Assessing cell counts by imaging has distinct advantages over these other methods. This system provides top-of-the-line imaging capabilities for cell-based immaging, such as live-cell analysis, image tiling, and Z-stacking. These scans create coherent images of each well, but analyzing these images manually (or semi-manually) was a long and tedious process. Equipment and Electronic Storage and Recycling, Chemiluminescent and GFP CCD Imager LAS4000, Site Development: Digital Strategies (Division of Communications). Because the brightness of the grids edges often approached that of cells, the software would repeatedly incorrectly detect the grid as many cells, skewing cell counts dramatically. Growth was assessed weekly by imaging the plates using the Scan function of the EVOS FL Auto imaging system. government site. Media used in the imaging experiments include: Imaging DMEM (Fluorobrite DMEM, Gibco), DMEM/F12 (Thermo), and custom breast cell medium M87 [7], as indicated in figure legends. To capture the entirety of each well, we set the machine to scan Center Region Only, typically at a setting of 75% or higher that guided the system to capture adjacent images roughly centered on each well, creating a 3x3 or 4x3 tiled image for each at these settings. Although applying this function to the images did remove the grid, it unfortunately also dimmed the H2B-GFP signal, causing the software to overlook large numbers of cells. All rights reserved. This function directs the microscope to capture neighboring microscopic fields at each well location, and combines neighboring images together to create a composite, tiled, image of each well. Biocompare is the leading resource for up-to-date product information, product reviews, Versions of all the analysis CellProfiler3 pipelines and Matlab code are available for download from: BreastCancerLab.com, http://breastcancerlab.com/evos-cell-counting-pipeline/. Finding the ideal combination of these featuresthat avoids artifacts and produces accurate cell countsoften requires a significant investment of time. Stay up to date on news and special offers related to these products. Shop for the latest new and used equipment designed to meet the challenges of this emerging and promising field. We discovered that if we treated the well halo as a single object of interest (much like we do when we identify cells), it would direct the program to isolate the well no matter its position in the image. Additional data and detailed instructions are available on our website: http://breastcancerlab.com/evos-cell-counting-pipeline/. To facilitate automated counting, we needed to first optimize experimental conditions to create high quality images of uniform intensity and high contrast. Software packages used in this work include CellProfiler 3.1.9 [1, 2], ImageJ [3], and Matlab R2018b (Mathworks) [4] (or GNU Octave [5] -an open-source Matlab alternative). We found that after this log-transformation, all cells were readily identified by CellProfiler, which we confirmed by manual counting random images. official website and that any information you provide is encrypted The EVOS imaging system is such a device and is capable of scanning multi-well dishes and stitching together multiple adjacent fields to produce coherent individual images of each well. We found the cell lines tolerated the imaging DMEM well, and it led to acquisition of high contrast images with readily detectable H2B-GFP+ cells. The final processing pipeline contains three distinct processing groups: Cropping (top), Log transformation (middle), and Counting (bottom). National Library of Medicine The long-term goal of our laboratory is aimed at defining the cellular and biochemical microenvironment of tissues and tumors; and determining how cells coordinate and communicate to maintain tissue homeostasis. We ultimately found a viable solution in CellProfiler, via the Identify Primary Objects module. Not surprisingly, the DMEM designed for fluorescent imaging performed the best, generating minimal background fluorescence that was roughly equivalent to PBS (Fig 2). We use cookies on this site to ensure the best service possible. Manual M. Version 9.5. Although the EVOS tiling function produces a composite image of each well, the brightness in the contributing images is not homogeneous. Data is expressed as population doublings. Research reported in this publication was supported by the University of New Mexico Cancer Center and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103451. From cell culture to complex protein analysis and multichannel fluorescence imaging, EVOS imaging systems help you perform a variety of routine and specialty applications. We thus needed a creative solution to crop the wells edge and extract the growth area within each image. Cells were then stained with NucBlue Live and imaged in Live Cell Imaging Solution using 40x objective (, Multichannel time-lapse live-cell imaging. An official website of the United States government. Shop for forensic lab equipment and get the latest trends on new products in forensic science. Unfortunately, adding FBS to the medium did introduce insoluble particulates that interfered with image processing (Fig 3). Establishing cell counts in each wellusually to assess cell proliferationcan be performed several ways, most of which require killing the cells (e.g., MTT assay) or disrupting growth (e.g., cell dissociation via trypsinization). This page is dedicated to featured products, resources, and valuable information pertaining to this rapidly developing area. To circumvent this, a simple solution was to