Effects of TGF‐β1 on mineralization mediated by rat calvaria‐derived osteogenic cells

In this study, we have analyzed the viability and cell growth, as well as, the mineralization of extracellular matrix (ECM) by alizarin red and von Kossa staining of calvaria‐derived osteogenic cultures, treated with TGF‐β1 alone or associated with Dex comparing with acid ascorbic (AA) + β‐glicerophosphate (βGP) (positive mineralization control). The expression of the noncollagenous proteins bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN) were evaluated by indirect immunofluorescence. In addition, the main ultrastructural morphological findings were assessed by transmission electron microscopy. Osteogenic cells were isolated of calvaria bone from newborn (2‐day‐old) Wistar rats were treated with TGF‐β1 alone or with dexamethasone for 7, 10, and 14 days. As positive mineralization control, the cells were supplemented only with AA+ βGP. As negative control, the cells were cultured with basal medium (α‐MEM + 10%FBS + 1%gentamicin). The treatment with TGF‐β1, even when combined with Dex, decreased the viability and cell growth when compared with the positive control. Osteoblastic cell cultures were positive to alizarin red and von Kossa stainings after AA + βGP and Dex alone treatments. Positive immunoreaction was found for BSP, OPN and FN in all studied treatments. Otherwise, when the cell cultures were supplemented with TGF‐β1 and TGF‐β1 + Dex, no mineralization was observed in any of the studied periods. These present findings suggest that TGF‐β1, in the studied in vitro doses, inhibits the proliferation and differentiation of osteoblastic cells by impairment of nodule formation.     

Effects of human umbilical cord mesenchymal stem cells on co‐cultured ovarian carcinoma cells

Ovarian carcinoma is mainly treated by surgery aided by chemotherapy. If supplemented by stem cells treatment, its recurrence rate and mortality rate will be decreased. This is a new therapy. In this study, ovarian cancer cells were cultured together with umbilical cord mesenchymal stem cells (UCMSCs), and the interactions between them were observed. The results showed that the survival rates of UCMSCs increased to 83.8 ± 2.2% from 56.5 ± 5.5%, and the survival rates of ovarian cancer cells decreased to 16.2 ± 2.2% from 43.5 ± 5.5% with the progression of the cultural time from 24 to 96 hr. There was a significant difference between them (p < .05). It revealed that UCMSCs could inhibit the proliferation of ovarian cancer cells.     

Embossed‐carving processing of cytoskeletons of cultured cells by using focused ion beam technology

The focused ion beam (FIB) technology has drawn considerable attention in diverse research fields. FIB can be used to mill samples at the nanometer scale by using an ion beam derived from electrically charged liquid gallium (Ga). This powerful technology with accuracy at the nanometer scale is now being applied to life science research. In this study, we show the potential of FIB as a new tool to investigate the internal structures of cells. We sputtered Ga⁺ onto the surface or the cross section of animal cells to emboss the internal structures of the cell. Ga⁺ sputtering can erode the cell surface or the cross section and thus emboss the cytoskeletons quasi‐3 dimensionally. We also identified the embossed structures by comparing them with fluorescent images obtained via confocal laser microscopy because the secondary ion micrographs did not directly provide qualitative information directly. Furthermore, we considered artifacts during the FIB cross sectioning of cells and propose a way to prevent undesirable artifacts. We demonstrate the usefulness of FIB to observe the internal structures of cells. Microsc. Res. Tech. 76:290–295, 2013. © 2013 Wiley Periodicals, Inc.     

Curcumin disturbed cell‐cycle distribution of HepG2 cells via cytoskeletal arrangement

