Bright Field Microscopy as an Alternative to Whole Cell Fluorescence in Automated Analysis of Macrophage Images

Background

Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity.

Methodology

We here present and validate a method to automatically detect cell population outlines directly from bright field images. By imaging samples with several focus levels forming a bright field -stack, and by measuring the intensity variations of this stack over the -dimension, we construct a new two dimensional projection image of increased contrast. With additional information for locations of each cell, such as stained nuclei, this bright field projection image can be used instead of whole cell fluorescence to locate borders of individual cells, separating touching cells, and enabling single cell analysis. Using the popular CellProfiler freeware cell image analysis software mainly targeted for fluorescence microscopy, we validate our method by automatically segmenting low contrast and rather complex shaped murine macrophage cells.

Significance

The proposed approach frees up a fluorescence channel, which can be used for subcellular studies. It also facilitates cell shape measurement in experiments where whole cell fluorescent staining is either not available, or is dependent on a particular experimental condition. We show that whole cell area detection results using our projected bright field images match closely to the standard approach where cell areas are localized using fluorescence, and conclude that the high contrast bright field projection image can directly replace one fluorescent channel in whole cell quantification. Matlab code for calculating the projections can be downloaded from the supplementary site: http://sites.google.com/site/brightfieldorstaining     

Multi-Scale and Shape Constrained Localized Region-Based Active Contour Segmentation of Uterine Fibroid Ultrasound Images in HIFU Therapy

Purpose

To overcome the severe intensity inhomogeneity and blurry boundaries in HIFU (High Intensity Focused Ultrasound) ultrasound images, an accurate and efficient multi-scale and shape constrained localized region-based active contour model (MSLCV), was developed to accurately and efficiently segment the target region in HIFU ultrasound images of uterine fibroids.

Methods

We incorporated a new shape constraint into the localized region-based active contour, which constrained the active contour to obtain the desired, accurate segmentation, avoiding boundary leakage and excessive contraction. Localized region-based active contour modeling is suitable for ultrasound images, but it still cannot acquire satisfactory segmentation for HIFU ultrasound images of uterine fibroids. We improved the localized region-based active contour model by incorporating a shape constraint into region-based level set framework to increase segmentation accuracy. Some improvement measures were proposed to overcome the sensitivity of initialization, and a multi-scale segmentation method was proposed to improve segmentation efficiency. We also designed an adaptive localizing radius size selection function to acquire better segmentation results.

Results

Experimental results demonstrated that the MSLCV model was significantly more accurate and efficient than conventional methods. The MSLCV model has been quantitatively validated via experiments, obtaining an average of 0.94 for the DSC (Dice similarity coefficient) and 25.16 for the MSSD (mean sum of square distance). Moreover, by using the multi-scale segmentation method, the MSLCV model’s average segmentation time was decreased to approximately 1/8 that of the localized region-based active contour model (the LCV model).

Conclusions

An accurate and efficient multi-scale and shape constrained localized region-based active contour model was designed for the semi-automatic segmentation of uterine fibroid ultrasound (UFUS) images in HIFU therapy. Compared with other methods, it provided more accurate and more efficient segmentation results that are very close to those obtained from manual segmentation by a specialist.     

Spatiotemporal variation of leaf epidermal cell growth: a quantitative analysis of Arabidopsis thaliana wild-type and triple cyclinD3 mutant plants

Background and Aims

The epidermis of an expanding dicot leaf is a mosaic of cells differing in identity, size and differentiation stage. Here hypotheses are tested that in such a cell mosaic growth is heterogeneous and changes with time, and that this heterogeneity is not dependent on the cell cycle regulation per se.

Methods

Shape, size and growth of individual cells were followed with the aid of sequential replicas in expanding leaves of wild-type Arabidopsis thaliana and triple cyclinD3 mutant plants, and combined with ploidy estimation using epi-fluorescence microscopy.

Key Results

Relative growth rates in area of individual epidermal cells or small cell groups differ several fold from those of adjacent cells, and change in time. This spatial and temporal variation is not related to the size of either the cell or the nucleus. Shape changes and growth within an individual cell are also heterogeneous: anticlinal wall waviness appears at different times in different wall portions; portions of the cell periphery in contact with different neighbours grow with different rates. This variation is not related to cell growth anisotropy. The heterogeneity is typical for both the wild type and cycD3.

Conclusions

Growth of leaf epidermis exhibits spatiotemporal variability.     

