Computer automated cell size and shape analysis in cryomicroscopy

Computer vision techniques have been developed for quantitative analysis of size and shape changes in cells frozen on a cryomicroscope. The analysis is based on implementation of standard serial edge detection algorithms in conjunction with a shape transform to isolate individual cells in complex scenes which may include adjacent and overlying ice crystals. In the present study the sensitivity of the automated analysis procedure is evaluated for images obtained by various microscope optical systems for progressive degrees of subject blurring by defocusing. Size measurements in calibration trials for freezing latex spheres with extracellular ice in the field of view were least sensitive for bright field images, although the most consistent data was obtained by differential interference contrast microscopy. In all cases phase contrast images produced the least accurate data. An example analysis is presented for the freezing of pancreas β-cells.     

Segmentation of ascidian notochord cells in DIC timelapse images

We have developed a method to automatically segment notochord cell boundaries from differential interference contrast (DIC) timelapse images of the elongating ascidian tail. The method is based on a specialized parametric active contour, the network snake, which can be initialized as a network of arbitrary but fixed topology and provides an effective framework for simultaneously segmenting multiple touching cells. Several modifications to the original network snake were necessary for high-quality segmentation, including linear Gaussian derivative filtering to reconstruct edge maps from DIC images and a new energy function to improve the segmentation of critical cell-cell vertices. We find that post-intercalation ascidian notochord cells exhibit two distinct cell behaviors: lateral cell edges expand along the AP axis while showing a rapid pulsatile behavior, whereas anterior and posterior cell edges contract smoothly.     

A method for quantifying cell size from differential interference contrast images: validation and application to osmotically stressed chondrocytes

An automatic image analysis method was developed to determine the shape and size of spheroidal cells from a time series of differential interference contrast (DIC) images. The program incorporates an edge detection algorithm and dynamic programming for edge linking. To assess the accuracy and working range of the method, results from DIC images of different focal planes and resolutions were compared to confocal images in which the cell membrane was fluorescently labelled. The results indicate that a 1‐µm focal drift from the in‐focus plane can lead to an overestimation of cell volume up to 14.1%, mostly due to shadowing effects of DIC microscopy. DIC images allow for accurate measurements when the focal plane lies in a zone slightly above the centre of a spherical cell. In this range the method performs with 1.9% overall volume error without taking into account the error introduced by the representation of the cell as a sphere. As a test case, the method was applied to quantify volume changes due to acute changes of osmotic stress.     

Quantifying cell–matrix adhesion dynamics in living cells using interference reflection microscopy

Focal adhesions and podosomes are integrin‐mediated cell‐substratum contacts that can be visualized using interference reflection microscopy (IRM). Here, we have developed automated image‐processing procedures to quantify adhesion turnover from IRM images of live cells. Using time sequences of images, we produce adhesion maps that reveal the spatial changes of adhesions and contain additional information on the time sequence of these changes. Such maps were used to characterize focal adhesion dynamics in mouse embryo fibroblasts lacking one or both alleles of the vinculin gene. Loss of vinculin expression resulted in increased assembly, disassembly and/or in increased translocation of focal adhesions, suggesting that vinculin is important for stabilizing focal adhesions. This method is also useful for studying the rapid dynamics of podosomes as observed in primary mouse dendritic cells.     

Image processing nanoparticle size measurement for determination of density values to correct the ELPI measures

The ELPI is an electrical low-pressure impactor that classifies aerosol particles according to their aerodynamic diameter, and generates the amount of particles impacted on each of the 12 stages. This number depends on the measured current induced by the pre-charged particles and on the density which is given by the user and may not be a priori known. In addition, the density used by the software is considered to be similar for all stages. In this paper, a method to evaluate the density of the particles on each stage is proposed in order to consequently increase the accuracy of the results given by the software. The data needed are the aerodynamic diameter, the equivalent diameter and information on the form. The aerodynamic diameter is a range defined by the cut-off aerodynamic diameter of the stages of the ELPI. To measure the equivalent diameter and evaluate the form, an adapted procedure that used microscopy and image processing tools was set up with the study of two different polydispersed aerosols, silica and fly ash particles from wood combustion. This method was validated with Silica particles (ρ = 2.5 g cm⁻³ with the pycnometer): the density was found to be 2.2 g cm⁻³ and 2.4 g cm⁻³ for stages 2 (d_ae around 76 nm) and 3 (d_ae around 127 nm), respectively. The results match reality for fly ashes from wood combustion as well: ρ = 1.0 g cm⁻³ for the stage 2 and 1.9 for stage 5.     

