Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images

The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non‐invasive determination of these characteristics. We present an image‐processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (<1 s per 1,208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source‐code for MATLAB and ImageJ is freely available under a permissive open‐source license. Biotechnol. Bioeng. 2014;111: 504–517. © 2013 Wiley Periodicals, Inc.     

Zero-cost modification of bright field microscopes for imaging phase gradient on cells: Schlieren optics

A simple, zero-cost, reversible modification of a bright field microscope permits visualization of phase gradients in cells by transmitted illumination, yielding a Nomarski-like effect. This modification, based on schlieren optics, is simultaneously compatible with high-aperture epi-illumination fluorescence excitation. For many objectives that are intended for use in fluorescence work, but are unavailable in phase contrast versions, the modification provides a simple means for locating cells in culture with good image contrast and resolution.     

液晶空间光调制器在生物光学显微中的应用

利用液晶空间光调制器(LCSLM)对光学显微中的成像光进行实时的相位/振幅调制,不仅可以实现各种传统的生物样品相位显微,而且能够以更复杂的相位调制方式,如螺旋相位滤波,得到新的显微图像。该方式已经和荧光显微、光镊技术结合,丰富了生物显微技术。     

Unsupervised organization of cervical cells using bright‐field and single‐shot digital holographic microscopy

We report results on unsupervised organization of cervical cells using microscopy of Pap‐smear samples in brightfield (3‐channel color) as well as high‐resolution quantitative phase imaging modalities. A number of morphological parameters are measured for each of the 1450 cell nuclei (from 10 woman subjects) imaged in this study. The principal component analysis (PCA) methodology applied to this data shows that the cell image clustering performance improves significantly when brightfield as well as phase information is utilized for PCA as compared to when brightfield‐only information is used. The results point to the feasibility of an image‐based tool that will be able to mark suspicious cells for further examination by the pathologist. More importantly, our results suggest that the information in quantitative phase images of cells that is typically not used in clinical practice is valuable for automated cell classification applications in general.      

Cytophotometric studies of cell populations

Technologic advances in the recording of digitized imagery have made the study of large cell populations by image analytical methods feasible. Computed image information provides quantitative, and novel, information that allows an exact measurement of minute changes in the chromatin distribution of cell nuclei, and the detection of subpopulations of cells or changes in the functional state of the cells. The sensitivity of the detection exceeds that of human observers; the specificity of the measured changes must be the subject of basic cell biologic research.     

Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast

We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire four images from which phase contrast images can be calculated. This polarisation-resolved differential phase contrast (pDPC) microscopy technique can be easily integrated with fluorescence microscopy.     

两种微体化石三维无损成像技术的对比

近年来,各种X射线三维无损成像技术在古生物学领域的应用越来越广泛。但是,不同的X射线三维无损成像技术针对不同保存类型和尺寸的化石标本在成像效果上各有利弊。本文以埃迪卡拉纪陡山沱组磷酸盐化的动物胚胎化石为研究对象,将目前应用最广的两种X射线三维无损成像方法,即基于实验室X光源的吸收衬度显微断层成像技术和基于同步辐射光源的相位衬度显微断层成像技术进行了对比分析。通过对两种技术的原理、效率、空间分辨率和图像衬度的对比,认为基于同步辐射光源的相位衬度显微断层成像技术是目前对于均一矿化的微体化石最佳的三维无损成像解决方案。     

Improved visualization of X-ray phase contrast volumetric data through artifact-free integrated differential images

Artifacts arising when differential phase images are integrated is a common problem to several X-ray phase-based experimental techniques. The combination of noise and insufficient sampling of the high-frequency differential phase signal leads to the formation of streak artifacts in the projections, translating into poor image quality in the tomography slices. In this work, we apply a non-iterative integration algorithm proven to reduce streak artifacts in planar (2D) images to a differential phase tomography scan. We report on how the reduction of streak artifacts in the projections improves the quality of the tomography slices, especially in the directions different from the reconstruction plane. Importantly, the method is compatible with large tomography datasets in terms of computation time.     

Dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by the digestive fluid of Trichoplusia ni (Lepidoptera:Noctuidae) larvae

The dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus by digestive fluid collected from 5th stage Trichoplusia ni larvae was studied in vitro. Observations were made at timed intervals using phase contrast microscopy, and scanning and transmission electron microscopy. Dissolution occurred rapidly and in a detectable sequence. Under phase contrast, most polyhedra lost their refringence by 0.5 min. The polyhedra became rounded in appearance with small protuberances on the surface and Brownian movement was observed within. After 1 min, the envelope of most polyhedra had ruptured, releasing the enclosed virions. The protuberances were also observed under the scanning electron microscope after digestion for 0.5 min. Many shell fragments devoid of internal contents were seen after more lengthy digestion. Internal structural changes were revealed by electron microscopy. After 1 min of exposure, polyhedra were observed in all stages of dissolution. By 3 min, only virions, scattered about in heterogeneous material, could be distinguished.     

