Application of high-content cell imaging system in cell proliferation detection

KAI-01050 is a high speed face-to-face interlinear transfer CCD produced by KODAK, USA, whose drive circuit has not only a high-speed horizontal transfer signal of up to 40 MHz, but also a vertical transfer signal with three level steps and an electronic shutter signal with high voltage pulses. The design of the history and development of CCD imaging systems, introduction of Kodak’s CCD image sensor KAI-01050 chip and its main features, structure, operating timing and timing drive mode, applications in imaging systems, as well as its external circuit design and other aspects of a detailed introduction.

Ubiquitin-proteasome system (UPS) plays an important role in neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important. In this study, we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD. At first, stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells, together with G418 screening. The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1. By employing the high-content fluorescence imaging system, together with stable YFP-CL1 HT22 cells, the UPS activators were successfully screened from our established TCM library. The representative images were captured and analyzed, and quantification of the YFP fluorescence intensity was performed by flow cytometry. Then, the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP (APP), pRK5-EGFP-Tau P301L (Tau P301L), or pRK5-EGFP-Tau (Tau) transiently transfected HT22 cells using fluorescence imaging, flow cytometry, and Western blot. In conclusion, our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the high-throughput screening of the UPS activators. Three compounds, namely salvianolic acid A (SAA), salvianolic acid B (SAB), and ellagic acid (EA), were identified to significantly decrease YFP fluorescence intensity, which suggested that these three compounds are UPS activators. The identified UPS activators were demonstrated to clear AD-related proteins, including APP, Tau, and Tau P301L. Therefore, these findings provide a novel insight into the discovery and development of anti-AD drugs.

Cultured human bone marrow stromal (mesenchymal) stem cells (hBM‐MSCs) are heterogenous cell populations exhibiting variable biological properties. Quantitative high‐content imaging technology allows identification of morphological markers at a single cell resolution that are determinant for cellular functions. We determined the morphological characteristics of cultured primary hBM‐MSCs and examined their predictive value for hBM‐MSC functionality. BM‐MSCs were isolated from 56 donors and characterized for their proliferative and differentiation potential. We correlated these data with cellular and nuclear morphological features determined by Operetta; a high‐content imaging system. Cell area, cell geometry, and nucleus geometry of cultured hBM‐MSCs exhibited significant correlation with expression of hBM‐MSC membrane markers: ALP, CD146, and CD271. Proliferation capacity correlated negatively with cell and nucleus area and positively with cytoskeleton texture features. In addition, in vitro differentiation to osteoblasts as well as in vivo heterotopic bone formation was associated with decreased ratio of nucleus width to length. Multivariable analysis applying a stability selection procedure identified nuclear geometry and texture as predictors for hBM‐MSCs differentiation potential to osteoblasts or adipocytes. Our data demonstrate that by employing a limited number of cell morphological characteristics, it is possible to predict the functional phenotype of cultured hBM‐MSCs and thus can be used as a screening test for “quality” of hBM‐MSCs prior their use in clinical protocols.

Backgrounds: Localization of non-palpable breast lesions is important for obtaining tumor-free resection margin and achieving better cosmetic outcome. Near infra-red (NIR) imaging system has been introduced for localization in breast surgery. This study aimed to evaluate the feasibility of localization using NIR imaging system in breast conserving surgery (BCS) (ClinicalTrials.gov identifier: NCT 02172989/ NCT02473159). Materials and Methods: Between June 2014 and October 2015, 20 women with benign neoplasm and 5 patients with early breast cancer were enrolled and underwent BCS using NIR imaging system. Before surgery, Indocyanine green was injected intratumoraly in benign lesions and peritumoraly for resection margins in early breast cancer. Their pathologic results for resection volume and re-excision rate were compared with those of the patients with 99 benign neoplasm and 203 early breast cancer who were treated with BCS by conventional method, respectively. Results: In the patients with benign lesions, the mean size in sonography was 1.48±0.98 cm in NIR group and 1.32±0.96cm in conventional group (P=0.4). There was no difference of pathologic lesion size and excised specimen size between these groups (1.57±0.78 cm vs 1.42±0.80 cm in pathologic lesion size; P=0.63, 4.28± 0.48 cm vs 4.15±1.37 cm in specimen size; P=0.73). In NIR group, the ratio of excised specimen/lesion was lesser than that of conventional group (3.19 ±1.40 vs 4.31±3.86; P=0.008). In addition, positive margin rate after BCS in early breast cancer patients was 0% in NIG group and 19.7% in conventional group. And re-excision rate was 0% and 4.4%, respectively. Conclusions: This study showed that localization using NIR image system could be a feasible method to obtain safe resection margins and optimum resection volumes in patients undergoing breast conserving surgery. This research was supported by National Cancer Center Grant NCC-1410202-1 & NCC-1410202-2 by the National Cancer Center, Republic of Korea. Citation Format: Jung S-Y, Lee S, Kim SK. Clinical applications of near infra-red imaging system for localization of non-palpable breast lesions in breast conserving surgery [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-11-10.