数字化适形铅模系统中靶区轮廓提取方法

根据开发数字化适形铅模系统的需要,研究了由X光片中提取出靶区轮廓的方法。对于标有靶区轮廓的X光片扫描图像,首先进行窗口变换,然后进行阈值变换使其二值化,接着对图像进行增强处理,最后进行细化,最终提取靶区轮廓坐标数据。     

An orientation‐independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model

Epithelial cell dynamics can be difficult to study in intact animals or tissues. Here we use the medusa form of the hydrozoan Clytia hemisphaerica, which is covered with a monolayer of epithelial cells, to test the efficacy of an orientation‐independent differential interference contrast microscope for in vivo imaging of wound healing. Orientation‐independent differential interference contrast provides an unprecedented resolution phase image of epithelial cells closing a wound in a live, nontransgenic animal model. In particular, the orientation‐independent differential interference contrast microscope equipped with a 40x/0.75NA objective lens and using the illumination light with wavelength 546 nm demonstrated a resolution of 460 nm. The repair of individual cells, the adhesion of cells to close a gap, and the concomitant contraction of these cells during closure is clearly visualized.     

Laser interference microscopy of amphibian erythrocytes: impact of cell volume and refractive index

This study examined the action of anisosmotic media on the volume of nucleated erythrocytes isolated from Rana temporaria. Elevation of medium osmolarity from 100 to 345 mOsm resulted in attenuation of mean cell volume by more than 3-fold, estimated by hematocrit measurement. By contrast to this 'classic' erythrocyte volume evaluation technique, we did not observe any significant cell volume modulation by examining the 3D reconstruction of erythrocyte interference images obtained by laser interference microscopy. Comparative analysis of mean cell volume, phase height and cell area appraised by laser interference microscopy showed that the lack of visible alterations of phase image geometry was caused by sharp elevation of the average refractive index of the cytoplasm in shrunken cells. Thus, our results show for the first time that laser interference microscopy in combination with a direct method for cell volume measurement may be employed for estimation of the refractory index of intracellular milieu and for assessment of changes of physical chemical properties of the cytoplasm evoked by diverse stimuli including osmotic stress.     

LIP-model-based three-dimensional reconstruction and visualization of HIV-infected entire cells

This paper presents a global solution from acquisition to visualization for the three-dimensional reconstruction of cell sections. Original techniques are proposed for the correct handling of the geometrical section distortions, and a new interpretation based on the logarithmic image processing (LIP) model is applied in order to create normalized grey-level sections where these are missing. Finally, a new method for generating a mesh of triangles to describe the envelope of the reconstructed cell is proposed, as well as a visualization mixing image synthesis and grey-level information. The product allows the user to explore the reconstructed cellular block in any desired direction, by showing user-defined grey-level sections inside the block mixed to a synthetic view of the cell envelope.     

Automatic segmentation,counting, size determination and classification of white blood cells

The counts, the so-called differential counts, and sizes of different types of white blood cells provide invaluable information to evaluate a wide range of important hematic pathologies from infections to leukemia. Today, the diagnosis of diseases can still be achieved mainly by manual techniques. However, this traditional method is very tedious and time-consuming. The accuracy of it depends on the operator’s expertise. There are laser based cytometers used in laboratories. These advanced devices are costly and requires accurate hardware calibration. They also use actual blood samples. Thus there is always a need for a cost effective and robust automated system. The proposed system in this paper automatically counts the white blood cells, determine their sizes accurately and classifies them into five types such as basophil, lymphocyte, neutrophil, monocyte and eosinophil. The aim of the system is to help for diagnosing diseases. In our work, a new and completely automatic counting, segmentation and classification process is developed. The outputs of the system are the number of white blood cells, their sizes and types.     

The use of ArcGIS for determination of quartz optical axis orientation in thin section images

In this paper, a new toolbox for petrographic thin section analysis is presented. It was designed using ArcGIS ModelBuilder to extract quartz grain boundary and determine its optical axis orientation from a set of thin section images. It can perform these analyses with little or no operator intervention and convert the boundary and optical axis data into a digital database with preserved spatial relationship. In order to test the validity of toolbox's outputs, an assessment was performed using three natural quartzite thin sections. The results indicate a close relationship (>0.8) between manual and model‐base extraction of grain boundary and optical axis orientation data. This study demonstrates the feasibility and usefulness of performing thin section image analysis within the GIS framework.     

