color of the colony. The plate imaging technique provides numeration and quantitative spectral reflectance information for each colony, while Raman spectroscopic analysis of bacteria colonies relates the Raman-shifted peaks to specific chemical bonding. Finally, the elastic-light-scatter technique provides a volumetric interaction of the whole colony through laser-bacteria interactions, instantly capturing the morphological traits of the colony and allowing quantitative classifications.

Mitochondrial dysfunction has been increasingly implicated as an important mechanism for chemical-induced toxicity. In the present unit, we describe a multi-parametric flow cytometry assay to assess the effects of drug or chemical-induced mitochondrial dysfunction in cells. Cells are cultured in a glucose-supplemented medium and exposed to increasing concentrations of various chemicals. Several key mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction, such as mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS) production, intracellular reduced glutathione (GSH) level, and cell viability, are simultaneously measured by flow cytometry.

Biofilm is a formation of microbial material on tooth substrata. Several methods to quantify dental biofilm coverage have recently been reported in the literature, but at best they provide a semi-automated approach to quantification with significant input from a human grader that comes with the graders bias of what are foreground, background, biofilm, and tooth. Additionally, human assessment indices limit the resolution of the quantification scale; most commercial scales use five levels of quantification for biofilm coverage (0%, 25%, 50%, 75%, and 100%). On the other hand, current state-of-the-art techniques in automatic plaque quantification fail to make their way into practical applications owing to their inability to incorporate human input to handle misclassifications. This paper proposes a new interactive method for biofilm quantification in Quantitative light-induced fluorescence (QLF) images of canine teeth that is independent of the perceptual bias of the grader. The method partitions a QLF image into segments of uniform texture and intensity called superpixels; every superpixel is statistically modeled as a realization of a single 2D Gaussian Markov random field (GMRF) whose parameters are estimated; the superpixel is then assigned to one of three classes (background, biofilm, tooth substratum) based on the training set of data. The quantification results show a high degree of consistency and precision. At the same time, the proposed method gives pathologists full control to post-process the automatic quantification by flipping misclassified superpixels to a different state (background, tooth, biofilm) with a single click, providing greater usability than simply marking the boundaries of biofilm and tooth as done by current state-of-the-art methods., 10 pages, 7 figures, Journal of Biomedical and Health Informatics 2014. keywords: {Biomedical imaging;Calibration;Dentistry;Estimation;Image segmentation;Manuals;Teeth}, http://ieeexplore.ieee.org/stamp/stamp.jsp?tp=&arnumber=6758338&isnumber=6363502

Lagrangian actinometry represents a new method of photochemical reactor characterization. The method is based on an application of dyed microspheres, which were developed by attachment of (E)-5-[2-(methoxycarbonyl)ethenyl]cytidine (hereafter referred to as S) to polystyrene microspheres. S is a nonfluorescent molecule that when subjected to ultraviolet (UV) irradiation yields a single product, 3-beta-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine (hereafter referred to as P), which displays a strong fluorescence signal. Dyed microspheres were subjected to UV irradiation under a collimated beam and using a single-lamp, monochromatic (low pressure Hg), continuous-flow reactor. In parallel with these experiments, a biodosimetry experiment was conducted using Bacillus subtilis spores as the challenge organism. Particle-specific fluorescence intensity measurements were conducted on samples from the collimated-beam experiments and the flow-through reactor experiments by flow cytometry. Estimates of the dose distribution delivered by the flow-through reactor for each operating condition were developed by deconvolution of data resulting from flow cytometry analysis of these samples. In conjunction with these experiments, a numerical model was developed to simulate the behavior of the reactor system. A commercially available computational fluid dynamics package was used to simulate the flow field, while line-source integration was used to simulate the irradiance field. A particle-tracking algorithm was employed to interrogate the flow and irradiance field simulations for purposes of developing particle-specific (Lagrangian) estimates of dose delivery. Dose distribution estimates from the microspheres assays and the numerical simulations were combined with the measured dose-response behavior of B. subtilis spores to yield estimates of spore inactivation in the flow-through experiments. For the range of operating conditions used in these experiments, predictions of spore inactivation based on dose distribution estimates from both methods were in good agreement with each other, and with the measured spore inactivation behavior. Lagrangian actinometry is capable of yielding accurate, detailed measurements of dose delivery by continuous-flow UV systems. This method represents a substantial improvement over existing experiment-based methods of UV reactor characterization (e.g., biodosimetry) in that it yields a measurement of the dose distribution for a given operating condition. This method also represents an improvement over existing methods for validation of numerical simulations. Specifically, because this method yields a measurement of the dose distribution, it is possible to compare these measurements with predicted dose distributions from the numerical simulation. The combined application of biodosimetry, numerical modeling, and Lagrangian actinometry represents an extremely robust approach to reactor characterization and validation.

Modern microscopic techniques like high-content screening (HCS), high-throughput screening, 4D imaging, and multispectral imaging may involve collection of thousands of images per experiment. Efficient image-compression techniques are indispensable to manage these vast amounts of data. This goal is frequently achieved using lossy compression algorithms such as JPEG and JPEG2000. However, these algorithms are optimized to preserve visual quality but not necessarily the integrity of the scientific data, which are often analyzed in an automated manner. Here, we propose three observer-independent compression algorithms, designed to preserve information contained in the images. These algorithms were constructed using signal-to-noise ratio (SNR) computed from a single image as a quality measure to establish which image components may be discarded. The compression efficiency was measured as a function of image brightness and SNR. The alterations introduced by compression in biological images were estimated using brightness histograms (earth's mover distance (EMD) algorithm) and textures (Haralick parameters). Furthermore, a microscope test pattern was used to assess the effect of compression on the effective resolution of microscope images.

