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). These included a phenol-red free DMEM (ClearDMEM), DMEM/F12, M87 (specific for mammary epithelial cells), and a medium designed specifically for fluorescent imaging (FluoroBrite DMEM, Thermo). 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. This was tested on images produced by the EVOS system, but we predict it can be easily adapted to images acquired by similar systems. However, we were able to set a compromising focal plane that produced acceptable images for cell identification. Continuous pH monitoring in a perfused bioreactor system using an optical pH sensor, HistoneGFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells, NIH Image to ImageJ: 25 years of image analysis, https://cellprofiler.org/examples/published_pipelines, http://breastcancerlab.com/evos-cell-counting-pipeline/, Prefilter medium (0.2 um); apply gaussian blur (step 12), Position of well varies (in tiled images), Identify well as object; apply mask (steps 16), Illumination artifact compounded by tiling, Apply illumination correction (steps 910), Dim cells after illumination correction, (steps 78), Applied mask didnt remove the entire well, Too many cells being detected by CellProfiler, Manually adjust (optimize) threshold setting (step 13). 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. The new PMC design is here! McQuin C, Goodman A, Chernyshev V, Kamentsky L, Cimini BA, Karhohs KW, et al. Accessibility Received 2020 Feb 21; Accepted 2020 Jul 6. A common practice when scanning multi-well dishes is to image random fields, which produces results that are relative. 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. Furthermore, contrary to traditonal mercury-based light sources, LEDs do not require special disposal and are thus more environmentally friendly and energy-efficient. HHS Vulnerability Disclosure, Help Free subscription! The https:// ensures that you are connecting to the The center is brightest and dims towards the edges. The first pdf is the light cube/fluorophore compatibility list. 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. Manual M. Version 9.5. To help set the correct focal plane, we seeded the corner wells with extra cells (~500) and manually focused on these. official website and that any information you provide is encrypted On left, two replicate wells captured using EVOS autoexposure setting. 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. On right, a saved image of a well (from the same plate/scan) that has compressed the well into an ovoid shape. This page is dedicated to featured products, resources, and valuable information pertaining to this rapidly developing area. 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. However, it took over an hour to manually crop an entire plates worth of images, so this was not practical. Plates were cultured at 37C in a humidified incubator (5% CO2) and were fed/imaged weekly. Because of this analysis bottleneck, we decided to develop an automated pipeline to accelerate the process. All equipment available for sale has been carefully tested for workability and is accompanied by a minimum 30-day functional warranty. 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. The growth area of each well is indicated by the color outlines (left). This is an open access article distributed under the terms of the, GUID:03D76FC0-FDB3-4A9D-9DDC-A932C8024D66. 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. Using multi-well plates to culture cells can substantially increase statistical power of experiments by supporting addition of numerous biological replicates. symbol emphasizes difference in mean fluorescence intensities between replicates (introduced by the automatic exposure setting). 0.944444 (R2018b), 2018. Stay up to date on news and special offers related to these products. We ultimately found a viable solution in CellProfiler, via the Identify Primary Objects module. 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. 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). This problem was corrected by increasing the computers virtual memory allocation. Equipment and Electronic Storage and Recycling, Chemiluminescent and GFP CCD Imager LAS4000, Site Development: Digital Strategies (Division of Communications). Defined cell numbers were deposited into 96 multi-well dishes using a Sony iCyt SY3200 flow sorter. This was puzzling, and occurred only when we imaged an entire plate, making it that much more difficult to diagnose. The top and bottom groups of steps are performed in CellProfiler (red boxes), whereas the log transformation (middle blue box) is performed in Matlab. Finally, the third processing group contains steps that correct illumination variance, removes random noise, identifies cell nuclei, and overlays outlines of the identified nuclei onto the greyscale image. Our original plan was to create a mask that could be applied universally to each image, but we discovered the precise location of the well (within each image) varied across the 96 acquired images (Fig 7). * p<0.0001 (t-test of random background fields (free of cells), n = 18 fields for each replicate). Comparing well A1 from eight 96-well plates shows the variability of the well location in the respective images. and new technologies for life scientists. LabX hosts an expansive portfolio of microscopy and imaging solutions. This however would result in a loss of more than 25% of the growth area of each well. To circumvent this, a simple solution was to replace the DMEM with phosphate buffered saline (PBS). An annotate pipeline is available at https://cellprofiler.org/examples/published_pipelines. When these are overlaid, the variability is evident (right). 