Due to its extensive antitumor activity, curcumin has been focused on by more researchers. But, its antiproliferative mechanisms are still unknown. Here we studied the antiproliferative activity of curcumin in human liver cancer HepG2 cells. In order to analyze the cytotoxic activity and anticancer mechanisms of curcumin, we carried out cytotoxicity tests using 3‐[4,5‐dimethyl‐2‐thiazolyl]‐2,5 diphenyltetrazolium bromide (MTT) assay. The HepG2 cell cycle distribution and the expression of tubulin were detected by flow cytometry. Alterations in morphological and cytoskeletal properties of HepG2 cells were investigated using atomic force microscopy (AFM). Simultaneously, the effects of curcumin on the growth and proliferation of HepG2 cells were also assayed by MTT method. Cells were incubated with different doses of curcumin (0–80 μmol/l) for 24 h, the cell viability decreased from 91.10 ± 3.2% to 10.84 ± 4.0%, and the 50% inhibiting concentration (IC₅₀) was 23.15 ± 0.37 μmol/l. Moreover, flow cytometry quantitatively detected that curcumin treatment resulted in a dose‐dependent accumulation of HepG2 cells in G2/M phase with concomitant losses from G0/G1 phase, so curcumin caused cell‐cycle arrest at G2/M phase. Furthermore, we discovered that curcumin was able to upregulate the expression of tubulin in HepG2 cells. In addition, AFM analysis including cell‐membrane structure and cytoskeleton networks is helpful to explain the relationship between the changes of cells and external pharmacologic stimulation. SCANNING 35: 253‐260, 2013. © 2012 Wiley Periodicals, Inc.     

Three‐dimensional Co‐culture of hepatic progenitor cells and mesenchymal stem cells in vitro and in vivo

Introduction: Here we co‐cultured hepatic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) to investigate whether the co‐culture environments could increase hepatocytes form. Methods: Three‐dimensional (3D) co‐culture model of HPCs and MSCs was developed and morphological features of cells were continuously observed. Hepatocyte specific markers Pou5f1/Oct4, AFP, CK‐18 and Alb were analyzed to confirm the differentiation of HPCs. The mRNA expression of CK‐18 and Alb was analyzed by RT‐PCR to investigate the influence of co‐culture model to the terminal differentiation process of mature hepatocytes. The functional properties of hepatocyte‐like cells were detected by continuously monitoring the albumin secretion using Gaussia luciferase assays. Scaffolds with HPCs and MSCs were implanted into nude mouse subcutaneously to set up the in vivo co‐culture model. Results: Although two groups formed smooth spheroids and high expressed of CK‐18 and Alb, hybrid spheroids had more regular structures and higher cell density. CK‐18 and Alb mRNA were at a relatively higher expression level in co‐culture system during the whole cultivation time (P < 0.05). Albumin secretion rates in the hybrid spheroids had been consistently higher than that in the mono‐culture spheroids (P < 0.05). In vivo, the hepatocyte‐like cells were consistent with the morphological features of mature hepatocytes and more well‐differentiated hepatocyte‐like cells were observed in the co‐culture group. Conclusions: HPCs and MSCs co‐culture system is an efficient way to form well‐differentiated hepatocyte‐like cells, hence, may be helpful to the cell therapy of hepatic tissues and alleviate the problem of hepatocytes shortage. Microsc. Res. Tech. 78:688–696, 2015. © 2015 Wiley Periodicals, Inc.     

激光共聚焦显微成像技术在植物细胞微丝骨架三维动态观察中的应用

激光扫描共聚焦显微镜(laser scanning confocal microscope,LSCM)是目前生物学领域应用最广泛、分辨率高的仪器。可用于细胞内形态结构观察及三维重建、组分空间定位、实时动态变化监测等研究,图像分析软件还能提供荧光强度、空间距离定量测定的丰富信息。本文以携带GFP融合拟南芥丝束蛋白1(AtFIM1)的肌动蛋白结合结构域2(fABD2)基因的B Y-2转基因细胞系为材料,运用LSCM技术观察到间期细胞的网络状微丝结构并重构出胞内微丝的三维网络结构;实时动态监测细胞有丝分裂过程中微丝骨架的动态变化;通过细胞内荧光强度的分布直观地看出BY-2细胞胞质分裂过程中微丝骨架的动态变化。这些结果显示出LSCM在研究植物细胞微丝骨架的三维网络动态结构及图像荧光强度分析与统计方面的优越性。     

AFM Investigation on Ox‐LDL‐Induced Changes in Cell Spreading and Cell‐Surface Adhesion Property of Endothelial Cells

The integrity and adhesion properties of endothelium play vital roles during atherosclerosis. It is well known that oxidized low‐density lipoprotein (Ox‐LDL) influences many physiological activities or mechanical properties of endothelial cells. However, the effects of Ox‐LDL on the integrity and nonspecific adhesion properties of endothelial cells are still unclear. In this study, using the topographical imaging and force measurement functions of atomic force microscopy (AFM), we found that Ox‐LDL can transiently weaken the integrity of endothelium by impairing cell spreading of endothelial cells and decrease the attachment of irrelevant blood cells to endothelium by impairing the nonspecific adhesion property of endothelial cells. The AFM‐based data provide important information for understanding the effects of Ox‐LDL on endothelial cells or during atherogenesis. SCANNING 35: 119‐126, 2013. © 2012 Wiley Periodicals, Inc.     