Two-component systems (TCS) are the most prevalent gene regulatory mechanism in bacteria. A typical TCS is comprised of a histidine kinase (HK) and a partner response regulator (RR). Specific environment signals lead to autophosphorylation of different HKs, which in turn act as phosphoryl donors for autophosphorylation of their partner RRs. Nonpartner HKs and RRs also interact, giving rise to cross regulation among TCSs in response to diverse signals.PhoR (HK) and PhoB (RR) constitute the TCS for detection of environmental (extracellular) inorganic phosphate (Pi). The PhoR/PhoB TCS controls the expression of a large number of genes for acquisition of alternative phosphorus sources, including phoA, which encodes the non-specific phosphohydrolase bacterial alkaline phosphatase (Bap). Cross activation of PhoB by the nonpartner HK CreC is now a classic example of cross regulation among TCSs. A systematic search for other cross talking HKs revealed five additional HKs that activate (phosphorylate) PhoB (J. M. B. and B. L. W., unpublished data).Examination of cross activation of PhoB by these non-partner HKs by flow cytometry at the single-cell level revealed a bimodal, “all-or-none,” distribution pattern for expression of a phoAp-gfp (green fluorescent protein) reporter fusion. Although the basis of the observed stochastic behavior is unclear, it seems to reflect an inherent property of TCSs. We propose that cells exploit the stochastic character of TCSs to achieve nongenetic (epigenetic) diversity within genetically homogeneous cell populations in order to facilitate adaptation to environmental changes.

The influence of an exercise training program and age on inflammatory cytokine production and CD14+cell-surface expression of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) was examined in 60 younger and older subjects. Subjects were assigned to: young physically active (YPA, n = 15; 25.2 +/- 5.0 years), young physically inactive (YPI, n = 14; 24.9 +/- 4.7 years), older physically active (OPA, n = 14; 71.2 +/- 4.4 years) or older physically inactive (OPI, n = 17; 71.0 +/- 4.3 years) groups. YPI and OPI completed 12 weeks (3 days/week) of endurance (20 min) and resistance exercise (eight exercises, two sets). YPA and OPA groups were instructed to continue their normal activity for 12 weeks. Blood was collected at rest, before and after the 12-week training and control period. A whole blood method was used to determine lipopolysaccharide-(LPS) and peptidoglycan-(PGN) stimulated IL-6, IL-1beta, and TNF-alpha production with supernatants analyzed using ELISA. CD14+ cell-surface expression of TLR2 and TLR4 were measured using flow cytometry. Training increased estimated VO(2max) by 10.4% and increased strength by an average of 38.1%. YPI and OPI had a post-training reduction in LPS-stimulated IL-6 production (P < .01), but LPS-stimulated IL-1beta and TNF-alpha and PGN-stimulated cytokines were not changed. CD14+ cell TLR4 was significantly reduced (P < .05) in YPI and OPI groups after training, but TLR2 was not significantly changed. An exercise training program reduced LPS-stimulated IL-6, concomitant with lower TLR4. These results provide further support for a training- or physical activity-induced lowering of TLR4 and inflammation.

An important step in the induction of an immune response to vaccines is the internalization of antigens by antigen presenting cells, such as dendritic cells (DCs). Many current vaccines are formulated with antigens adsorbed to an aluminum-containing adjuvant. Following injection of the vaccine the antigens may either elute or stay adsorbed to the adjuvant surface. Antigens, which elute from the adjuvant surface, are internalized by dendritic cells through macropinocytosis while those that remain adsorbed are internalized with the adjuvant particle by phagocytosis. The relative efficiency of these two routes of internalization was studied. Alpha casein (AC) labeled with a green fluorescent dye was selected as the model antigen. In order to model vaccine antigens that elute from aluminum-containing adjuvants following administration, dendritic cells were incubated with a solution of fluorochrome-labeled alpha casein. To model vaccine antigens that do not elute from aluminum-containing adjuvants following administration, dendritic cells were exposed to fluorochrome-labeled alpha casein adsorbed to aluminum hydroxide adjuvant (AH). Alpha casein has eight phosphate groups and adsorbs to aluminum hydroxide adjuvant through ligand exchange. Alpha casein does not elute from aluminum hydroxide adjuvant upon exposure to cell culture media. The uptake of antigen by dendritic cells was determined at 0.5, 1, 2 and 3h by confocal microscopy and flow cytometry. Dendritic cells internalized both alpha casein in solution and alpha casein adsorbed to aluminum hydroxide adjuvant. However, the mean fluorescence intensity of dendritic cells incubated with adsorbed alpha casein was four times greater than dendritic cells incubated with alpha casein in solution. In addition, the internalization of alpha casein was enhanced when the mean aggregate diameter of the adjuvant in the cell culture media was reduced from 17 microm to 3 microm. It was concluded that antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed to the aluminum-containing adjuvant following administration and the aggregate size of the adjuvant was smaller than dendritic cells which are approximately 10 microm in diameter.