Ultrasonic NDI methods have an impressive record of applications on metallic and composite structures. However, limitations arise from the need for a wet couplant between the specimen and the transducer and the rather long inspection times necessitated by point-by-point scanning of large structures. To overcome these constraints, a dry-contact large-area ultrasonic imaging system is being developed for real-time high-resolution NDI applications. This system includes the following: a large ultrasonic source, either piezoelectric or laser-based, a polymer dry-couplant, and a commercially available real-time ultrasonic CCD camera displaying easy-to-interpret images rather than A-scans. Applications of this real-time high-resolution ultrasonic imaging system on metallic and composite structures, using either PZT or laser-based ultrasound generation as the source, are presented. Aluminum and unidirectional and woven composites have been investigated. Images acquired in both through-transmission and pulse-echo modes are presented. Images of artificial defects of different types and shapes in the investigated materials will be demonstrated. The latest developments of the imaging system, with laser-based ultrasound generation as the source, are also reported. The laser-based source provides an efficient solution for some applications of the imaging system. In this configuration, the ultrasound is generated in a 1in. diameter area by an expanded laser beam which heats a constrained absorbing polymer layer. The soft polymer layer is also used as dry couplant to transmit the ultrasound between the test sample and the imaging system.

For plasma diagnostic imaging systems such as the electron cyclotron emission imaging (ECEI) system, spurious rf heating power may saturate or even damage the mixer arrays. Without protection, the sensitivity of the mixers can significantly decrease or in the extreme case, the diodes can even be burnt. A metallic dichroic plate is usually used to rejection the spurious rf heating power. However, as a high pass filter, the dichroic plate can not be used when the frequency of the heating power is in the middle of the frequency range of interest. Consequently, a frequency selective surface (FSS) has been introduced as a planar filter in ECEI systems. FSSs can work as low pass, high pass, and band stop filters according to the various system requirements. Also, as a thin, light, planar filter, it is very easy to mount in imaging systems. This paper will focus on the design and fabrication of the FSS notch filter applied in TEXTOR, which is used to protect the imaging array from stray 140 GHz ECRH power. The filter is used in TEXTOR due to its deep rejection, and excellent angle insensitivity. The design procedure will be presented. More FSS applications will be talked in this paper. The new fabrication technique Electro Fine Forming (EF2) technology will also be introduced. FSS filters in the millimeter wave range also have possible applications in imaging systems in other fusion machines such as KSTAR, DIIID, and LHD.

Objective: To construct a high-content imaging system analysis model to study the effects of the traditional Chinese medicine compounds puerarin on proliferation and migration of cervical cancer (HeLa) cells. Methods: The digital phase-contrast mode unique to the high-content imaging system was used to study the proliferation and migration effects of puerarin on HeLa cells. Results: Puerarin inhibited HeLa cell proliferation and migration. The high-content analysis model constructed in this study enabled efficiently and accurately determining cell proliferation and migration parameters in batches and allowed detecting multiple phenotypic parameters in one experiment. These parameters provide a theoretical basis for subsequent research. Conclusion: Puerarin inhibited HeLa cell proliferation and migration. The high-content analysis model constructed in this study uses a digital phase-contrast mode rather than traditional fluorescence staining and cell scratch tests for label-free analysis, thus providing a more rapid, efficient and accurate analysis method for tumor research.

