Classification of C2C12 cells at differentiation by convolutional neural network of deep learning using phase contrast images

In the field of regenerative medicine, tremendous numbers of cells are necessary for tissue/organ regeneration. Today automatic cell-culturing system has been developed. The next step is constructing a non-invasive method to monitor the conditions of cells automatically. As an image analysis method, convolutional neural network (CNN), one of the deep learning method, is approaching human recognition level. We constructed and applied the CNN algorithm for automatic cellular differentiation recognition of myogenic C2C12 cell line. Phase-contrast images of cultured C2C12 are prepared as input dataset. In differentiation process from myoblasts to myotubes, cellular morphology changes from round shape to elongated tubular shape due to fusion of the cells. CNN abstract the features of the shape of the cells and classify the cells depending on the culturing days from when differentiation is induced. Changes in cellular shape depending on the number of days of culture (Day 0, Day 3, Day 6) are classified with 91.3% accuracy. Image analysis with CNN has a potential to realize regenerative medicine industry.     

Image processing for accurate cell recognition and count on histologic slides

OBJECTIVE: To design an automatic system for recognition and count of two different cell families on histologic slides. STUDY DESIGN: The segmentation strategy uses color information on the image. The morphologic operations and Support Vector Machine approaches are used for each color to obtain precise segmentation of the image into separate cells for recognition. RESULTS: A large set of histologic slides of bone marrow was assessed byour system and the results compared to the score of a human expert. The results are in good agreement. The difference is within acceptable limits (below 10%). CONCLUSION: The automatic system of cell recognition and extraction is accurate and provides a useful tool for cell recognition and count on histologic slides.     

Segmentation of overlapping leucocyte images with phase detection and spiral interpolation

Leucocyte segmentation is one of the most crucial functionalities for an automatic leucocyte recognition system. In this paper, an algorithm is proposed to segment the leucocytes from the overlapping cell images. It consists of two main steps. The first step involves generation of a combined image based on the saturation and green channels (CIBSGC) by means of the different distribution characteristics of the leucocyte nucleus. A weight coefficient is used to adjust the CIBSGC for extracting the nucleus and estimating the location of the leucocyte. Second, a method of phase detection and spiral interpolation identifies the overlapping regions of cells and determines the leucocyte edge curve. The performance is evaluated by three parameters: sensitivity, positive predictive value and pixel number error. Experimental results validate that the proposed algorithm can successfully segment the overlapping leucocyte with the satisfactory performance for two cell image datasets under different recording conditions.     

Segmentation of overlapping leucocyte images with phase detection and spiral interpolation

Leucocyte segmentation is one of the most crucial functionalities for an automatic leucocyte recognition system. In this paper, an algorithm is proposed to segment the leucocytes from the overlapping cell images. It consists of two main steps. The first step involves generation of a combined image based on the saturation and green channels (CIBSGC) by means of the different distribution characteristics of the leucocyte nucleus. A weight coefficient is used to adjust the CIBSGC for extracting the nucleus and estimating the location of the leucocyte. Second, a method of phase detection and spiral interpolation identifies the overlapping regions of cells and determines the leucocyte edge curve. The performance is evaluated by three parameters: sensitivity, positive predictive value and pixel number error. Experimental results validate that the proposed algorithm can successfully segment the overlapping leucocyte with the satisfactory performance for two cell image datasets under different recording conditions.     

Imaging cytometry by multiparameter fluorescence.

A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.     

Design of a Cryogenic Videocamera-Recorder and Image Processing System and Calculation of Volumes of Red Cells

Since Rowe reported that the storage technique for red cells at very low temperatures had been realized successfully (4, 5), many experts who work in the fields of cryobiology and medicine have turned their attention to this storage technique for tissues and organs (3). Since the first quantitative cryomicroscope was made successfully about 20 years ago (1), it has been possible to observe changes in shape and phase. Particularly the image processing technique has laid the foundation for quantitative analysis of the relationship between changes in shape and damage from freezing of cells and cooling rate and storage temperature. In this work, we constructed a system consisting of a cryomicroscope, a videocamera-recorder, and an image processor. We carried out many experiments with red cells in cold storage and have established a model for calculating the volumes of red cells. In our experiments we also dynamically traced the shape changes of cells with various experimental parameters.     