PROLOGUEI met Mack Fulwyler only once, in 1999. We spent aweek together in May at Hortobagy-EPONA, Hungary, dur-ing the Hungarian Society for Cytometry meeting run byJanos Szollosi. During that time, I had a wonderful oppor-tunity to talk with Mack about the history of flow cytome-try and why he had developed the things that he hadinvented. He told me many stories and I actually tooknotes! I also found out that the Smithsonian had made ahistoric video in 1991, in which Mack had been inter-viewed. I purchased copies of the eight videos made byRamunas Kondratas and started to wade through them.I never made it to the end (14 h), but for some reason, justprior to Christmas in 2004, I came across the videos in myoffice and started watching them again. After wadingthrough 10 h of tape, I came to the interview with MackFulwyler, which was conducted in the laboratory of Dr.Boris Rotman, at Brown University. What struck me wasthat Dr. Rotman had one of Mack’s original instruments inhis laboratory and it was right there in the video. At about4A

The ability to create extracellular matrix (ECM) constructs that are mechanically and biochemically similar to those found in vivo and to understand how their properties affect cellular responses will drive the next generation of tissue engineering strategies. To date, many mechanisms by which cells biochemically communicate with the ECM are known. However, the mechanisms by which mechanical information is transmitted between cells and their ECM remain to be elucidated. “Self-assembled” collagen matrices provide an in vitro-model system to study the mechanical behavior of ECM. To begin to understand how the ECM and the cells interact mechanically, the three-dimensional (3D) mechanical properties of the ECM must be quantified at the micro-(local) level in addition to information measured at the macro-(global) level. Here we describe an incremental digital volume correlation (IDVC) algorithm to quantify large (>0.05) 3D mechanical strains in the microstructure of 3D collagen matrices in response to applied mechanical loads. Strain measurements from the IDVC algorithm rely on 3D confocal images acquired from collagen matrices under applied mechanical loads. The accuracy and the precision of the IDVC algorithm was verified by comparing both image volumes collected in succession when no deformation was applied to the ECM (zero strain) and image volumes to which simulated deformations were applied in both 1D and 3D (simulated strains). Results indicate that the IDVC algorithm can accurately and precisely determine the 3D strain state inside largely deformed collagen ECMs. Finally, the usefulness of the algorithm was demonstrated by measuring the microlevel 3D strain response of a collagen ECM loaded in tension.

A common observation about confocal microscopy images is that lower image stacks have lower voxel intensities and are usually blurred in comparison with the upper ones. The key reasons are light absorption and scattering by the objects and particles in the volume through which light passes. This report proposes a new technique to reduce such noise impacts in terms of an adaptive intensity compensation and structural sharpening algorithm. With these image-processing procedures, effective 3D rendering techniques can be applied to faithfully visualize confocal microscopy data.

Lysozyme placed on the SiO2 surfaces that have previously been derivatized with C18 coating will capture both Escherichia coli and Listeria monocytogenes cells from PBS buffer at pH 7.2. This phenomenon is of significance for the design and fabrication of protein biochips that are designed to capture bacteria from buffer or water so that these can be further interrogated with respect to possible pathogenicity. Fluorescent microscopy shows that two types of bacteria (gram-negative E. coli and gram-positive Listeria spp.) will be adsorbed by lysozyme placed on the surface of the biochip but that strong adsorption of the bacteria is reduced but not eliminated when Tween 20 is present (at 0.5%) in the PBS buffer in which the cells are suspended. In comparison, Tween 20 and Bovine Serum Albumin (BSA) almost completely block adsorption of these bacteria on C18 coated surfaces. The combination of a lysozyme surface with Tween 20 gives a greater degree of adsorption of L. monocytogenes than E. coli, and hence suggests selectivity for the more hydrophobic E. coli may be reduced by the Tween 20. This paper presents protocols for preparing protein-coated, SiO2 surfaces and the effect of buffer containing Tween 20 on adsorption of bacteria by SiO2 surfaces coated with C18 to which BSA, lysozyme or C11E9 antibody is immobilized at pH 7.2 and ambient temperature.

Recent evidence supports the notion that biological functions of extracellular matrix (ECM) are highly correlated to not only its composition but also its structure. This article integrates confocal microscopy imaging and image-processing techniques to analyze the microstructural properties of ECM. This report describes a two- and three-dimensional fiber middle-line tracing algorithm that may be used to quantify collagen fibril organization. We utilized computer simulation and statistical analysis to validate the developed algorithm. These algorithms were applied to confocal images of collagen gels made with reconstituted bovine collagen type I, to demonstrate the computation of orientations of individual fibers.

This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C(18)-coated SiO(2) substrate via specific or non-specific bio-mediated interactions. Biotinylated bovine serum albumin pre-adsorbed onto a C(18) surface was used to mediate the surface assembly of streptavidin-coated microbeads (2.8 microm), while a bare C(18) surface was used to immobilize anti-Listeria antibody-coated microbeads (2.8 microm) through hydrophobic interactions. For a C(18) surface pre-adsorbed with bovine serum albumin, hydrophobic polystyrene microbeads (0.8 microm) and positively charged dimethylamino microbeads (0.8 microm) were allowed to self-assemble onto the surface. A monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads. The adsorption characteristics of Escherichia coli and Listeria monocytogenes on these microbead-based surfaces were studied using fluorescence microscopy. Both streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody-coated microbeads showed specific capture of L. monocytogenes, while polystyrene and dimethylamino microbeads captured both E. coli and L. monocytogenes non-specifically. The preparation of microbead-based surfaces for the construction of microfluidic devices for separation, detection, or analysis of specific biological species is discussed.