replace the DMEM with phosphate buffered saline (PBS). However, without an automated tool to count cells in acquired images, it becomes the most tedious method of them all. Other instrument accessories included. The top and bottom groups of steps are performed in CellProfiler (red boxes), whereas the log transformation (middle blue box) is performed in Matlab. After this step, we added a few standard clean-up processes to facilitate cell counting, such as masking the illumination corrected images, and applying a mild gaussian blur to remove artifacts and random noise (each step is described in S1 Fig). The resulting images were then imported back into CellProfiler, and the illumination function (described above) was applied. Epifluorescence and transmitted light (bright field and phase-contrast), Simultaneously accommodates up to 4 fluorescent light cubes, Mechanical stage with X-Y axis fine-positioning controls, High-sensitivity interline CCD color camera, Height 58 cm (23 in); Depth 47 cm (19 in); Width 36 cm (14 in), LPLAN PH2 4x/0.13; PLAN Fluor 10x/0.3; PLAN Fluor 20x/0.45; PLAN Fluor 40x/0.65, Zeiss ID03 Inverted Phase Contrast Microscope. Based on these results, we performed all future cell imaging experiments using FluoroBrite imaging DMEM. The Equipment Manager can provide any initial training for free which would get you up and running. All equipment available for sale has been carefully tested for workability and is accompanied by a minimum 30-day functional warranty. London, Ontario, Canada, N6A 5B7Tel: (519) 931-5777 x 24441 or 24151LRMF@robarts.caPrivacy | Having optimized culture conditions (medium), computer settings (virtual memory allocation), and EVOS acquisition options (4x objective, focus and exposure settings), we were confident that the acquired tiled images were ready to be analyzed by CellProfiler. Because of this analysis bottleneck, we decided to develop an automated pipeline to accelerate the process. This worked well, however its not unexpected that the cells didnt tolerate being in PBS over long periodsespecially the hour or more required to image an entire plate. will also be available for a limited time. However, there are instances where using random fields for quantification may be confounding. We found the log transform in Matlab performed the best (S1 Fig steps 78). The https:// ensures that you are connecting to the LabX has a large variety of resources for the expanding world of materials science. Because an entire well cannot fit into a single microscopic field, imaging multiple overlapping fields is required. sharing sensitive information, make sure youre on a federal Find products and equipment for pharma and biopharma research, development, and processing all in one place. In this case, cells occupy only a small fraction of the available growth area, so its essential the entire well be imaged to allow for an accurate count. To remove these particulates, we it was crucial we filter the FBS-supplemented FluoroBrite medium through a 0.2um filtration unit. On left, two replicate wells captured using EVOS autoexposure setting. The EVOS Automated Cell Imaging System (ThermoFisher Scientific) is such a device, and we frequently use our machine for high-throughput imaging applications and experiments that require quantifying absolute numbers of cells in multi-well dishes. All images were assembled into a single folder, and the pipeline was applied to generate growth curves for each cell type. about navigating our updated article layout. The LabX marketplace has all the laboratory products, equipment, and accessories for your cell culture needs. Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, et al. This system was used to analyzed in vitro cultures of differentiated pluripotent stem cells. Plates were cultured at 37C in a humidified incubator (5% CO2) and were fed/imaged weekly. During development of the pipeline, we encountered many obstacles. This strategy worked and allowed us to create a template mask for each image, allowing the exterior portion of the well to be removed. The new PMC design is here! The exploding cannabis industry has created the need for high quality processing and testing products for laboratory operations of all scales. Identification of a signalling mechanism by which the microbiome regulates Th17 cell-mediated depressive-like behaviors in mice, Effects of mir-128a on the invasion and proliferation of glioma U251 cells, Long-Term In Vitro Expansion of Epithelial Stem Cells Enabled by Pharmacological Inhibition of PAK1-ROCK-Myosin II and TGF- Signaling, Using Mouse Oocytes to Assess Human Gene Function During Meiosis I, Cell Culture Fluorescent Analysis with the Evos FL Digital Inverted Microscope From ThermoFisher Scientific. Web Standards | LabX keeps pace with this industry by offering a wide range of instruments and solutions for the most demanding questions. This was tested on images produced by the EVOS system, but we predict it can be easily adapted to images acquired by similar systems. The trick was to direct the software to look for a very large object. After imaging, counting an entire set of images from a dish (96 wells) was consistently taking us between 78 hours to complete in ImageJ, largely because of the manual cropping required. Furthermore, contrary to traditonal mercury-based light sources, LEDs do not require special disposal and are thus more environmentally friendly and energy-efficient. We traced this problem to the autoexposure setting in the EVOS software and found this could be prevented if we set the exposure manually, using the softwares Actual