The intranuclear bodies of Entamoeba

Intranuclear bodies which are spherical in shape are clearly seen by ‘Anoptral’ (negative) phase microscopy in the nucleus of Entamoeba. These bodies vary in size and numbers from cell to cell. With interference microscopy the intranuclear bodies appear as spherical granules in the nucleus of the cell. Their distribution and numbers are again very variable. With electron microscopy the bodies can be clearly seen inside the nucleus. They are always spherical in shape but vary in size from 0·1 to 1·5 μm. They may be empty or contain granular or membranous material. They display the capacity to move out of the nucleus.     

Quantitative Analysis of Centromeric FISH Spots during the Cell Cycle by Image Cytometry

Two-color fluorescence in situ hybridization (FISH) with chromosome enumeration DNA probes specific to chromosomes 7, 11, 17, and 18 was applied to CAL-51 breast cancer cells to examine whether the fluorescence intensity of FISH spots was associated with cell cycle progression. The fluorescence intensity of each FISH spot was quantitatively analyzed based on the cell cycle stage determined by image cytometry at the single-cell level. The spot intensity of cells in the G₂ phase was larger than that in the G_0/1 phase. This increased intensity was not seen during the early and mid S phases, whereas the cells in the late S phase showed significant increases in spot intensity, reaching the same level as that observed in the G₂ phase, indicating that alpha satellite DNA in the centromeric region was replicated in the late S phase. Thus, image cytometry can successfully detect small differences in the fluorescence intensities of centromeric spots of homologous chromosomes. This combinational image analysis of FISH spots and the cell cycle with cell image cytometry provides insights into new aspects of the cell cycle. This is the first report demonstrating that image cytometry can be used to analyze the fluorescence intensity of FISH signals during the cell cycle.     

Methods for acquisition of quantitative data from confocal images of gene expression in situ

In this review, we summarize original methods for the extraction of quantitative information from confocal images of gene-expression patterns. These methods include image segmentation, the extraction of quantitative numerical data on gene expression, and the removal of background signal and spatial registration. Finally, it is possible to construct a spatiotemporal atlas of gene expression from individual images recorded at each developmental stage. Initially all methods were developed to extract quantitative numerical information from confocal images of segmentation gene expression in Drosophila melanogaster. The application of these methods to Drosophila images makes it possible to reveal new mechanisms in the formation of segmentation gene expression domains, as well as to construct a quantitative atlas of segmentation gene expression. Most image processing procedures can be easily adapted to process a wide range of biological images.     

Noise suppressed,multifocus image fusion for enhanced intraoperative navigation

Current intraoperative imaging systems are typically not able to provide ‘sharp’ images over entire large areas or entire organs. Distinct structures such as tissue margins or groups of malignant cells are therefore often difficult to detect, especially under low signal‐to‐noise‐ratio conditions. In this report, we introduce a noise suppressed multifocus image fusion algorithm, that provides detailed reconstructions even when images are acquired under sub‐optimal conditions, such is the case for real time fluorescence intraoperative surgery. The algorithm makes use of the Anscombe transform combined with a multi‐level stationary wavelet transform with individual threshold‐based shrinkage. While the imaging system is integrated with a respiratory monitor triggering system, it can be easily adapted to any commercial imaging system. The developed algorithm is made available as a plugin for Osirix. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Inline characterization of cell concentration and cell volume in agitated bioreactors using in situ microscopy: application to volume variation induced by osmotic stress

A new in situ microscope (ISM) was developed and tested to perform in-line monitoring of average cell volume and cell concentration in agitated cultures subjected to osmotic stress. The ISM is directly immersed into the agitated broth in a bioreactor and generates still images of cells by using pulsed luminescent diode illumination and a virtual probe volume defined by depth of focus. This technique allows the acquisition of microscopic still images without mechanical sampling techniques. The front end of the sensor fits into a standard 25-mm port and it can be steam sterilized together with the bioreactor. The automatic image evaluation generates signals of the cell concentration and the average cell volume with a time resolution of a few minutes per data point (if a 200 MHz PC is used). Without the need for evaluation, the images can be acquired and stored at a rate of one image per 0.6 s. Hansenula anomala was cultivated as batch fermentation and monitored inline with the ISM. The ISM signal of the cell concentration agreed well with referential growth curves that were obtained from counting with a hemocytometer. The ISM signal of the average cell volume shows a gradual volume reduction as a result of the aging of the culture, and it monitors an abrupt and strong cell contraction if osmotic shocks are generated in the bioreactor. Systematic in vitro studies of osmotic shocks were performed by applying the ISM to agitated culture samples of H. anomala. The volume signal of H. anomala during osmotic shocks showed a very fast cell contraction within less than a second. Within half an hour after the shocks, no signal drifts were observed, which would indicate volume restoration. These findings suggest that the ISM volume signal can be used as an inline indicator of osmotic stress in cell cultures.     