The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.

The oxidative burst is frequently evaluated by the conversion of dihydrorhodamine 123 (DHR) to rhodamine 123 (R123) and hydroethidium (HE) to ethidium with the use of flow cytometry (FCM). Added R123 accumulates in mitochondria, but during phagocytosis R123 originating from DHR has been observed in neutrophil granules. The present study was designed to identify the site of reactive oxygen species (ROS) formation and the intracellular traffic of R123 in neutrophils by using mitochondrial membrane potential probes and the lysosomotropic probe LysoTracker Red, which have not previously been applied to neutrophils. Quiescent and phagocytosing human peripheral blood neutrophils were incubated with DHR, HE, R123, MitoTracker Green (MTG), MitoTracker Red (CMX-Ros), and LysoTracker Red alone and in all combinations of red and green probes, and studied by FCM and confocal laser scanning microscopy (CLSM). Phagosomes were filled with R123 originating from DHR. Phagocytosis also triggered the oxidative burst in oxidative response granules that differed from acidic granules. All the neutrophils stained with mitochondrial and lysosomotropic dyes. Added R123 and MTG selectively accumulated in mitochondria. Added R123, MTG, and DHR increased the fluorescence of CMX-Ros and LysoTracker Red. This is the first FCM and CLSM demonstration of ROS formation in phagosomes. A distinct subpopulation of neutrophil granules, termed oxidative response granules, also was identified. Neutrophil mitochondrial membrane potential may be evaluated by incubating the cells with R123 and MTG, but results with CMX-Ros should be interpreted with caution. HE and DHR seem to measure a common pathway in the oxidative burst. The simultaneous application of several probes for investigations of organelles carries the risk of probe interference.

The monitoring of reactive oxygen species (ROS) levels in injured nervous tissue is critical for both studying the mechanism of secondary damage and evaluating the effectiveness of antioxidants. Flow cytometry is an excellent method to detect ROS in cultured cells and naturally suspended individual cells. However, its use in nervous tissue is limited due to the difficulties in obtaining single cells in suspension. We have developed a new method which minimizes the error during conventional dissociation. Specifically, we introduced a fixation step (with formaldehyde) between the dye loading and dissociation. As a result, the post-injury ROS signals detected by flow cytometry increase significantly when using hydroethidine as superoxide indicator. The injury-induced elevation of ROS obtained from this new method was also in better agreement with the two other standard ROS detection methods, fluorescence microscopy and lipid peroxidation assay. Furthermore, more pronounced decrease of ROS was found in this improved method in response to treatment with a superoxide scavenger, manganese(III)tetrakis(4-benzoic acid)porphyrin. Based on these observations, we suggest that the data obtained from the cells by this new method are more accurate than those from the classic cell dissociation method that dissociates cells directly from fresh tissues.

The importance and priority of specific micro-structural and mechanical design parameters must be established to effectively engineer scaffolds (biomaterials) that mimic the extracellular matrix (ECM) environment of cells and have clinical applications as tissue substitutes. In this study, three-dimensional (3-D) matrices were prepared from type I collagen, the predominant compositional and structural component of connective tissue ECMs, and structural-mechanical relationships were studied. Polymerization conditions, including collagen concentration (0.3–3 mg/mL) and pH (6–9), were varied to obtain matrices of collagen fibrils with different microstructures. Confocal reflection microscopy was used to assess specific micro-structural features (e.g., diameter and length) and organization of component fibrils in 3-D. Microstructural analyses revealed that changes in collagen concentration affected fibril density while maintaining a relatively constant fibril diameter. On the other hand, both fibril length and diameter were affected by the pH of the polymerization reaction. Mechanically, all matrices exhibited a similar stress-strain curve with identifiable “toe,” “linear,” and “failure” regions. However, the linear modulus and failure stress increased with collagen concentration and were correlated with an increase in fibril density. Additionally, both the linear modulus and failure stress showed an increase with pH, which was related to an increased fibril length and a decreased fibril diameter. The tensile mechanical properties of the collagen matrices also showed strain rate dependence. Such fundamental information regarding the 3-D microstructural-mechanical properties of the ECM and its component molecules are important to our overall understanding of cell-ECM interactions (e.g., mechanotransduction) and the development of novel strategies for tissue repair and replacement.