The field of cell biology has seen major advances in both cellular imaging modalities and the development of automated image analysis platforms that increase rigor, reproducibility, and throughput for large imaging data sets. However, there remains a need for tools that provide accurate morphometric analysis of single cells with complex, dynamic cytoarchitecture in a high-throughput and unbiased manner. We developed a fully automated image-analysis algorithm to rapidly detect and quantify changes in cellular morphology using microglia cells, an innate immune cell within the central nervous system, as representative of cells that exhibit dynamic and complex cytoarchitectural changes. We used two preclinical animal models that exhibit robust changes in microglia morphology: (1) a rat model of acute organophosphate intoxication, which was used to generate fluorescently labeled images for algorithm development; and (2) a rat model of traumatic brain injury, which was used to validate the algorithm using cells labeled using chromogenic detection methods. All ex vivo brain sections were immunolabeled for IBA-1 using fluorescence or diaminobenzidine (DAB) labeling, images were acquired using a high content imaging system and analyzed using a custom-built algorithm. The exploratory data set revealed eight statistically significant and quantitative morphometric parameters that distinguished between phenotypically distinct groups of microglia. Manual validation of single-cell morphology was strongly correlated with the automated analysis and was further supported by a comparison with traditional stereology methods. Existing image analysis pipelines rely on high-resolution images of individual cells, which limits sample size and is subject to selection bias. However, our fully automated method integrates quantification of morphology and fluorescent/chromogenic signals in images from multiple brain regions acquired using high-content imaging. In summary, our free, customizable image analysis tool provides a high-throughput, unbiased method for accurately detecting and quantifying morphological changes in cells with complex morphologies.

Liquid crystal (LC) lens has the characteristic of variable focal length. Many studies have demonstrated that electronically controlled focusing without mechanical movements can be realized by using LC lenses in imaging system. In this paper, a four-electrodes LC lens with a rectangular aperture is applied to focus. The aperture’s aspect ratio of the LC lens can be designed arbitrarily so that installing it with the periscope lens in phones becomes possible.

Objective To investigate the application of the ultrasound diffusion breast imaging(UDBI) system in the diagnosis of T1 stage breast cancer. Methods Totally 2 245 patients with ultrasound grade 3 and above (the patients in grade 6 were excluded) in the Chengdu Women’s and Children’s Central Hospital from April 2016 to April 2018 were included in this study. The patients underwent both ultrasonography and UDBI. Among them, 1 157 patients underwent biopsy, with complete pathological data, so they were retrospectively analyzed. According to the maximum diameter of the tumor, 1 157 cases were divided into 3 groups: 452 cases in S1 group (≤1.0 cm), 489 cases in S2 group (1.1-2.0 cm), and 216 cases in S3 group (>2.0 cm). The receiver operating characteristic (ROC) curve was used to analyze the diagnostic performance of the two methods. The area under the ROC curve and 95%CI were calculated accordingly. The area under the ROC curve was compared by Z test using the Medcalc V19.0.4 software. The sensitivity and specificity of the two methods were compared by χ2 test or Fisher exact test. The paired χ2 test (McNemar test) was used to compare the two methods with pathological results. Results Among the 1 157 cases, there were 144 cases of malignant tumor (12.4%), confirmed as invasive ductal carcinoma, 1 013 cases of benign disease (87.6%), including 852 cases of fibroadenoma, 49 intraductal papilloma and 112 adenosis. The sensitivity, specificity and diagnostic accuracy in the diagnosis of breast cancer were 90.2% (130/144), 67.7% (686/1 013) and 70.5 (816/1 157) by ultrasound, 94.4% (136/144), 81.8% (829/1 013) and 83.4% (965/1 157) by UDBI, respectively. The diagnostic accuracy and specificity of UDBI were significantly higher than those of ultrasound (χ2=55.148, P 2.0 cm, there was no significant difference in the diagnostic performance between UDBI and ultrasound (Z=1.911, P=0.056). Subgroup analysis showed that for the tumor with the diameter of 0-1.0 cm, the specificity of UDBI was significantly higher than that of ultrasound [85.7% (359/419) vs 72.3% (303/419), χ2=22.555, P 2.0 cm, the specificity of UDBI was significantly higher than that of ultrasound [73.1% (117/160) vs 58.1% (93/160), χ2=7.979, P=0.005]. There was no significant difference in the sensitivity between UDBI and ultrasound in each subgroup (P=0.427; χ2=0.374, P=0.541; P=1.000). Conclusion The UDBI is superior to ultrasound in the diagnosis of breast cancer of T1 stage, which can be used as an effective method for early breast cancer diagnosis. Key words: Ultrasonography; Breast neoplasms; Early diagnosis; Ultrasound diffusion breast imaging system