Cell counting tool parameters optimization approach for electroporation efficiency determination of attached cells in phase contrast images

Categorizing biologic signals by analysis of symbolic sequences was employed in the study of prostate microvessels. The estimates of the volume fraction of the vessels immunostained to Factor-VIII was mapped to binary sequences. The distance between sequences was estimated by comparing the rank and frequency of repetitive elements. These measurements were applied to detect whether there are unique microvascular patterns for each individual, and to search for patterns describing prostate microvessels of different conditions. Normal prostate, benign prostate hyperplasia and prostate carcinoma groups were studied. All the specimens were immunostained to F-VIII and strips formed by adjacent quadrats were explored. At each point of the long axis of the strip, the V(V) F-VIII was calculated. These values were processed with the information-based similarity software to estimate the dissimilarity between two space series. The following comparisons were carried out: intrasubject versus intragroup distances; intragroup distances among the groups studied and intergroup distances. The distance defined between a vessels immunostained to Factor-VIII space series and its randomized surrogate was considered as an index of the nonrandomness of the space series. These indices were compared for all the groups. We conclude that (a) The information-based similarity analysis can be adapted to vessels immunostained to Factor-VIII space series from prostate microvessels. (b) There are no unique microvascular patterns associated with each individual. (c) There are characteristic patterns describing the microvessels from normal prostate, benign prostate hyperplasia and carcinoma. (d) This method is able to account for the differences between prostate cancer and both normal and benign prostate conditions, with respect to the microvessel patterns.     

Determining image distortion and PBS (point of best symmetry) in digital images using straight line matrices

It is impossible to take accurate measurements in photogrammetry without first removing the distortion in images. This paper presents a methodology for correcting radial and tangential distortion and for determining the PBS (Point of Best Symmetry) without knowledge of the interior orientation parameters (IOPs). An analytical plumb-line calibration method is used, measuring only the coordinates of points on straight lines, regardless of the position and direction of these lines within the image. Points belonging to multiple lines can also be used since the effects on their X and Y coordinates are calculated independently. The results obtained on an image of a common scene, taken with a handheld non-metric camera, show a high degree of accuracy even with a minimum number of observables. And its application on a calibrated grid for engineering purposes with a semi-metric camera, results optimal even using a single image.     

MRT letter: Nanoscopy of protein colocalization in living cells by STED and GSDIM

We report the use of superresolution fluorescence microscopy for studying the nanoscale distribution of protein colocalization in living mammalian cells. Nanoscale imaging is attained both by a targeted and a stochastic fluorescence on-off switching superresolution method, namely by stimulated emission depletion (STED) and ground state depletion microscopy followed by individual molecular return (GSDIM), respectively. Analysis of protein colocalization is performed by bimolecular fluorescence complementation (BiFC). Specifically, a nonfluorescent fragment of the yellow fluorescent protein Citrine is fused to tubulin while a counterpart nonfluorescent fragment is fused to the microtubulin-associated protein MAP2 such that fluorescence is reconstituted on contact of the fragment-carrying proteins. Images with resolution down to 65 nm prove a powerful new way for studying protein colocalization in living cells at the nanoscale.     

An integrated environmental perfusion chamber and heating system for long-term, high resolution imaging of living cells

This communication presents the design and application of an integrated environmental perfusion chamber and stage heating blanket suitable for time-lapse video microscopy of living cells. The system consists of two independently regulated components: a perfusion chamber suitable for the maintenance of cell viability and the variable delivery of environmental factors, and a separate heating blanket to control the temperature of the microscope stage and limit thermal conduction from the perfusion chamber. Two contrasting experiments are presented to demonstrate the versatility of the system. One long-term sequence illustrates the behaviour of cells exposed to ceramic fibres. The other shows the shrinking response of cultured articular cartilage chondrons under dynamic hyper-osmotic conditions designed to simulate joint loading. The chamber is simple in design, economical to produce and permits long-term examination of dynamic cellular behaviour while satisfying the fundamental requirements for the maintenance of environmental factors that influence cell viability.     