The marriage between immunology and cytometry is one of the most stable and productive in the recent history of science. A rapid search in PubMed shows that, as of July 2017, using “flow cytometry immunology” as a search term yields more than 68 000 articles, the first of which, interestingly, is not about lymphocytes. It might be stated that, after a short engagement, the exchange of the wedding rings between immunology and cytometry officially occurred when the idea to link fluorochromes to monoclonal antibodies came about. After this, recognizing different types of cells became relatively easy and feasible not only by using a simple fluorescence microscope, but also by a complex and sometimes esoteric instrument, the flow cytometer that is able to count hundreds of cells in a single second, and can provide repetitive results in a tireless manner. Given this, the possibility to analyse immune phenotypes in a variety of clinical conditions has changed the use of the flow cytometer, which was incidentally invented in the late 1960s to measure cellular DNA by using intercalating dyes, such as ethidium bromide. The epidemics of HIV/AIDS in the 1980s then gave a dramatic impulse to the technology of counting specific cells, since it became clear that the quantification of the number of peripheral blood CD4+ T cells was crucial to follow the course of the infection, and eventually for monitoring the therapy. As a consequence, the development of flow cytometers that had to be easy-to-use in all clinical laboratories helped to widely disseminate this technology. Nowadays, it is rare to find an immunological paper or read a conference abstract in which the authors did not use flow cytometry as the main tool to dissect the immune system and identify its fine and complex functions. Of note, recent developments have created the sophisticated technology of mass cytometry, which is able to simultaneously identify dozens of molecules at the single cell level and allows us to better understand the complexity and beauty of the immune system.

Flow cytometry is one of the most important technologies for high-throughput single-cell analysis. Fluorescent labeling acts as the primary approach for cellular analysis in flow cytometry. Nevertheless, the fluorescent tags are not applicable to all cases, especially to small molecules, for which labeling may significantly perturb the biological functionality. Spontaneous Raman scattering flow cytometry offers the capability to non-invasively detect chemical contents of cells but suffers from slow data acquisition. In order to achieve label-free high-throughput single-particle analysis using Raman scattering, we developed a 32-channel multiplex stimulated Raman scattering flow cytometry (SRS-FC) technique that can measure chemical contents of single particles at a speed of 5 μs per Raman spectrum. Using mixed polymer beads, we demonstrate the discrimination of different particles at a throughput of up to 11,000 particles per second. This is a four orders of magnitude improvement in throughput compared to conventional spontaneous Raman flow cytometry. As a proof of concept, we show the differentiation of 3T3-L1 cells at different states by SRS-FC according to the difference in cellular chemical content. The SRS-FC technique opens new opportunities for high-throughput and high-content chemical analysis of live cells in a label-free manner.

Dental biofilm is the deposition of microbial material over a tooth substratum. Several methods have recently been reported in the literature for biofilm quantification; however, at best they provide a barely automated solution requiring significant input needed from the human expert. On the contrary, state-of-the-art automatic biofilm methods fail to make their way into clinical practice because of the lack of effective mechanism to incorporate human input to handle praxis or misclassified regions. Manual delineation, the current gold standard, is time consuming and subject to expert bias. In this paper, we introduce a new semi-automated software tool, BiofilmQuant, for dental biofilm quantification in quantitative light-induced fluorescence (QLF) images. The software uses a robust statistical modeling approach to automatically segment the QLF image into three classes (background, biofilm, and tooth substratum) based on the training data. This initial segmentation has shown a high degree of consistency and precision on more than 200 test QLF dental scans. Further, the proposed software provides the clinicians full control to fix any misclassified areas using a single click. In addition, BiofilmQuant also provides a complete solution for the longitudinal quantitative analysis of biofilm of the full set of teeth, providing greater ease of usability., 4 pages, 4 figures, 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC 2014)

Significant multiplexing capacity of optical time-domain coding has been recently demonstrated by tuning luminescence lifetimes of the upconversion nanoparticles called ‘τ-Dots’. It provides a large dynamic range of lifetimes from microseconds to milliseconds, which allows creating large libraries of nanotags/microcarriers. However, a robust approach is required to rapidly and accurately measure the luminescence lifetimes from the relatively slow-decaying signals. Here we show a fast algorithm suitable for the microsecond region with precision closely approaching the theoretical limit and compatible with the rapid scanning cytometry technique. We exploit this approach to further extend optical time-domain multiplexing to the downconversion luminescence, using luminescence microspheres wherein lifetimes are tuned through luminescence resonance energy transfer. We demonstrate real-time discrimination of these microspheres in the rapid scanning cytometry, and apply them to the multiplexed probing of pathogen DNA strands. Our results indicate that tunable luminescence lifetimes have considerable potential in high-throughput analytical sciences., Accurately determining luminescence lifetimes for slow-decaying signals can be challenging. Here, the authors report a fitting algorithm and subsequently experimentally show a method for the rapid measurement of luminescence lifetimes in the microsecond region.