Short‐Term Response of Human Osteoblast‐Like Cells on Titanium Surfaces With Micro‐ and Nano‐Sized Features

Since the way that human bone cells behave on contact with different surfaces topographies seems to be crucial to osseointegration, the aim of the present study is to evaluate the participation of some micro‐ and nanosized features of Ti surfaces in the short‐term response of primary human osteoblast‐like cells (HOC). Surfaces were prepared as ground (G‐Ti), hydrofluoric acid etched (HF‐Ti), and sandblasted/HF‐etched (SLA‐Ti), and analyzed using both three‐dimensional (3D) profilometer and atomic force microscope (AFM). Cell morphology was assessed using scanning electron microscopy (SEM) after 4 and 24 h in culture. Cell viability, adhesion, and spreading were also evaluated 4 and 24 h after seeding over each surface. Data were compared by analysis of variance (ANOVA) complemented by Duncan test. Cell morphology, cell counting, and membrane integrity (Neutral Red, NR) were not affected by surface treatment at any time. However, HF‐Ti presented the smallest surface area and did not increase tetrazolium hydroxide (XTT) reduction from 4 to 24 h. On the other hand, a higher level of spreading was only found on the rougher and isotropic SLA‐Ti at 4 h. In conclusion, although all evaluated Ti surfaces allowed HOC short‐term adhesion, the finer topography introduced by HF as single treatment did not favor HOC mitochondrial activity and spreading. The rougher and more complex SLA surface seems to provide a better substrate for HOC short‐term response. SCANNING 34: 378‐386, 2012. © 2012 Wiley Periodicals, Inc.     

Extremely thin layer plastification for focused‐ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells

Since the recent boost in the usage of electron microscopy in life‐science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused‐ion beam scanning electron microscopy (FIB‐SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold‐labelled breast cancer SKBR3 cells was to visualise gold‐labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 m² cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back‐scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB‐SEM. Cross‐sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.     

Three‐dimensional morphometric comparison of normal and apoptotic endothelial cells based on laser scanning confocal microscopy observation

Three‐dimensional (3D) morphometric analysis of cellular and subcellular structures provides an effective method for spatial cell biology. Here, 3D cellular and nuclear morphologies are reconstructed to quantify and compare morphometric differences between normal and apoptotic endothelial cells. Human umbilical vein endothelial cells (HUVECs) are treated with 60 μM H₂O₂ to get apoptotic cell model and then a series of sectional images are acquired from laser scanning confocal microscopy. The 3D cell model containing plasma membrane and cell nucleus is reconstructed and fused utilizing three sequential softwares or packages (Mimics, Geomagic, and VTK). The results reveal that H₂O₂ can induce apoptosis effectively by regulating the activity of apoptosis‐related biomolecules, including pro‐apoptotic factors p53 and Bax, and anti‐apoptotic factor Bcl‐2. Compared with the normal HUVECs, the apoptotic cells exhibit significant 3D morphometric parameters (height, volume and nucleus‐to‐cytoplasm ratio) variation. The present research provides a new perspective on comparative quantitative analysis associated with cell apoptosis and points to the value of LSCM as an objective tool for 3D cell reconstruction. Microsc. Res. Tech. 76:1154–1162, 2013. © 2013 Wiley Periodicals, Inc.     

