For example, we frequently perform experiments where we deposit a limiting number of cells (e.g., 1,10, or 50) into individual wells of a 96-well culture plate. Before This system provides top-of-the-line imaging capabilities for cell-based immaging, such as live-cell analysis, image tiling, and Z-stacking. We tested several contrast adjustments in CellProfiler and Matlab to increase the visibility of cell nuclei. EVOS systems were designed to allow researchers to focus on their data rather than worrying about the operation of a microscope. Self-contained imaging systems are versatile instruments that are becoming a staple in cell culture laboratories. Vanderbilt University is committed to principles of equal opportunity and affirmative action. Automated batch analysis and quantification of these tiled images does however require off-loading files to other software platforms. Seeking an alternative solution, we measured the background fluorescence generated by four different types of media (Fig 2). All equipment available for sale has been carefully tested for workability and is accompanied by a minimum 30-day functional warranty. However, it cannot be used on tiled images. Stay up to date on news and special offers related to these products. From monoclonal and polyclonal antibodies to reagents, buffers and sera all spanning a wide range of applications. Whether molecular cloning, sequencing, or exploring genomes, LabX has the products required for your cutting-edge research. Using this manual mode indeed created tiled images of consistent brightness that, in turn, was critical for CellProfiler to batch process and detect GFP positive cells. We found manually cropping the images in ImageJ [11] (shift+oval selection for circular ROI, edit > selection > make inverse, delete selection) produced accurate results. 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). Growth was assessed weekly by imaging the plates using the Scan function of the EVOS FL Auto imaging system. This combination of steps produced finely polished images containing distinct cell nuclei that CellProfiler could readily identify and count (Fig 8). Proprietary LED light cube technology is designed to minimize photo-bleaching, offers >50,000 hours of LED illumination, and allows adjustable intensitywith no darkroom and no consumable costs. Cells were then stained with NucBlue Live and imaged in Live Cell Imaging Solution using 40x objective (, Multichannel time-lapse live-cell imaging. We submit the current pipeline in hopes that it will assist others with similar applications and goals. 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. However, there are instances where using random fields for quantification may be confounding. 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. The third pdf is a Quick Start Guide to provide you with the basic information on operating the microscope. Manual M. Version 9.5. sharing sensitive information, make sure youre on a federal The current pipeline has already demonstrated applications beyond our original intent, and we continue to seek ways to further refine and advance its sensitivity to input data. 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). Analysis of stitched images also presented a unique problem, in that the illumination artifact created a grid pattern in the composite image (Fig 1; S1 Fig, steps 910). These included: high background, illumination and stitching artifacts, low contrast, noise, focus inconsistencies, and image distortionall of which negatively impacted processing efficiency. There are five objectives available for use: 100X Plan FL 1.28NA Oil, Coverslip Corrected. Because of this analysis bottleneck, we decided to develop an automated pipeline to accelerate the process. Shop for the latest new and used equipment designed to meet the challenges of this emerging and promising field. Because an entire well cannot fit into a single microscopic field, imaging multiple overlapping fields is required. We thank members of our laboratory for their critical reading of this manuscript and useful discussions. Outlines of the identified nuclei are overlaid on the original input image and summarized into a .csv output file. 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. Other instrument accessories included. 1878 - document.write(new Date().getFullYear()) Western University, Robarts Research Institute1151 Richmond Street North, London, Ontario, Canada As anticipated, the first problem we encountered was the need for a different imaging medium. The composite images that are produced contain a central circular growth area encircled by a bright fluorescent halo (produced by the perimeter of each well). will also be available for a limited time. We therefore sought to develop an automated analysis pipeline (S1 Fig). The bulk of processing is performed in CellProfiler, and our final pipeline is supplied in the S1 Fig that can be found also at https://cellprofiler.org/examples/published_pipelines. 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. As we began loading images into CellProfiler, we discovered a curious problem: a few of the acquired images were compressed along the y-axis, transforming the well into an ovoid shape (Fig 6). Natick, Massachusetts: The MathWorks Inc. Modelling breast cancer requires identification and correction of a critical cell lineage-dependent transduction bias. Data is expressed as population doublings. Learn More. 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]. This flexible approach and combination of settings prepared the images for cell detection, but there was another unique problem that wed need to overcome: an illumination artifact caused by the stitching algorithm. 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. 