We investigated the application capabilities of a laser optical sensor, BARDOT (bacterial rapid detection using optical scatter technology) to generate differentiating scatter patterns for the 20 most frequently reported serovars of Salmonella enterica. Initially, the study tested the classification ability of BARDOT by using six Salmonella serovars grown on brain heart infusion, brilliant green, xylose lysine deoxycholate, and xylose lysine tergitol 4 (XLT4) agar plates. Highly accurate discrimination (95.9%) was obtained by using scatter signatures collected from colonies grown on XLT4. Further verification used a total of 36 serovars (the top 20 plus 16) comprising 123 strains with classification precision levels of 88 to 100%. The similarities between the optical phenotypes of strains analyzed by BARDOT were in general agreement with the genotypes analyzed by pulsed-field gel electrophoresis (PFGE). BARDOT was evaluated for the real-time detection and identification of Salmonella colonies grown from inoculated (1.2 × 102 CFU/30 g) peanut butter, chicken breast, and spinach or from naturally contaminated meat. After a sequential enrichment in buffered peptone water and modified Rappaport Vassiliadis broth for 4 h each, followed by growth on XLT4 (~16 h), BARDOT detected S. Typhimurium with 84% accuracy in 24 h, returning results comparable to those of the USDA Food Safety and Inspection Service method, which requires ~72 h. BARDOT also detected Salmonella (90 to 100% accuracy) in the presence of background microbiota from naturally contaminated meat, verified by 16S rRNA sequencing and PFGE. Prolonged residence (28 days) of Salmonella in peanut butter did not affect the bacterial ability to form colonies with consistent optical phenotypes. This study shows BARDOT’s potential for nondestructive and high-throughput detection of Salmonella in food samples., IMPORTANCE High-throughput screening of food products for pathogens would have a significant impact on the reduction of food-borne hazards. A laser optical sensor was developed to screen pathogen colonies on an agar plate instantly without damaging the colonies; this method aids in early pathogen detection by the classical microbiological culture-based method. Here we demonstrate that this sensor was able to detect the 36 Salmonella serovars tested, including the top 20 serovars, and to identify isolates of the top 8 Salmonella serovars. Furthermore, it can detect Salmonella in food samples in the presence of background microbiota in 24 h, whereas the standard USDA Food Safety and Inspection Service method requires about 72 h.

Enterotoxigenic Escherichia coli (ETEC) induces severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa). We investigated the distribution and characteristics of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves. We found that density of the receptors and their affinity to STa were higher on enterocytes and BBMVs that were derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine. This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains.

Experiments were conducted in vitro to study the regulation of progesterone production in chicken granulosa cells by homologous basal lamina isolated from preovulatory follicles of chicken ovary. The majority of components of the basal lamina (90-95% by weight) were solubilized with guanidine-HCl (and designated fraction 1); the remaining components were solubilized with beta-mercaptoethanol containing guanidine-HCl (and designated fraction 2). The ability of fraction 1 to regulate progesterone production in granulosa cells obtained from the largest (F(1), mature), third largest (F(3), growing), fifth to seventh largest (F(5-7), growing) follicles and a pool of small yellow follicles (SYF, immature) of chicken ovary was assessed. Granulosa cells isolated from SYF follicles were in the least differentiated (undifferentiated) and those obtained from F(1) follicles were in the most differentiated state. The ability of fraction 1 to regulate progesterone production by chicken granulosa cells was influenced both by the state of cell differentiation and the form of the matrix material (whether solid or liquid). When fraction 1 was added as liquid to the incubation mixture, it promoted progesterone production by granulosa cells at all stages of differentiation; however, it caused a greater relative increase in the amount of progesterone produced by undifferentiated (SYF) and differentiating (F(3)) granulosa cells than by differentiated (F(1)) ones. In the presence of the liquid-form of fraction 1, luteinizing hormone (LH) stimulated progesterone production in differentiated (F(1)) and differentiating (F(5-7)) granulosa cells. Similarly, follicle-stimulating hormone (FSH) stimulated progesterone production by differentiating (F(3)) and undifferentiated (SYF) granulosa cells in the presence of the liquid-form of fraction 1 protein. In culture wells that had been pre-coated with fraction 1 (solid-form), progesterone production by less differentiated (SYF, F(5-7)) granulosa cells was enhanced, whereas progesterone production by differentiated (F(1)) cells was reduced. The solid-form of fraction 1 augmented LH-stimulated progesterone production by less differentiated (F(5-7)) granulosa cells however, it attenuated LH-induced progesterone production in differentiated (F(1)) cells. FSH-promoted progesterone production in granulosa cells from immature follicles (SYF) was augmented by solid-form of fraction 1 whereas the effect of FSH on cells obtained from older follicle (F(3)) was suppressed by solid-form of fraction 1. In experiments in which gonadotropin action was attenuated by solid-form of fraction 1, the amount of progesterone produced in the presence of maximally inhibiting concentrations of fraction 1 protein was greater than control values (no fraction 1, no gonadotropin). These results show that basal lamina of the ovarian follicle can regulate progesterone production by granulosa cells. The data demonstrate that the interactions between the components of basal lamina and LH or FSH on granulosa cell function were dependent on the stage of follicular development and were influenced by the form of the matrix material. It is concluded that the basal lamina of the chicken ovarian follicle is biologically active and regulates granulosa cell function.

Studies on the effects of time and passage on porcine primary muscle cell cultures and methods to purify myoblasts were conducted using flow cytometry and fluorescence-activated cell sorting (FACS). Primary muscle cells cultured on single plates revealed a small cell ( or = 10 mm diameter) population containing desmin-positive myoblasts and nonmyoblasts. The small myoblasts were detectable up to 28 days but after cell sorting and passage, they became indistinguishable from the large myoblast population. This indicates that pig muscle contains small self-renewing myoblasts similar to humans, that become larger when induced to proliferate. A human myoblast-specific monoclonal antibody allows FACS of both large and small myoblasts from primary cells within 2 days of culture and independent of passage. These characteristics of porcine myoblasts indicate that the pig may be a suitable large animal model for myoblast-mediated gene transfer.