Automatic digital image analysis for identification of mitotic cells in synchronous mammalian cell cultures

Mitotic frequency in a synchronous culture of mammalian cells was determined fully automatically and in real time using low-intensity phase-contrast microscopy and a newvicon video camera connected to an EyeCom III image processor. Image samples, at a frequency of one per minute for 50 hours, were analyzed by first extracting the high-frequency picture components, then thresholding and probing for annular objects indicative of putative mitotic cells. Both the extraction of high-frequency components and the recognition of rings of varying radii and discontinuities employed novel algorithms. Spatial and temporal relationships between annuli were examined to discern the occurrences of mitoses, and such events were recorded in a computer data file. At present, the automatic analysis is suited for random cell proliferation rate measurements or cell cycle studies. The automatic identification of mitotic cells as described here provides a measure of the average proliferative activity of the cell population as a whole and eliminates more than eight hours of manual review per time-lapse video recording.     

Image analysis of rat satellite cell proliferation in vitro

Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14. The cell cycle phases were determined by examining Feulgen-stained cultures with a cell image processor. The nuclei were automatically analyzed by calculating 18 parameters relating to the texture and densitometry of chromatin and the shape of each nucleus. Cell cycle phases were characterized (Moustafa and Brugal, 1984). The recognition methods made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes.After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of foetal calf serum, this percentage increased until day 3 after plating. At that time, the DNA content of 28.2% of the cell population was higher than 3C, whereas it is 2C in G1 or G0 nuclei; image analysis showed that 42% of these cells were in S or G2 phase. From day 4, the proliferation rate gradually slowed down until day 8. After day 8, when numerous myotubes differentiated, the percentage of S and G2 phase cells had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. Myotube nuclei were largely in G0. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed before cell fusion.These methods of analysis give the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscles.     

3-D image analysis system and megakaryocyte quantitation

A quasi-automatic computer image analysis system has been developed for 3-D reconstruction of stained serial sections and implemented on an IBAS system. Some new automatic image analysis techniques have been designed and incorporated into the system. For image segmentation, a transition region determination based thresholding method is introduced. Neither histogram calculation nor empirical parameters are needed in the automatic threshold selection. A two step 3-D reconstruction procedure--symbolic and pictorial reconstructions--is designed to improve the flexibility and the computational capability of the system. The global level registration and local level registration are separated. The former consists of establishing the relationship among a large numbers of profile pairs dispersed in adjacent sections. A pattern matching method based on pattern recognition principles is devised to exploit the information about the statistical character of mismatch caused by deformation of sections and about the relationship of nearby objects. For the latter, an equivalent elliptical approximation method based on the physical theory of the rotation of rigid bodies is proposed. The system has been used for 3-D reconstruction and quantitation of megakaryocytes in human bone marrow tissue. Features about individual 3-D megakaryocyte cell and the spatial distribution of megakaryocytes are determined. The latter is a new contribution to megakaryocyte quantitation and is not possible by using conventional stereologic techniques. These experimental results have demonstrated the ability of the system to perform quantitative analysis.     

Effects of ice-seeding temperature and intracellular trehalose contents on survival of frozen Saccharomyces cerevisiae cells

Freezing tolerance is an important characteristic for baker’s yeast, Saccharomyces cerevisiae, as it is used to make frozen dough. The ability of yeast cells to survive freezing is thought to depend on various factors. The purpose of this work was to study the viability of yeast cells during the freezing process. We examined factors potentially affecting their survival, including the growth phase, ice-seeding temperature, intracellular trehalose content, freezing period, and duration of supercooling. The results showed that the ice-seeding temperature significantly affected cell viability. In the stationary phase, trehalose accumulation did not affect the viability of yeast cells after brief freezing, although it did significantly affect the viability after prolonged freezing. In the log phase, the ice-seeding temperature was more important for cell survival than the presence of trehalose during prolonged freezing. The importance of increasing the extracellular ice-seeding temperature was verified by comparing frozen yeast survival rates in a freezing test with ice-seeding temperatures of −5 °C and −15 °C. We also found that the cell survival rates began to increase at 3 h of supercooling. The yeast cells may adapt to subzero temperatures and/or acquire tolerance to freezing stress during the supercooling.     