Imaging intracellular elemental distribution and ion fluxes in cultured cells using ion microscopy: a freeze-fracture methodology

A freeze-fracture methodology was standardized for tissue culture cells to study intracellular distribution of diffusible elements with ion microscopy. Chinese hamster ovary (CHO) and normal rat kidney (NRK) cells grown on a silicon substrate were sandwiched using another smooth surface (silicon, glass, mica) in the presence of spacers and fast frozen in liquid nitrogen slush. The sandwich was fractured by prying the two halves apart under liquid nitrogen. This procedure produced large areas on the silicon substrate containing hundreds of cells grouped together and fractured at the apical cell surface. After freeze-drying, these cells revealed a subcellular distribution of Na, K, Ca, Mg, P, Cl and S with the ～0·5 μm lateral resolution of the ion microscope. Between the nuclei and the cytoplasm of cells, no major differences were observed for Na, K, Mg, P, Cl and S intensities. Calcium alone, however, exhibited a remarkable distribution. Calcium accumulated more in the cytoplasm than in the nuclei of cells. Even within the cytoplasm its distribution was heterogeneous, suggesting Ca binding sites. The fractured cells consistently exhibited high K-low Na intensities. The injured or dead cells were easily recognized among the healthy ones due to their abnormal ion composition. This simple freeze-fracture methodology allowed fracturing of cells without removing the cells from the substrate. In addition, it eliminated the need for washing the nutrient media away and cryo-sectioning before ion microanalysis. The methodology was successfully extended to 3T3 mouse fibroblast, PtK₂ rat kangaroo and L5 rat myoblast cultures.     

Morphology and expression status investigations of specific surface markers on B‐cell chronic lymphocytic leukemia cells

The morphology of cells and expression status of specific surface markers [cluster of differentiation (CD)], such as CD5, CD19, CD20, CD38, and CD45, have long been considered as the essential indicators for the diagnosis and prognosis of B‐cell chronic lymphocytic leukemia (B‐CLL). Clinically, it is difficult to simultaneously obtain cell morphology and distribution of surface markers with flow cytometry, especially for some surrogate markers such as CD38. Here, as an alternative and complementary prognostic method, fluorescence microscopy and image processing method are introduced to directly visualize the cells from patients and to quantitatively determine the expression status of surface markers. In this study, the morphological parameters of B‐CLL cells were measured to establish the correlation between the cellular morphology and the surface marker expression. It was clear that the CD38+ and CD38? B‐CLL cells from the same CD38+ patients had hardly any size differences; however, an increase in perimeter was observed for CD38? patients. Moreover, the expression level of the receptors on the cell was independent of the cell size. There was no evidence showing that the expression intensities of CD19 and CD38 were related to each other for the CD38+ B‐CLL cells. On the same cells, CD5 was more selectively expressed on the cell membrane; however, the expression patterns suggested that the cell membrane of CD38? B‐CLL cells contained the least expression level of CD19. Microsc. Res. Tech. 76:1147–1153, 2013. © 2013 Wiley Periodicals, Inc.     

Structural organization of the cytoskeleton in SV40 human corneal epithelial cells cultured on nano- and microscale grooves

The basement membrane of human corneal epithelial cells (HCECs) has a three-dimensional nanoscale architecture, which includes pores, bumps and fibers that may influence cell-substrate adhesion and spreading in the overlying cells. We previously demonstrated that nano- and microscale groove and ridge patterns influence the morphological response and the adhesive response of HCECs to a nominal wall shear stress. Cell-substrate adhesion is mediated by adhesion receptors that bind to extracellular matrix components and anchor the cytoskeleton (CSK) of cells to extracellular elements. Here we investigate the CSK organization in SV40-transformed HCECs grown on nano- and microscale groove and ridge patterns. X-ray lithography was used to fabricate uniform groove and ridge patterns with features ranging in size from 200 nm to 2 microm grooves. Scanning electron microscopy and transmission electron microscopy were used to investigate CSK structure and the distribution of -beta1 integrin adhesion receptors. CSK elements aligned with the patterns; however, the spatial organization of these elements was influenced by feature size. Larger CSK bundles lay on top of the ridges and ran parallel to the patterns, whereas smaller CSK bundles, whose width was proportional to the groove size, spanned the grooves. -Beta1 integrins co-localized with the CSK and had a higher density at the poles of aligned spindle-shaped cells. Differences in organization seen on the different topographical feature sizes may be indicative of differences in extracellular matrix organization. This may explain, in part, previous observations regarding the dependence of cell adhesive responses on the size of topographic features in the substrate.     