The morphology of 2D cell colonies has been studied to understand tumor metastasis in the past decades. However, 2D cell cultures are lacking many features of 3D tissues, and their physiological behaviors are quite different from solid tumors in vivo. In this work, we studied the multi-cellular tumor spheroid (MCTS) spreading on the substrate, which keeps parts of 3D tissue characteristics and facilitates cell tracking through 2D imaging. By using a high content imaging system (HCS), we tracked multiple spheroids in one single 96-well plate for 36 h. An automated algorithm based on Otsu’s method was developed to investigate the morphological details of spheroids through the quantification of radius length and its coefficients of variation. Spheroid spreading is altered by the PIP-platin, which was a novel platinum based drug previously reported by us with an inhibitory effect on cell migration. All parameters showed dose dependent decreases when PIP-platin concentration increased, indicating the inhibition of spheroid expansion by this compound. To investigate the surface roughness of spheroids affected by the drug, we applied the Fourier parameter β and the normalized standard deviation of the radius STDr /, which were found inversely proportional to the concentrations of PIP-platin. Particularly at the low drug concentrations, the indices of contour roughness appeared to be more sensitive than spheroid sizes, which could be the potential morphological markers for high content screening of drugs.

Due to some problems (such as abnormal time consuming, low spatial resolution and large system volume) existing in the traditional passive millimeter wave (PMMW) imaging system, the application of traditional imaging system based on real aperture incoherent technology is limited on many occasion (security/medical check, etc). These defects can be overcame by replacing the traditional bulky imaging systems with two dimensional antenna arrays and electronic processors. This paper discusses preliminary design work for a demonstrator of a near range millimeter wave synthetic aperture passive imaging system. In the synthetic aperture system developed in this paper, the theory of near range synthetic aperture imaging technology is discussed, then a simple 2-d PMMW imaging prototype is designed for obtaining high-resolution PWMM images on the basis of this theory. Finally, we verify the feasibility of the system by numerical simulation. The experimental result demonstrates the superior performance in improving the spatial resolution of PMMW images.

P16-47: Lysosomal trapping measurement in multiple cell types using a High Content Imaging (HCI) system