Impact of cell cycle dynamics on pathology recognition: Raman imaging study

Confocal Raman imaging combined with fluorescence‐activated cell sorting was used for in vitro studies of cell cultures to look at biochemical differences between the cells in different cell phases. To answer the question what is the impact of the cell cycle phase on discrimination of pathological cells, the combination of several factors was checked: a confluency of cell culture, the cell cycle dynamics and development of pathology. Confluency of 70% and 100% results in significant phenotypic cell changes that can be also diverse for different batches. In 100% confluency cultures, cells from various phases become phenotypically very similar and their recognition based on Raman spectra is not possible. For lower confluency, spectroscopic differences can be found between cell cycle phases (G₀/G₁, S and G₂/M) for control cells and cells incubated with tumor necrosis factor alpha (TNF‐α), but when the mycotoxin cytochalasin B is used the Raman signatures of cell phases are not separable. Generally, this work shows that heterogeneity between control and inflamed cells can be bigger than heterogeneity between cell cycle phases, but it is related to several factors, and not always can be treated as a rule.      

The identification of myogenic cells in regenerating skeletal muscle. II. Early mammalian regenerates.

Most of the recent studies on skeletal muscle regeneration have used the criteria of cell shape and position as the primary means of identifying early presumptive myogenic elements or satellite cells. Studies of anuran muscle regeneration indicate, however, that macrophages can mimic early myogenic cells by adopting a fusiform shape and a sublaminar position during the initial stages of phagocytic invasion. The present study confirms these observations in injured mammalian muscle. Gastrocnemius muscle tissues from Sprague-Dawley rats were killed by lyophilization or repeated freezing and implanted subcutaneously to examine the cytology of the invading macrophages free from contamination by any endogenous myogenic cells. Within 2 days the implants are infiltrated by large numbers of fusiform macrophages. These cells form continuous cuffs around the degenerating myofibers but initially show little evidence of phagocytosis. They contain dense concentrations of free ribosomes but display few lysosomes, phagosomes, or pseudopodia. These distinctive phagocytic features do not appear until the macrophages penetrate the cores of the injured fibers and actually begin removal of the myofibrillar debris. These observations indicate that the criteria of cell shape and location cannot reliably distinguish between early mammalian macrophages and myogenic cells.     

基于知识的颅内出血（ICH）三维医学图像分割方法

本文描述一种基于知识的三维医学图像自动分割方法,用于进行人体颅内出血(Intracranial Hemorrhage,ICH)的分割和分析。首先,数字化CT胶片,并自动对数字化后的胶片按照有无异常分类。然后,阀值结合模糊C均值聚类算法将图像分类成多个具有统一亮度的区域。最后,在先验知识以及预定义的规则的基础上,借助基于知识的专家系统将各个区域标记为背景、钙化点、血肿、颅骨、脑干。     

Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses

BACKGROUND AND AIMS: Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. It has long been believed that cryopreservation of plant cell cultures is best performed with cells at the late lag or early exponential growth phase. At these stages the cells are small and non-vacuolated. This belief was based on studies using conventional slow prefreezing protocols and survival determined with fluorescein diacetate staining or 2,3,5-triphenyltetrazolium chloride assays. This classical issue was revisited here to determine the optimum growth phase for cryopreserving a bromegrass (Bromus inermis) suspension culture using more recently developed protocols and regrowth assays for determination of survival. METHODS: Cells at different growth phases were cryopreserved using three protocols: slow prefreezing, rapid prefreezing and vitrification. Stage-dependent trends in cell osmolarity, water content and tolerance to freezing, heat and salt stresses were also determined. In all cases survival was assayed by regrowth of cells following the treatments. KEY RESULTS: Slow prefreezing and rapid prefreezing protocols resulted in higher cell survival compared with the vitrification method. For all the protocols used, the best regrowth was obtained using cells in the late exponential or early stationary phase, whereas lowest survival was obtained for cells in the late lag or early exponential phase. Cells at the late exponential phase were characterized by high water content and high osmolarity and were most tolerant to freezing, heat and salt stresses, whereas cells at the early exponential phase, characterized by low water content and low osmolarity, were least tolerant. CONCLUSIONS: The results are contrary to the classical concept which utilizes cells in the late lag or early exponential growth phase for cryopreservation. The optimal growth phase for cryopreservation may depend upon the species or cell culture being cryopreserved and requires re-investigation for each cell culture. Stage-dependent survival following cryopreservation was proportionally correlated with the levels of abiotic stress tolerance in bromegrass cells.     