Synergistic effect of diisopropyl phosphite and over‐based calcium sulphonate additives on tribological properties of AISI 52100 steel/Al2O3 ceramics

The friction‐reducing and anti‐wear effect of the 500SN base oil containing diisopropyl phosphite (T451) and over‐based calcium sulphonate (KT5447) on AISI 52100 steel/Al₂O₃ ceramic were investigated with a ball‐on‐disc tribometer at a light load of 200 N and a high load of 400 N. The results indicate that the 500SN base oil containing T451 and KT5447 appears to have a synergistic effect on the pair. For the light load of 200 N, the effective composition is 3 wt% T451 + 2–3 wt% KT5447. For the high load of 400 N, the combination of T451 and KT5447 appears to have a synergistic friction‐reducing and anti‐wear effect. The scanning electron microscope images show that ploughed grooves, pitting, spalling and corrosion are the dominant wear modes for both 200 and 400 N. However, no evidence for the formation of the expected sulphur‐containing or phosphorus‐containing chemical compound is found according to X‐ray photoelectron spectroscopy analysis of the worn steel ball surface at both loads. Copyright © 2011 John Wiley & Sons, Ltd.     

Micro‐community cytometry: sensing changes in cell health and glycoconjugate expression by imaging and flow cytometry

Discerning the extent of biologically relevant heterogeneity presents unique challenges to both microscopy and flow cytometry. Micro‐environmental influences and stochastic changes in cellular behaviour can act to mask the origins of both progression and therapeutic resistance in tumour cell systems. In part the dimensionality of different and frequently metastable states can be assessed by multi‐parameter flow cytometry with unparalleled statistical robustness. Complementary application of imaging can provide valuable insights into the complex temporal changes that can occur in cell micro‐communities either spontaneously or in response to selection pressure. With an extensive range of methodologies for the labelling of cells there are multiple options for tracking cells, defining fate and the re‐construction of provenance and behavioural history. The challenge is highlighted by attempts to identify the critical glycosylation events modifying the function of cell surface proteins. Central to a cytometric approach is the availability of methods that reveal cell health and are compatible with the detection of cell surface changes within dynamic micro‐communities. The review briefly addresses the options for sensing cell health and the co‐application of an antibody mimetic for detection of cell surface glycoconjugate expression accessible for both imaging and flow cytometry.     

Effect of different irrigation protocols on the radicular dentin interface and bond strength with a metacrylate‐based endodontic sealer

This study assessed the influence of different endodontic chemical substances on the adhesion of the Epiphany SE/Resilon system (with and without resinous solvent) to radicular dentin walls, using the push‐out test and scanning electron microscopy (SEM). Forty‐eight root canals of human canines were prepared biomechanically with ProTaper rotary files (crown‐down technique) and the radicular dentin was treated with either 17% EDTA, 2% chlorhexidine gel (CHX) or 2.5% NaOCl (control). The root canals were filled with Resilon cones and Epiphany SE sealer with and without resinous solvent. Six groups of eight canals each had their roots sectioned transversally to obtain 1‐mm thick slices. Data were subjected to statistical analysis by ANOVA and Tukey's tests. The specimens treated with 17% EDTA (1.59 ± 0.91) presented higher bond strength (P < 0.05) than those treated with 2.5% NaOCl (0.93 ± 0.27) and 2% CHX (0.92 ± 0.22). Significantly higher bond strength (P < 0.05) was observed when the Epiphany SE was prepared with (1.37 ± 0.78) than without (0.92 ± 0.33) solvent. Adhesive failures were predominant in all groups. SEM analysis showed greater homogeneity of the filling mass when the solvent was added to the sealer. Treatment of root canal walls with 17% EDTA, and addition of a resinous solvent to Epiphany SE produced the highest adhesion to radicular dentin. Microsc. Res. Tech. 77:446–452, 2014. © 2014 Wiley Periodicals, Inc.     