mode. MDA-MB-231 and MCF-7 breast cancer cell lines were obtained directly from the American Type Culture Collection (ATCC) and were cultured in high glucose Dulbeccos modified eagles medium (DMEM, Sigma), supplemented with 10% FBS and 1x penicillin/streptomycin, as reported previously [6]. Other common methods and endpoint assays of cell quantification in these types of experiments are possible; e.g., MTT or ATP assays, but quantification afforded by imaging is non-destructive and allows an accurate and dynamic assessment of cell-division over the course of a months-long experiment. Although imaging DMEM drastically reduced background fluorescence, we noticed that there was still noticeable variation in image brightness between wellseven in replicate wells with identical treatments (Fig 4). The second pdf shows the various types of plates we have for holding a wide variety of samples (i.e., slides, round culture dishes, flasks). However, it cannot be used on tiled images. We do not have an onstage incubator for the microscope although it will do time-lapse imaging. This combination of steps produced finely polished images containing distinct cell nuclei that CellProfiler could readily identify and count (Fig 8). H2B-GFP was imaged with the GFP LED filter cube (470/22 excitation; 510/42 emission). Trusted diagnostic technologies and emerging analytical techniques form the backbone of this important industry. We were able to overcome these problems and have outlined, in detail, an automatic analysis pipeline that can be used to quantify cell nuclei within tiled images. On right, a saved image of a well (from the same plate/scan) that has compressed the well into an ovoid shape. Accessibility However, high-throughput analysis of these mosaic images requires use of other software platforms for quantification, such as: CellProfiler [1, 2], ImageJ [3], or Matlab/Octave [4, 5]. Vanderbilt University is committed to principles of equal opportunity and affirmative action. LabX is a marketplace with new, surplus, and used equipment for sale from a variety of vendors. Vanderbilt, Vanderbilt University and the Vanderbilt logos are trademarks of The Vanderbilt University. Accessibility to these systems has increased the sheer volume of images that can be generated in short time, yielding densely layered data that becomes unwieldly without assistance of computational algorithms. However, we were able to set a compromising focal plane that produced acceptable images for cell identification. Buy and sell today! EVOS systems were designed to allow researchers to focus on their data rather than worrying about the operation of a microscope. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. The perimeter of the well appears as a bright green fluorescent, labeled well halo (indicated by the yellow dashed line). Site Development: Digital Strategies (Division of Communications) However, when we created these masks, we found the positions of wells across different plates was also not consistent. Vanderbilt University does not discriminate against individuals on the basis of their race, sex, sexual orientation, gender identity, religion, color, national or ethnic origin, age, disability, military service, or genetic information in its administration of educational policies, programs, or activities; admissions policies; scholarship and loan programs; athletic or other University-administered programs; or employment. This instrument is in immaculate condition and is being sold with three light cubes (DAPI, GFP and TX RED) along with a universal vessel holder for the stage. Our first objective was to isolate the growth area by removing the fluorescent border produced by the plastic wall that circumscribes each well (well halo, Fig 1). On left, is a 4x tiled scan (a composite of 4x3 images) of a single well of a 96-well plate containing H2B-GFP labeled MCF-7 cancer cells. Using multi-well plates to culture cells can substantially increase statistical power of experiments by supporting addition of numerous biological replicates. Shop now for new and used products for materials research and testing applications. Before The complete medium we use for these cells is supplemented with fetal bovine serum (FBS). It is essential that manual exposure (Actual mode) is used to acquire images to restrict the EVOS systems autonomy for varying exposure time. As anticipated, the first problem we encountered was the need for a different imaging medium. We used H2B-GFP because it causes the cells nuclei to fluoresce and helps the computer identify and distinguish between neighboring cells. The resulting image sets are then imported back into CellProfiler 3 to a) remove illumination artifacts caused by the EVOS stitching algorithm, b) remove boarder pixels (erosion), and c) reduce noise within the images by smoothing with a gaussian blur. PMC legacy view From pipettors and pipette tips, to CO2 incubators, fume hoods and cell imaging systems, you can shop all the essentials of the modern cell culture laboratory on LabX. A common practice when scanning multi-well dishes is to image random fields, which produces results that are relative. Miniaturization of advanced microscopy equipment and automation of data collection now permit acquisition of high-quality fluorescent images within the confines of a typical cell laboratory. MCF-7 (left) and MBA-MD-231 (right) cells were seeded at low density (100 cells per well, 96-well plate). These software programs are powerful tools, but they do have many parameters that can be applied and/or adjusted. Join our monthly newsletter for expert lab tips and a