This study aims to investigate the effect of ethoxysanguinarine(Eth) on cisplatin(DDP)-resistant human gastric cancer cells and decipher the underlying mechanism. The human gastric cancer cell line SGC7901 and the DDP-resistant cell line SGC7901/DDP were used as the cell models. Western blot was employed to determine the expression levels of multidrug resistance-related proteins, and methyl thiazolyl tetrazolium(MTT) assay to detect the proliferation of SGC7901 and SGC7901/DDP cells exposed to DDP. After treatment with different concentrations of Eth, the proliferation of SGC7901 and SGC7901/DDP cells was detected by MTT assay, trypan blue exclusion assay, colony formation assay, and high-content imaging and analysis system. The apoptosis of SGC7901/DDP cells was detected by flow cytometry with Annexin V-FITC/PI staining. GFP-LC3 transfection was carried out to detect the effect of Eth on the autophagy of SGC7901/DDP cells. The expression levels of the multidrug resistance-related protein P-glycoprotein(P-gp), the apoptosis-related proteins [caspase-9, caspase-3, and poly(ADP-ribose) polymerase(PARP)], the autophagy-related protein light chain 3-Ⅱ(LC3-Ⅱ), the key effectors [mammalian target of rapamycin(mTOR), 70 kDa ribosomal protein S6 kinase(P70 S6 K), and 4 E binding protein 1(4 E-BP1)] of the mammalian target of rapamycin complex 1(mTORC1) signaling pathway, cancerous inhibitor of protein phosphatase 2A(CIP2A), and protein kinase B(Akt) were measured by Western blot. The mRNA level of CIP2A in the SGC7901/DDP cells exposed to Eth for 24 h was analyzed by RT-qPCR. After SGC7901/DDP cells were transfected with CIP2A expression vector pcDNA3.1-HA-CIP2A and treated with different concentrations of Eth, MTT assay was used to determine the prolife-ration of SGC7901/DDP cells and Western blot to detect the expression levels of related proteins. The interaction sites of Eth and CIP2A were predicted by molecular docking. The affinity between Eth and CIP2A was determined by drug affinity responsive target stability(DARTS) assay. The pharmacokinetic properties and drug-like activity of Eth were predicted by SwissADME. The results indicated that SGC7901/DDP cells were more sensitive to Eth than SGC7901 cells. Eth significantly inhibited proliferation and colony formation and changed the morphology, roundness, and area of SGC7901/DDP cells. Eth treatment caused the nucleus shrinking and significantly increased the apoptosis rate of the cells. Furthermore, Eth down-regulated the expression of caspase-9 and caspase-3 precursors and promoted the cleavage of PARP, which suggested that Eth induced the apoptosis of SGC7901/DDP cells. The GFP-LC3 in Eth-treated cells showed speckled aggregation. The up-regulated expression of LC3-Ⅱ by Eth indicated that Eth activated the autophagy of SGC7901/DDP cells. Eth down-regulated the expression of P-gp, the phosphorylation of mTOR, P70 S6K, and 4E-BP1, the expression of CIP2A, and the phosphorylation of Akt. Additionally, it increased the activity of PP2A, and had no significant effect on the expression of CIP2A in SGC7901/DDP cells. CIP2A overexpression antagonized the inhibition of cell proliferation and the activation of autophagy by Eth. Molecular docking suggested that Eth bound to CIP2A. The results of DARTS assay further proved the above binding effect. Eth has potential drug-like activity. The above results demonstrated that Eth inhibited the proliferation, induced the apoptosis, and activated the autophagy of SGC7901/DDP cells by targeting CIP2A and then down-regulating PP2A/mTORC1 signaling pathway. This study provided a new target for the treatment of cisplatin-resistant gastric cancer.

The purpose of this study is to develop a method for characterisation of time-of-flight (ToF) imaging system for application in deep inspiration breath-hold radiotherapy (DIBH-RT). The performance of an Argos 3D P330 ToF camera (Bluetechnix, Austria) was studied for patient surface monitoring during DIBH-RT using a phantom to simulate the intra-patient and inter-patient stability of the camera. Patient setup error was also simulated by positioning the phantom at predefined shift positions (2, 5 and 10 mm) from the isocentre. The localisation accuracy of the phantom was measured using ToF imaging system and repeated using CBCT imaging alone (CBCT) and simultaneously using ToF imaging during CBCT imaging (ToF-CBCT). The mean and SD of the setup errors obtained from each of the imaging methods were calculated. Student t-test was used to compare the mean setup errors. Correlation and Bland-Altman analysis were also performed. The intra-and inter-patient stability of the camera were within 0.31 mm and 0.74 mm, respectively. The localisation accuracy in terms of the mean ±SD of the measured setup errors were 0.34 ± 0.98 mm, 0.12 ± 0.34 mm, and −0.24 ± 1.42 mm for ToF, CBCT and ToF-CBCT imaging, respectively. A strong correlation was seen between the phantom position and the measured position using ToF (r = 0.96), CBCT (r = 0.99) as well as ToF-CBCT (r = 0.92) imaging. The limits of agreement from Bland Altman analysis between the phantom position and ToF, CBCT and ToF-CBCT measured positions were −1.52, 2.31 mm, −0.55, 0.78 mm; and −3.03, 2.55 mm, respectively. The sensor shows good stability and high accuracy comparable to similar sensors in the market. The method developed is useful for characterisation of an optical surface imaging system for application in monitoring DIBH-RT.