Joint level-set and spatio-temporal motion detection for cell segmentation

Background

Cell segmentation is a critical step for quantification and monitoring of cell cycle progression, cell migration, and growth control to investigate cellular immune response, embryonic development, tumorigenesis, and drug effects on live cells in time-lapse microscopy images.

Methods

In this study, we propose a joint spatio-temporal diffusion and region-based level-set optimization approach for moving cell segmentation. Moving regions are initially detected in each set of three consecutive sequence images by numerically solving a system of coupled spatio-temporal partial differential equations. In order to standardize intensities of each frame, we apply a histogram transformation approach to match the pixel intensities of each processed frame with an intensity distribution model learned from all frames of the sequence during the training stage. After the spatio-temporal diffusion stage is completed, we compute the edge map by nonparametric density estimation using Parzen kernels. This process is followed by watershed-based segmentation and moving cell detection. We use this result as an initial level-set function to evolve the cell boundaries, refine the delineation, and optimize the final segmentation result.

Results

We applied this method to several datasets of fluorescence microscopy images with varying levels of difficulty with respect to cell density, resolution, contrast, and signal-to-noise ratio. We compared the results with those produced by Chan and Vese segmentation, a temporally linked level-set technique, and nonlinear diffusion-based segmentation. We validated all segmentation techniques against reference masks provided by the international Cell Tracking Challenge consortium. The proposed approach delineated cells with an average Dice similarity coefficient of 89 % over a variety of simulated and real fluorescent image sequences. It yielded average improvements of 11 % in segmentation accuracy compared to both strictly spatial and temporally linked Chan-Vese techniques, and 4 % compared to the nonlinear spatio-temporal diffusion method.

Conclusions

Despite the wide variation in cell shape, density, mitotic events, and image quality among the datasets, our proposed method produced promising segmentation results. These results indicate the efficiency and robustness of this method especially for mitotic events and low SNR imaging, enabling the application of subsequent quantification tasks.

     

Early cell death detection with digital holographic microscopy

Background

Digital holography provides a non-invasive measurement of the quantitative phase shifts induced by cells in culture, which can be related to cell volume changes. It has been shown previously that regulation of cell volume, in particular as it relates to ionic homeostasis, is crucially involved in the activation/inactivation of the cell death processes. We thus present here an application of digital holographic microscopy (DHM) dedicated to early and label-free detection of cell death.

Methods and Findings

We provide quantitative measurements of phase signal obtained on mouse cortical neurons, and caused by early neuronal cell volume regulation triggered by excitotoxic concentrations of L-glutamate. We show that the efficiency of this early regulation of cell volume detected by DHM, is correlated with the occurrence of subsequent neuronal death assessed with the widely accepted trypan blue method for detection of cell viability.

Conclusions

The determination of the phase signal by DHM provides a simple and rapid optical method for the early detection of cell death.     

A time-series method for automated measurement of changes in mitotic and interphase duration from time-lapse movies

Background

Automated time-lapse microscopy can visualize proliferation of large numbers of individual cells, enabling accurate measurement of the frequency of cell division and the duration of interphase and mitosis. However, extraction of quantitative information by manual inspection of time-lapse movies is too time-consuming to be useful for analysis of large experiments.

Methodology/Principal Findings

Here we present an automated time-series approach that can measure changes in the duration of mitosis and interphase in individual cells expressing fluorescent histone 2B. The approach requires analysis of only 2 features, nuclear area and average intensity. Compared to supervised learning approaches, this method reduces processing time and does not require generation of training data sets. We demonstrate that this method is as sensitive as manual analysis in identifying small changes in interphase or mitotic duration induced by drug or siRNA treatment.

Conclusions/Significance

This approach should facilitate automated analysis of high-throughput time-lapse data sets to identify small molecules or gene products that influence timing of cell division.     

From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy

Background

In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM).

Methodology

To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression.

Conclusion

Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM.     

Distinct Cell Clusters Touching Islet Cells Induce Islet Cell Replication in Association with Over-Expression of Regenerating Gene (REG) Protein in Fulminant Type 1 Diabetes

Background

Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs), extracellular matrix (ECM), and possible cell clusters, are unclear.

Procedures

The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans.

Result

Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG) Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells.

Conclusion

The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells.     

BactImAS: a platform for processing and analysis of bacterial time-lapse microscopy movies

Background

The software available to date for analyzing image sequences from time-lapse microscopy works only for certain bacteria and under limited conditions. These programs, mostly MATLAB-based, fail for microbes with irregular shape, indistinct cell division sites, or that grow in closely packed microcolonies. Unfortunately, many organisms of interest have these characteristics, and analyzing their image sequences has been limited to time consuming manual processing.