first-look at our new warehouse arrivals: Publication quality images are always available, High quality camera and optics to give maximum resolution, LED light source is used to replace the traditional Mercury light source, Hard-coated filter sets are used to enable higher transmission efficiencies, Use of the light cubes means brighter fluorescence, higher transmission efficiencies, the ability to detect faint fluorescence signals, and better signal-to-noise ratios, Integrated software serves as a key component to all EVOS systems, Illumination adjustable-intensity LED ( 50,000 hour life per light cube), Interchangeable vessel holder available with instrument. The high number of replicates afforded by this approach reveals the phenotypic variation between wells, as well as between both cell types. This inverted imaging system can be used for four-color fluorescence and transmitted-light applications. We found manually cropping the images in ImageJ [11] (shift+oval selection for circular ROI, edit > selection > make inverse, delete selection) produced accurate results. Through this exercise, we encountered multiple problems that severely affected results (Table 1)not all of which we predicted from the outset. Our standard DMEM produced high levels of fluorescence background, likely from the vitamins and phenol red in the medium, as phenol red absorbs light across a broad spectrum (400-500nm at pH 7) [9] overlapping with GFP [10]. Accessibility information. Cropping the images consists steps that open the image, converts the color image to grey and masks the grey image to remove areas exterior to the well. a) Representative tiled-images of wells containing different media (phenol red-free ClearDMEM; standard DMEM containing phenol red; DMEM/F12; phosphate buffered saline PBS; M87 breast epithelial medium; and FlouroBrite Imaging DMEM b) Fluorescent intensity within the colored circles (shown in 2a) was measured by ImageJ (n = 6 for each condition). Standard techniques were then applied to advance the pipeline towards automated cell detection. Comparing well A1 from eight 96-well plates shows the variability of the well location in the respective images. The powerful yet easy-to-use EVOS FL Cell Imaging System provides the flexibility to fit most epifluorescence microscopy applications. Microscopy, Live Cell Imaging, Fluorescence, Time Lapse, Eva M. Medina-Rodriguez, Derik Madorma, Gregory OConnor, Brittany L. Mason, Dongmei Han, Sapna K. Deo, Mark Oppenheimer, Charles B. Nemeroff, Madhukar H. Trivedi, Sylvia Daunert, Elonore Beurel, Guozhang Hu, Wei Fang, Naijie Liu, Chang Li, Chengkang Zhang, Hyung Joo Lee, Anura Shrivastava, Ruipeng Wang, Travis J. McQuiston, Sharon S. Challberg, Brian A. Pollok, Ting Wang, Diego Marin, Alexandra L. Nguyen, Richard T. Scott,, Karen Schindler. Defined cell numbers were deposited into 96 multi-well dishes using a Sony iCyt SY3200 flow sorter. This problem was corrected by increasing the computers virtual memory allocation. EVOS images were imported into CellProfiler 3 from their raw acquired state, and processed to convert the images to greyscale, then inverted and smoothened to identify the well as an object. The EVOS imaging system contains a scanning algorithm that screens culture plates and creates individual tiled images of each well. Using manual focus in this way reduced the scan time by more than two hours, meaning a single 96-well plate could now be imaged in just over one hour (Fig 5). However, it expectedly took the machine a considerable amount of time to adjust and find the correct focal plane. On right, Matlab histogram analysis of pixel count and fluorescence intensity of these two replicates. However, with each added replicate comes an increased demand on time needed to maintain the culture, perform experiments, and analyze results. This lighting artifact is common to fluorescent microscopy, and CellProfiler does contain an algorithm to remove this radial illumination artifact from single fields. With this and the above parameters set, we were ready to begin analyzing images. We thank members of our laboratory for their critical reading of this manuscript and useful discussions. 1878 - document.write(new Date().getFullYear()) Western University, Robarts Research Institute1151 Richmond Street North, London, Ontario, Canada Increasing the operating systems virtual memory allocation alleviated the problem. Whether molecular cloning, sequencing, or exploring genomes, LabX has the products required for your cutting-edge research. From monoclonal and polyclonal antibodies to reagents, buffers and sera all spanning a wide range of applications. To overcome this problem, we needed a strategy to enhance the GFP cell signal. The final step outlines each cell nuclei and generates a merged image of the outlines(purple outlines) and H2B-GFP fluorescent nuclei (green). Additionally, we thank the users and team members of the CellProfiler community help forum for their guidance with the software and feedback in its application. Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America. In early experiments, we had EVOS autofocus the microscope for each image, which indeed produced crisp images. With positions for 5 objectives and 3 fluorescent light cubes, the EVOS FL Imaging System provides the flexibility to help meet most imaging research applications. Received 2020 Feb 21; Accepted 2020 Jul 6. The second processing group performs a log contrast transformation of each pixel, which is essential to increase the cell nuclei signal relative to background.

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