Improvements to differential voltage contrast and light and electron scanning beam analysis of solar cells

Differential voltage contrast (DVC) in conjunction with light and electron beam scanning (LEBEAMS) technique were used for measuring the electric potential, field, and charge distribution in solar cells. The DVC is based on enhancement or retardation of secondary electron emission, generated by an electron beam, due to local changes in the potential of a semiconductor device. The information provided by this technique is invaluable to the development of any device. Solar cells have been studied by the DVC technique, both under electrical bias (DVC) and under illumination (DVC in conjunction with LEBEAMS); however, the conditions of the previous did not replicate the normal illumination conditions of a solar cell. The goal of this research was to redesign and expand the previous LEBEAMS experiments to produce accurate profiles of quasi Fermi energies on solar cells.     

Calibration and quantification of fast intracellular motion (FIM) in living cells using correlation analysis

Video rate confocal laser scanning microscopy at the highest spatial and temporal resolution of backscattered light (BSL) imaging allowed for regular observation of fast intracellular motion (FIM) first revealed in living neoplastic cells. However, the absence of an objective evaluation has hampered further study of the mechanisms and biological significance of FIM. Particularly, a quantification of apparent differences in velocities that would complement and improve the current demonstration of FIM by color coding using the combination of red-green-blue (RGB) images had been missing. Standard methods of tracking or pattern recognition could not be applied because of the fuzzy nature of images of FIM. A search for a suitable method led to correlation analysis. It was calibrated on Brownian motion and a known type of motion, such as cell marginal ruffling, compared with FIM. Results approved its explanatory potential. Therefore, several crucial incidences of FIM could be analyzed. Apart from an argument against viewing FIM as a manifestation of simple Brownian motion, the correlation analysis of FIM in the adjacent peripheries of a rat fibroblast and a K4 rat sarcoma cell confirmed the notion of higher and uneven distribution of velocity of FIM in a tumor cell so far shown in color-coded images only. This result and other yet unpublished observations indicate that the velocity and topology of FIM can also contribute to a biological distinction between neoplastic and normal cells. Regular application of the correlation analysis should further expand the study of FIM for its mechanisms and predictive value. Such an approach should be thoroughly examined for a contribution to the knowledge of cancer cells.     

Differential interference contrast and confocal reflectance imaging of collagen organization in three-dimensional matrices

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the process of 3-D extracellular matrix (ECM) remodeling at the cellular and subcellular level. In this study, we directly compare two imaging modalities for both quantitative and qualitative imaging of 3-D collagen organization in vitro: differential interference contrast (DIC) and confocal reflectance imaging. The results demonstrate that two-dimensional (2-D) DIC images allow visualization of the same population of collagen fibrils as observed in 2-D confocal reflectance images. Thus, DIC can be used for qualitative assessment of fibril organization, as well as tracking of fibril movement in sequential time-lapse 2-D images. However, we also found that quantitative techniques that can be applied to confocal reflectance images, such as Fourier transform analysis, give different results when applied to DIC images. Furthermore, common techniques used for 3-D visualization and reconstruction of confocal reflectance datasets are not generally applicable to DIC. Overall, obtaining a complete understanding of cell-matrix mechanical interactions will likely require a combination of both wide-field DIC imaging to study rapid changes in ECM deformation which can occur within minutes, and confocal reflectance imaging to assess more gradual changes in cell-induced compaction and alignment of ECM which occur over a longer time course.     

Enhancement of contrast in living and fixed specimens by the use of fibre optics

A method is described which enhances the contrast of living and fixed specimens examined with the stereomicroscope. It consists of immersing the ends of flexible fibre optic light sources together with the specimen in the fluid used for examination. It is reported that not only does this method increase the contrast of living specimens but that it may also be applied to specimens being prepared as thin sections or freeze fracture surfaces for examination with the transmission electron microscope. A further method of enhancement of contrast is suggested which involves the fitting of light filters of complementary colours, one to each of the fibre optic light sources, before immersion with the specimen.     

Stereological estimation of average cell volume in monolayer culture by combined light and electron microscopy

In order to convert stereological ratio estimates into ‘absolute’ values (‘per cell’ values) the average cell volume must be estimated. The present paper describes a stereological method based on well-known point counting procedures for the estimation of average cell volumes in monolayer cultures fixed in situ. This method involves estimation of the average attachment area per cell by light microscopy combined with estimation of the attachment membrane surface density by electron microscopy. There is no need for any assumption as to cellular or nuclear shape. The method has been tested on an established cell line, NHIK 3025, and shows a good accuracy. It has also been used to analyse the volume changes that take place in human monocytes during monolayer culture, demonstrating a 28-fold increase of the average cell volume over 10 days.