Bones Morphogenic Protein‐4 and retinoic acid combined treatment comparative analysis for in vitro differentiation potential of murine mesenchymal stem cells derived from bone marrow and adipose tissue into germ cells

Nowadays, infertility is no longer considered as an unsolvable disorder due to progresses in germ cells derived from stem lineage with diverse origins. Technical and ethical challenges push researchers to investigate various tissue sources to approach more efficient gametes. The purpose of the current study is to investigate the efficacy of a combined medium, retinoic acid (RA) together with Bone Morphogenic Protein‐4 (BMP4), on differentiation of Bone Marrow Mesenchymal Stem Cells (BMMSCs) and adipose‐derived mesenchymal stem cells (ADMSCs) into germ cells. Murine MSCs were obtained from both Bone Marrow (BM) and Adipose Tissue (AT) samples and were analyzed for surface markers to get further verification of their nature. BMMSCs and ADMSCs were induced into osteogenic and adipogenic lineage cells respectively, to examine their multipotency. They were finally differentiated into germ cells using media enriched with BMP4 for 4 days followed by addition of RA for 7 days (11 days in total). Analyzing of differentiation potential of BMMSCs‐ and ADMSCs were performed via Immunofluorescence, Flowcytometry and Real time‐PCR techniques for germ cell‐specific markers (Mvh, Dazl, Stra8 and Scp3). Mesenchymal surface markers (CD90 and CD44) were expressed on both BMMSCs and ADMSCs, while endothelial and hematopoietic cell markers (CD31 and CD45) had no expression. Finally, all germ‐specific markers were expressed in both BM and AT. Although germ cells differentiated from ADMSCs showed faster growth and proliferation as well as easy collection, they significantly expressed germ‐specific markers lower than BMMSCs. This suggests stronger differentiation potential of murine BMMSCs than ADMSCs.     

Development and plasticity of adrenal chromaffin cells: Cues based on in vitro studies

Neural crest derived precursors of the sympathoadrenal cell lineage give rise to two major cell types that differ in a number of morphological, ultrastructural, and biochemical characteristics: principal sympathetic neurons and chromaffin cells of the adrenal medulla. The present article reviews experimental studies performed on cultured adrenal medullary cells and designed to unravel the nature of epigenetic signals governing the developmental choice between the endocrine chromaffin and the neuronal sympathetic phenotype. Emphasis is placed on the role of glucocorticoids in initiation, development, and maintenance of the endocrine chromaffin phenotype and apparently antagonistic influences exerted by nerve growth factor (NGF) in vitro, resulting in the acquisition of neuronal properties by differentiated chromaffin cells. Experimental data from in vitro studies are compatible with the following conclusions. Glucocorticoids represent the decisive signal for the initial induction of endocrine differentiation. Moreover, high steroid hormone concentrations, as present in the adrenal medulla, are a prerequisite for the maturation of chromaffin cells. Even in a differentiated state, the endocrine phenotype is unstable in the absence of glucocorticoids, and the cells seem to reenter the neuronal developmental pathway. Under these conditions, cellular survival and differentiation into sympathetic neurons become NGF-dependent, as in normal sympathetic development. Thus, the effects of NGF survival, neurite outgrowth, and transmitter synthesis of cultured chromaffin cells probably do not reflect the induction of a specific phenotype, but they may be interpreted as a general neurotrophic support observable with other responsive cell types.     

Influence of the phase effect on gradient‐based and statistics‐based focus measures in bright field microscopy

Autofocusing is essential to high throughput microscopy and live cell imaging and requires reliable focus measures. Phase objects such as separated single Chinese hamster ovary cells are almost invisible at the optical focus position in bright field microscopy images. Because of the phase effect, defocused images of phase objects have more contrast. In this paper, we show that widely used focus measures exhibit an untypical behaviour for such images. In the case of homogeneous cells, that is, when most cells tend to lie in the same focal plane, both gradient‐based and statistics‐based focus measures tend to have a local minimum instead of a global maximum at the optical focus position. On the other hand, if images show inhomogeneous cells, gradient‐based focus measures tend to yield typical focus curves, whereas statistics‐based focus measures deliver curves similar to the case of homogeneous cells. These results were interpreted using the equation describing the phase effect and patch‐wise analysis of the focus curves. Bioprocess engineering experts are also influenced by the phase effect. Forty‐four focus positions selected by them led to the conclusion that they prefer to look at defocused images instead of those at the optical focus.