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. When these are overlaid, the variability is evident (right). Defined cell numbers were deposited into 96 multi-well dishes using a Sony iCyt SY3200 flow sorter. We have since overcome these obstacles and have created a rigorous cell counting pipeline for analyzing images captured by the EVOS scan function. and new technologies for life scientists. To determine the effects of growth factors (or other media components) on cell proliferation, we often seed cells into 96-well dishes at clonal densitiesreserving several rows for multiple biological replicates. We present development and optimization of this automated pipeline and submit it as an effective and facile tool for accurately counting cells from tiled images. 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). LabX has a large variety of resources for the expanding world of materials science. Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, et al. Here, we present these problems, as well as solutions to each. Bethesda, MD 20894, Web Policies We use cookies on this site to ensure the best service possible. *Note that PBS is not recommended for long scan times. 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. The Invitrogen EVOS FL Imaging System was designed for a broad range of applications including, but not limited to, multiple-channel fluorescence imaging, protein analysis, pathology, cell culture, and in situ imaging. 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). Web Standards | Finally, there is a .jpg at the bottom which demonstrates the potential image quality using the 40x coverslip corrected objective. At the end of the scan, the software produces 96 stitched image files in a lossless format (*.PNG) that were saved and used for analysis and cell quantification. We have overlaid a gray dashed line to indicate the edge of each image. Equipment and Electronic Storage and Recycling, Chemiluminescent and GFP CCD Imager LAS4000, Site Development: Digital Strategies (Division of Communications). The EVOS imaging system contains a scanning algorithm that screens culture plates and creates individual tiled images of each well. From fluorescence and electron microscopes, to gel imagers and more, shop LabX for a wide range of products and accessories to fit your needs. There is some slight variance in the thickness of the plates, so perfectly focused images were not expected. An annotate pipeline is available at https://cellprofiler.org/examples/published_pipelines. The high number of replicates afforded by this approach reveals the phenotypic variation between wells, as well as between both cell types. To demonstrate the cell counting pipeline, weve assessed the growth of MCF-7 and MDA-MB-231 breast cancer cells lines over a two-week period, imaging every two days (n = 12 wells each, Fig 9). 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. 0.944444 (R2018b), 2018. MCF-7 cells were processed via the optimized imaging pipeline. The new PMC design is here! Buy and sell today! Cells were then stained with NucBlue Live and imaged in Live Cell Imaging Solution using 40x objective (Thermo Fisher Scientific). All rights reserved. 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). 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 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. * statistically significant (p<0.05, t-student test). The https:// ensures that you are connecting to the On left, an image of a 96-well produced by EVOS. 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. Our initial attempts to quantify tiled images captured on an EVOS device was plagued by some expectedand other unforeseeableissues that arose at nearly every stage of analysis. This strategy worked and allowed us to create a template mask for each image, allowing the exterior portion of the well to be removed. 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. Accessibility, a high-sensitivity monochrome camera optimized for fluorescence imaging and quantitation, and a high-resolution color camera optimized for colorimetric imaging, HeLa cells expressing Mitochondria RFP were plated on a 6-well Falcon plate. The EVOS fluorescence microscope provides users withexceptional signal-to-noise ratios with its groundbreaking LED illumination system. The Equipment Manager can provide any initial training for free which would get you up and running. Currently, we do not have a fee for using the EVOS as we wanted to provide an opportunity for labs to explore the instrument; however, this may change in the future. Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America. 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). CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Learn more One solution we considered was to create cropping masks unique to each well position. Images were processed via the developed pipeline, as described in methods. Standard techniques were then applied to advance the pipeline towards automated cell detection. This problem was corrected by increasing the computers virtual memory allocation. Although this does require importing images into another software platform, the calculations were quick (taking only 12 minutes for 96 images) and provided images with readily detectable nuclei (S1 Fig steps 78). The exploding cannabis industry has created the need for high quality processing and testing products for laboratory operations of all scales. Biocompare is the leading resource for up-to-date product information, product reviews,
8600 Rockville Pike It is essential that manual exposure (Actual mode) is used to acquire images to restrict the EVOS systems autonomy for varying exposure time. This system was used to analyzed in vitro cultures of differentiated pluripotent stem cells. On right, a saved image of a well (from the same plate/scan) that has compressed the well into an ovoid shape. Increasing the operating systems virtual memory allocation alleviated the problem.