This month marks the 50th anniversary of the birth of flow cytometry. One would imagine that a technology invented so long ago would be radically different today—yet it has changed remarkably little in its fundamentals. It allows rapid and simultaneous analysis of multiple parameters of live cells in a heterogeneous mix as they flow in a stream through photonic detectors. The data are integrated to give a comprehensive view of the sample. Flow cytometry has become a staple of biological research as well as clinical diagnostics, and its application has been crucial to innumerable advances in immunology and cell biology, and for understanding diseases such as AIDS and cancer. It has spawned the development of dozens of other key technologies and its application continues to expand, ranging from single-cell to cell population analysis.

The importance of understanding cell–extracellular matrix (ECM) interaction is now evident as scientists, engineers, and physicians search for novel scaffolds that support and maintain tissue-specific cellular growth and function both in vivo and in vitro. Small intestinal submucosa (SIS) represents an ECM that has been derived from porcine intestine while preserving its natural composition and architecture. More recently, an extract of this physiologic ECM, which forms a three-dimensional gel in vitro, has been developed. When compared to routinely used culture substrata (e.g., plastic, Vitrogen, and Matrigel), intact SIS and SIS-derived gel possess unique compositional and architectural features. Simple squamous epithelial (pulmonary artery), fibroblastic (Swiss 3T3), glandular epithelial (adenocarcinoma), and smooth muscle-like (urinary bladder) cells were seeded upon intact SIS and SIS-derived gel and their morphologic response evaluated. For each of the four cell types studied, intact SIS and SIS-der...

The existence of phenotypic differences in the drug responses of 3D tissue relative to 2D cell culture is a concern in high-content drug screening. Biodynamic imaging is an emerging technology that probes 3D tissue using short-coherence dynamic light scattering to measure the intracellular motions inside tissues in their natural microenvironments. The information content of biodynamic imaging is displayed through tissue dynamics spectroscopy (TDS) but has not previously been correlated against morphological image analysis of 2D cell culture. In this article, a set of mitochondria-affecting compounds (FCCP, valinomycin, nicardipine, ionomycin) and Raf kinase inhibitors (PLX4032, PLX4720, GDC, and sorafenib) are applied to multicellular tumor spheroids from two colon adenocarcinoma cell lines (HT-29 and DLD-1). These were screened by TDS and then compared against conventional image-based high-content analysis (HCA). The responses to the Raf inhibitors PLX4032 and PLX4720 are grouped separately by cell line, reflecting the Braf/Kras difference in these cell lines. There is a correlation between TDS and HCA phenotypic clustering for most cases, which demonstrates the ability of dynamic measurements to capture phenotypic responses to drugs. However, there are significant 2D versus 3D phenotypic differences exhibited by several of the drugs/cell lines.

Hyperspectral cytometry is an emerging technology for single-cell analysis that combines ultrafast optical spectroscopy and flow cytometry. Spectral cytometry systems utilize diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels (organic dyes, nanoparticles, or fluorescent proteins) present in each analyzed bioparticle onto linear detector arrays such as multianode photomultipliers or charge-coupled device sensors. The resultant data, consisting of a series of characterizing every analyzed cell, are not compensated by employing the traditional cytometry approach, but rather are spectrally unmixed utilizing algorithms such as constrained Poisson regression or non-negative matrix factorization. Although implementations of spectral cytometry were envisioned as early as the 1980s, only recently has the development of highly sensitive photomultiplier tube arrays led to design and construction of functional prototypes and subsequently to introduction of commercially available systems. This chapter summarizes the historical efforts and work in the field of spectral cytometry performed at Purdue University Cytometry Laboratories and describes the technology developed by Sony Corporation that resulted in release of the first commercial spectral cytometry system-the Sony SP6800. A brief introduction to spectral data analysis is also provided, with emphasis on the differences between traditional polychromatic and spectral cytometry approaches.

Inorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic/soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immunoprotection of islets in transplantation therapies for type 1 diabetes. The new method takes advantage of the islet capsule surface as a template for silica formation. Mouse and human islets were exposed to medium containing saturating silicic acid levels for 9–15 min. The resulting tissue constructs were then cultured for up to 4 wk under normal conditions. Scanning electron microscopy and energy dispersive X-ray spectroscopy was used to monitor the morphology and elemental composition of the material at the islet surface. A cytokine assay was used to assess biocompatibility with macrophages. Islet survival and function were assessed by confocal microscopy, glucose-stimulated insulin release assays, oxygen flux at the islet surface, expression of key genes by RT-PCR, and syngeneic transplant into diabetic mice.

Only a few inventors can be said to have made as great an impact on mankind as Wallace Coulter. His inquisitive mind and ability to see well beyond what existed served him well for 40 years of inventing. So many of the fundamental tools that exist today in the area of hematology were derived from or driven by Coulter’s inventions that he could be called the most technological innovator in the field of modern hematology. In achieving these discoveries Wallace Coulter was clearly capable of visualizing future opportunities that few others recognized. His vision was combined with an uncanny ability to translate his ideas into products. He developed a large number of tools that shaped the fields of cytometry, image analysis, and industrial materials. His understanding of the future power of computation drove him to link these technologies in a unique way. In the end, Coulter shaped the technologies that ultimately drove hematology in a new direction, one that remains on a critical pathway linking technology innovation all the way to true translational impact. It was said of Henry Ford that ‘‘[h]e has no notion that wealth has made him great, and any one who is imprest merely by his wealth bores him. In his personal contacts he likes to dodge the subject. He would prefer to talk with a machinist about machinery, or with somebody who likes birds about birds. In these contacts, he asks no deference; and if he gets it, he suspects it is mere deference to wealth, and that ends his interest.’’ 1 The same could be said of Wallace Coulter, who, like Ford, understood the concepts of mass production and customer service. Coulter had the ability to recognize the opportunity and fulfill the need for development of a blood-cell counter that could be placed in every pathology laboratory, and in so doing transformed a field from a qualitative to a quantitative environment. Every person who has ever entered a medical lab, hospital, or doctor’s office has felt the impact of Coulter’s discovery. ' 2013 International Society for Advancement of Cytometry