Results

Here we describe BactImAS – a modular, multi-platform, open-source, Java-based software delivered both as a standalone program and as a plugin for Icy. The software is designed for extracting and visualizing quantitative data from bacterial time-lapse movies. BactImAS uses a semi-automated approach where the user defines initial cells, identifies cell division events, and, if necessary, manually corrects cell segmentation with the help of user-friendly GUI and incorporated ImageJ application. The program segments and tracks cells using a newly-developed algorithm designed for movies with difficult-to-segment cells that exhibit small frame-to-frame differences. Measurements are extracted from images in a configurable, automated fashion and an SQLite database is used to store, retrieve, and exchange all acquired data. Finally, the BactImAS can generate configurable lineage tree visualizations and export data as CSV files. We tested BactImAS on time-lapse movies of Mycobacterium smegmatis and achieved at least 10-fold reduction of processing time compared to manual analysis. We illustrate the power of the visualization tool by showing heterogeneity of both icl expression and cell growth atop of a lineage tree.

Conclusions

The presented software simplifies quantitative analysis of time-lapse movies overall and is currently the only available software for the analysis of mycobacteria-like cells. It will be of interest to the community of both end-users and developers of time-lapse microscopy software.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-251) contains supplementary material, which is available to authorized users.     

Structures formed by a cell membrane-associated arabinogalactan-protein on graphite or mica alone and with Yariv phenylglycosides

Background

Certain membrane-associated arabinogalactan-proteins (AGPs) with lysine-rich sub-domains participate in plant growth, development and resistance to stress. To complement fluorescence imaging of such molecules when tagged and introduced transgenically to the cell periphery and to extend the groundwork for assessing molecular structure, some behaviours of surface-spread AGPs were visualized at the nanometre scale in a simplified electrostatic environment.

Methods

Enhanced green fluorescent protein (EGFP)-labelled LeAGP1 was isolated from Arabidopsis thaliana leaves using antibody-coated magnetic beads, deposited on graphite or mica, and examined with atomic force microscopy (AFM).

Key Results

When deposited at low concentration on graphite, LeAGP can form independent clusters and rings a few nanometres in diameter, often defining deep pits; the aperture of the rings depends on plating parameters. On mica, intermediate and high concentrations, respectively, yielded lacy meshes and solid sheets that could dynamically evolve arcs, rings, ‘pores’ and ‘co-pores’, and pits. Glucosyl Yariv reagent combined with the AGP to make very large and distinctive rings.

Conclusions

Diverse cell-specific nano-patterns of native lysine-rich AGPs are expected at the wall–membrane interface and, while there will not be an identical patterning in different environmental settings, AFM imaging suggests protein tendencies for surficial organization and thus opens new avenues for experimentation. Nanopore formation with Yariv reagents suggests how the reagent might bind with AGP to admit Ca²⁺ to cells and hints at ways in which AGP might be structured at some cell surfaces.     

Function of Mouse Embryonic Stem Cell-Derived Supporting Cells in Neural Progenitor Cell Maturation and Long Term Cxpansion

Background

In the differentiation of mouse embryonic stem (ES) cells into neurons using the 5-stage method, cells in stage 4 are in general used as neural progenitors (NPs) because of their ability to give rise to neurons. The choice of stage 4 raises several questions about neural progenitors such as the type of cell types that are specifically considered to be neural progenitors, the exact time when these progenitors become capable of neurogenesis and whether neurogenesis is an independent and autonomous process or the result of an interaction between NP cells and the surrounding cells.

Methodology/Principal Findings

In this study, we found that the confluent monolayer cells and neural sphere like cell clusters both appeared in the culture of the first 14 days and the subsequent 6 weeks. However, only the sphere cells are neural progenitors that give rise to neurons and astrocytes. The NP cells require 14 days to mature into neural lineages fully capable of differentiation. We also found that although the confluent monolayer cells do not undergo neurogenesis, they play a crucial role in the growth, differentiation, and apoptosis of the sphere cells, during the first 14 days and long term culture, by secreted factors and direct cell to cell contact.

Conclusions/Significance

The sphere cells in stage 4 are more committed to developing into neural progenitors than monolayer cells. Interaction between the monolayer cells and sphere cells is important in the development of stage 4 cell characteristics.     

Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels

Background

The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years.

Methodology/Principal Findings

We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization.Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology.Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1–5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces.

Conclusions

To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions.     

Characteristic thickened cell walls of the bracts of the 'eternal flower' Helichrysum bracteatum

Background and Aims

Helichrysum bracteatum is called an ‘eternal flower’ and has large, coloured, scarious bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum.