A novel assay based on fluorescence microscopy and image processing for determining phenotypic distributions of rod-shaped bacteria

Cell population balance (CPB) models can account for the phenotypic heterogeneity that characterizes isogenic cell populations. To utilize the predictive power of these models, however, we must determine the single-cell reaction and division rates as well as the partition probability density function of the cell population. These functions can be obtained through the Collins-Richmond inverse CPB modeling methodology, if we know the phenotypic distributions of (a) the overall cell population, (b) the dividing cell subpopulation, and (c) the newborn cell subpopulation. This study presents the development of a novel assay that combines fluorescence microscopy and image processing to determine these distributions. The method is generally applicable to rod-shaped cells dividing through the formation of a characteristic constriction. Morphological criteria were developed for the automatic identification of dividing cells and validated through direct comparison with manually obtained measurements. The newborn cell subpopulation was obtained from the corresponding dividing cell subpopulation by collecting information from the two compartments separated by the constriction. The method was applied to E. coli cells carrying the genetic toggle network with a green fluorescent marker. Our measurements for the overall cell population were in excellent agreement with the distributions obtained via flow cytometry. The new assay constitutes a powerful tool that can be used in conjunction with inverse CPB modeling to rigorously quantify single-cell behavior from data collected from highly heterogeneous cell populations.     

Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits

Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential for rapid F-actin turnover, and most depolymerize actin in a pH-dependent manner. Complexes of human and plant ADF with F-actin at different pH were examined using electron microscopy and a novel method of image analysis for helical filaments. Although ADF changes the mean twist of actin, we show that it does this by stabilizing a preexisting F-actin angular conformation. In addition, ADF induces a large ( approximately 12 degrees ) tilt of actin subunits at high pH where filaments are readily disrupted. A second ADF molecule binds to a site on the opposite side of F-actin from that of the previously described ADF binding site, and this second site is only largely occupied at high pH. All of these states display a high degree of cooperativity that appears to be an integral part of F-actin.     

The mammalian cardiac muscle thick filament: crossbridge arrangement

Although skeletal muscle thick filaments have been extensively studied, information on the structure of cardiac thick filaments is limited. Since cardiac muscle differs in many physiological properties from skeletal muscle it is important to elucidate the structure of the cardiac thick filament. The structure of isolated and negatively stained rabbit cardiac thick filaments has been analyzed from computed Fourier transforms and image analysis. The transforms are detailed, showing a strong set of layer lines corresponding to a 42.9 nm quasi-helical repeat. The presence of relatively strong "forbidden" meridional reflections not expected from ideal helical symmetry on the second, fourth, fifth, seventh, eighth, and tenth layer lines suggest that the crossbridge array is perturbed from ideal helical symmetry. Analysis of the phase differences for the primary reflections on the first layer line of transforms from 15 filaments showed an average difference of 170 degrees, close to the value of 180 degrees expected for an odd-stranded structure. Computer-filtered images of the isolated thick filaments unequivocally demonstrate a three-stranded arrangement of the crossbridges on the filaments and provide evidence that the crossbridge arrangement is axially perturbed from ideal helical symmetry.     

Movement correction method for laser speckle contrast imaging of cerebral blood flow in cranial windows in rodents

Laser speckle contrast imaging (LSCI) is used in clinical research to dynamically image blood flow. One drawback is its susceptibility to movement artifacts. We demonstrate a new, simple method to correct motion artifacts in LSCI signals measured in awake mice with cranial windows during sensory stimulation. The principle is to identify a region in the image in which speckle contrast (SC) is independent of blood flow and only varies with animal movement, then to regress out this signal from the data. We show that (1) the regressed signal correlates well with mouse head movement, (2) the corrected signal correlates better with independently measured blood volume and (3) it has a (59 ± 6)% higher signal-to-noise ratio. Compared to three alternative correction methods, ours has the best performance. Regressing out flow-independent global variations in SC is a simple and accessible way to improve the quality of LSCI measurements.