Background Conventional diagnosis and identification of bacteria requires shipment of samples to a laboratory for genetic and biochemical analysis. This process can take days and imposes significant delay to action in situations where timely intervention can save lives and reduce associated costs. To enable faster response to an outbreak, a low-cost, small-footprint, portable microbial-identification instrument using forward scatterometry has been developed. Results This device, weighing 9 lb and measuring 12 × 6 × 10.5 in., utilizes elastic light scatter (ELS) patterns to accurately capture bacterial colony characteristics and delivers the classification results via wireless access. The overall system consists of two CCD cameras, one rotational and one translational stage, and a 635-nm laser diode. Various software algorithms such as Hough transform, 2-D geometric moments, and the traveling salesman problem (TSP) have been implemented to provide colony count and circularity, centering process, and minimized travel time among colonies. Conclusions Experiments were conducted with four bacteria genera using pure and mixed plate and as proof of principle a field test was conducted in four different locations where the average classification rate ranged between 95 and 100%.

Early evaluation of new drug entities for their potential to cause mitochondrial dysfunction is becoming an important task for drug development. Multi-parametric high-content screening (mp-HCS) of mitochondrial toxicity holds promise as a lead in-vitro strategy for drug testing and safety evaluations. In this study, we have developed a mp-HCS and multi-parametric data analysis scheme for assessing cell responses to induced mitochondrial perturbation. The mp-HCS measurements are shown to be robust enough to allow for quantitative comparison of biological systems with different metabolic pathways simulated by alteration of growth media. Substitution of medium glucose for galactose sensitized cells to drug action and revealed novel response parameters. Each compound was quantitatively characterized according to induced phenotypic changes of cell morphology and functionality measured by fluorescent biomarkers for mitochondrial activity, plasma membrane permeability, and nuclear morphology. Descriptors of drug effects were established by generation of a SCRIT (Specialized-Cell-Response-to-Induced-Toxicity) vector, consisting of normalized statistical measures of each parameter at each dose and growth condition. The dimensionality of SCRIT vectors depends on the number of parameters chosen, which in turn depends on the hypothesis being tested. Specifically, incorporation of three parameters of response into SCRIT vectors enabled clustering of 84 training compounds with known pharmacological and toxicological activities according to the degree of toxicity and mitochondrial involvement. Inclusion of 6 parameters enabled the resolution of more subtle differences between compounds within a common therapeutic class; scoring enabled a ranking of statins in direct agreement with clinical outcomes. Comparison of drug-induced changes required variations in glucose for separation of mitochondrial dysfunction from other types of cytotoxicity. These results also demonstrate that the number of drugs in a training set, the choice of parameters used in analysis, and statistical measures are fundamental for specific hypothesis testing and assessment of quantitative phenotypic differences.

We have developed an automated system for drug screening using a single-cell-multiple functional response technology. The approach uses a semiautomated preparatory system, high-speed sample collection, and a unique analytical tool that provides instantaneous results for compound dilutions using 384-well plates. The combination of automation and rapid robotic sampling increases quality control and robustness. High-speed flow cytometry is used to collect single-cell results together with a newly defined analytical tool for extraction of IC(50) curves for multiple assays per cell. The principal advantage is the extreme speed of sample collection, with results from a 384-well plate being completed for both collection and data processing in less than 10 min. Using this approach, it is possible to extract detailed drug response information in a highly controlled fashion. The data are based on single-cell results, not populations. With simultaneous assays for different functions, it is possible to gain a more detailed understanding of each drug/compound interaction. Combined with integrated advanced data processing directly from raw data files, the process from sampling to analytical results is highly intuitive. Direct PubMed links allow review of drug structure and comparisons with similar compounds.

Flow cytometry has been around for over 40 years, but only recently has the opportunity arisen to move into the high-throughput domain. The technology is now available and is highly competitive with imaging tools under the right conditions. Flow cytometry has, however, been a technology that has focused on its unique ability to study single cells and appropriate analytical tools are readily available to handle this traditional role of the technology.Expansion of flow cytometry to a high-throughput (HT) and high-content technology requires both advances in hardware and analytical tools. The historical perspective of flow cytometry operation as well as how the field has changed and what the key changes have been discussed. The authors provide a background and compelling arguments for moving toward HT flow, where there are many innovative opportunities. With alternative approaches now available for flow cytometry, there will be a considerable number of new applications. These opportunities show strong capability for drug screening and functional studies with cells in suspension.There is no doubt that HT flow is a rich technology awaiting acceptance by the pharmaceutical community. It can provide a powerful phenotypic analytical toolset that has the capacity to change many current approaches to HT screening. The previous restrictions on the technology, based on its reduced capacity for sample throughput, are no longer a major issue. Overcoming this barrier has transformed a mature technology into one that can focus on systems biology questions not previously considered possible.