Methods

DAPI (4''6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized light microscopy were used for observation of cells, including cell wall morphology.

Key Results

Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not observed in the petal and bract tissues of Chrysanthemum morifolium.

Conclusions

Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves. The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they may be a new cell type.Key words: Helichrysum bracteatum, scarious bract, secondary cell wall, primary cell wall, cell morphology, birefringence, orientated cellulose microfibrils, cell death, DAPI, transmission electron microscopy, polarized light microscopy     

CellProfiler: Novel Automated Image Segmentation Procedure for Super-Resolution Microscopy

Background

Super resolution (SR) microscopy enabled cell biologists to visualize subcellular details up to 20 nm in resolution. This breakthrough in spatial resolution made image analysis a challenging procedure. Direct and automated segmentation of SR images remains largely unsolved, especially when it comes to providing meaningful biological interpretations.

Results

Here, we introduce a novel automated imaging analysis routine, based on Gaussian, followed by a segmentation procedure using CellProfiler software (www.cellprofiler.org). We tested this method and succeeded to segment individual nuclear pore complexes stained with gp210 and pan-FG proteins and captured by two-color STED microscopy. Test results confirmed accuracy and robustness of the method even in noisy STED images of gp210.

Conclusions

Our pipeline and novel segmentation procedure may benefit end-users of SR microscopy to analyze their images and extract biologically significant quantitative data about them in user-friendly and fully-automated settings.

     

Automatic identification and characterization of radial files in light microscopy images of wood

Background and Aims

Analysis of anatomical sections of wood provides important information for understanding the secondary growth and development of plants. This study reports on a new method for the automatic detection and characterization of cell files in wood images obtained by light microscopy. To facilitate interpretation of the results, reliability coefficients have been determined, which characterize the files, their cells and their respective measurements.

Methods

Histological sections and blocks of the gymnosperms Pinus canariensis, P. nigra and Abies alba were used, together with histological sections of the angiosperm mahogany (Swietenia spp.). Samples were scanned microscopically and mosaic images were built up. After initial processing to reduce noise and enhance contrast, cells were identified using a ‘watershed’ algorithm and then cell files were built up by the successive aggregation of cells taken from progressively enlarged neighbouring regions. Cell characteristics such as thickness and size were calculated, and a method was developed to determine the reliability of the measurements relative to manual methods.

Key Results

Image analysis using this method can be performed in less than 20 s, which compares with a time of approx. 40 min to produce the same results manually. The results are accompanied by a reliability indicator that can highlight specific configurations of cells and also potentially erroneous data.

Conclusions

The method provides a fast, economical and reliable tool for the identification of cell files. The reliability indicator characterizing the files permits quick filtering of data for statistical analysis while also highlighting particular biological configurations present in the wood sections.     

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

Background

The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen.

Results

Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion.

Conclusions

The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0039-2) contains supplementary material, which is available to authorized users.     

Ultrastructural Features of Ischemic Tissue following Application of a Bio-Membrane Based Progenitor Cardiomyocyte Patch for Myocardial Infarction Repair

Background and Objective

Implantation of cell-sheets into damaged regions of the heart after myocardial infarction (MI) has been shown to improve heart function. However, the tissue morphology following application of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) has not been studied in detail at the level afforded by electron microscopy. We hypothesized that increasing the number of CM derived from iPSC would increase the effectiveness of cell-sheets used to treat ischemic cardiomyopathy. We report here on the ultrastructural features after application of a bio-membrane ‘cell patch’.

Methods

iPSC-derived progenitor cells were transduced using lentivirus vectors with or without NCX1 promoter. iPSC-CM sheets were transplanted over the transmural MI region in a mouse model of regional ischemic cardiomyopathy. Mice were divided into four groups, 1) Sham; 2) MI; 3) MI + iPSC without NCX1 treated cells (MI + iPSC^Null) and 4) MI + iPSC receiving NCX1 promoter treated cells (MI + iPSC^NCX1). Echocardiography was performed 4 weeks after cell patch application, followed by histological and transmission electron microscopy (TEM) analysis.

Results

Large numbers of transplanted CM were observed with significant improvements in left ventricular performance and remodeling in group 4 as compared with group 3. No teratoma formation was detected in any of the treatment groups.

Conclusion

Manipulation of iPSC yields large numbers of iPSC-CM and favorable morphological and ultrastructural tissue changes. These changes have the potential to enhance current methods used for restoration of cardiac function after MI.