Ras-dependent cell cycle commitment during G2 phase

Synchronization used to study cell cycle progression may change the characteristics of rapidly proliferating cells. By combining time-lapse, quantitative fluorescent microscopy and microinjection, we have established a method to analyze the cell cycle progression of individual cells without synchronization. This new approach revealed that rapidly growing NIH3T3 cells make a Ras-dependent commitment for completion of the next cell cycle while they are in G2 phase of the preceding cell cycle. Thus, Ras activity during G2 phase induces cyclin D1 expression. This expression continues through the next G1 phase even in the absence of Ras activity, and drives cells into S phase.     

A histological analysis of liver injury in freezing storage

As part of a more extensive study on the use of high subzero freezing for cryopreservation of mammalian livers we have tried to single out the effects of freezing and thawing on tissue damage. We compared the morphology of livers after freezing and thawing with what we considered an optimal high subzero cryopreservation protocol with the morphology of livers preserved under the same thermal conditions and in the same solution in a supercooled state, without freezing. The results show that while hepatocytes survive high subzero cryopreservation, detachment of endothelial cells occurs in every freezing experiment. On the other hand, the endothelial cells in livers that are not frozen are intact. This suggests that endothelial cell damage is caused by freezing and may be an important factor in high subzero freezing cryopreservation of the liver.     

Processing and characterization of porous structures from chitosan and starch for tissue engineering scaffolds

Natural biodegradable polymers were processed by different techniques for the production of porous structures for tissue engineering scaffolds. Potato, corn, and sweet potato starches and chitosan, as well as blends of these, were characterized and used in the experiments. The techniques used to produce the porous structures included a novel solvent-exchange phase separation technique and the well-established thermally induced phase separation method. Characterization of the open pore structures was performed by measuring pore size distribution, density, and porosity of the samples. A wide range of pore structures ranging from 1 to 400 microm were obtained. The mechanisms of pore formation are discussed for starch and chitosan scaffolds. Pore morphology in starch scaffolds seemed to be determined by the initial freezing temperature/freezing rate, whereas in chitosan scaffolds the shape and size of pores may have been determined by the processing route used. The mechanical properties of the scaffolds were assessed by indentation tests, showing that the indentation collapse strength depends on the pore geometry and the material type. Bioactivity and degradation of the potential scaffolds were assessed by immersion in simulated body fluid.     

The morphogenesis of cell hairs on Drosophila wings

We describe in this paper details of morphogenesis of wing hairs in Drosophila pupae. The ultimate objective is to relate specific protein components used in hair construction to specific components produced in the rapidly changing patterns of gene expression that are characteristic for the period of hair differentiation in wing cells (H. K. Mitchell and N. S. Petersen, 1981, Dev. Biol. 85, 233-242). Hair extrusion to essentially full size occurs quite suddenly at about 34 hr (postpupariation) and this is followed by deposition of a double-layer of cuticulin during the next 4 to 5 hr. Extreme changes in shape of cells and hairs, probably related to actin synthesis, then occur for the next 5 to 6 hr. Deposition of fibers within the hairs and on hair pedestals follows. Formation of cuticle on the cell surface begins and continues until some time in the 60-hr range. It appears that cuticle is formed only on the cell surface and not in hairs or on the top of hair pedestals. The protein synthesis patterns associated with these events are described.