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. However, it expectedly took the machine a considerable amount of time to adjust and find the correct focal plane. The Standard techniques were then applied to advance the pipeline towards automated cell detection. The field of Bioprocessing technology is expanding, with advances in upstream and downstream process development and innovations in scalable equipment. 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. This inverted imaging system can be used for four-color fluorescence and transmitted-light applications. With the help of Thermo Technical Services, we eventually traced this problem to the virtual memory of the PC controlling the EVOS system. We deemed this unacceptable because it ran contrary to our main goal of imaging every cell in the well. MCF-7 cells were processed via the optimized imaging pipeline. Learn more Not unexpectedly, MDA-MB-231 cellsknown for their fast doubling timesestablished growth almost immediately, whereas the MCF-7 cells exhibited a slower growth rate. Because an entire well cannot fit into a single microscopic field, imaging multiple overlapping fields is required. 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. Data is expressed as population doublings. In early experiments, we had EVOS autofocus the microscope for each image, which indeed produced crisp images. An Invitrogen EVOS Cell Imaging System is a must-have in your lab for cell imaging whether youre capturing images for publication, teaching, or research. Many of these machines possess motorized stages and on-stage incubators that permit programmable imaging of live cells that make them a sensible tool for high-throughput applications. H2B-GFP was imaged with the GFP LED filter cube (470/22 excitation; 510/42 emission). It is essential that manual exposure (Actual mode) is used to acquire images to restrict the EVOS systems autonomy for varying exposure time. 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. With the brightness inconsistencies solved, our next goal was to reduce the scan time for each 96-well plate, which at the time was taking several hours. We therefore sought to develop an automated analysis pipeline (S1 Fig). 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. Self-contained imaging systems are versatile instruments that are becoming a staple in cell culture laboratories. These steps included converting the color image to greyscale, inverting the image, applying a gaussian blur to smoothen edges, and contracting the well mask slightly (to prevent potential perturbances along the edge of the well from being incorrectly identified as cells, Fig 7). 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). Increasing the operating systems virtual memory allocation alleviated the problem. To determine if we could accelerate the imaging process, we deactivated the autofocus function. The LabX marketplace has all the laboratory products, equipment, and accessories for your cell culture needs. Automated batch analysis and quantification of these tiled images does however require off-loading files to other software platforms. Combining precision components with unique design functionality, the EVOS FL Cell Imaging System has revolutionized fluorescent microscopy. will also be available for a limited time. 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). London, Ontario, Canada, N6A 5B7Tel: (519) 931-5777 x 24441 or 24151LRMF@robarts.caPrivacy | 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. The resulting images were then imported back into CellProfiler, and the illumination function (described above) was applied. With this and the above parameters set, we were ready to begin analyzing images. Applying this function to all 96 wells took the software about 15 minutes to complete, and this became our first processing step after image acquisition (S1 Fig, Steps 16). 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. Another option we explored was to create a uniform mask that would be small enough to fit inside the well area and not intersect with any of the variable well positions (Fig 7). The final processing pipeline contains three distinct processing groups: Cropping (top), Log transformation (middle), and Counting (bottom). The EVOS fluorescence microscope provides users withexceptional signal-to-noise ratios with its groundbreaking LED illumination system. 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]. 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. Federal government websites often end in .gov or .mil. MCF-7 (left) and MBA-MD-231 (right) cells were seeded at low density (100 cells per well, 96-well plate). A 4x3 tiled image is illustrated in Fig 1. Cells were then stained with NucBlue Live and imaged in Live Cell Imaging Solution using 40x objective (Thermo Fisher Scientific). Biocompare is the leading resource for up-to-date product information, product reviews, An official website of the United States government. FOIA sharing sensitive information, make sure youre on a federal We tested several contrast adjustments in CellProfiler and Matlab to increase the visibility of cell nuclei. Although the EVOS tiling function produces a composite image of each well, the brightness in the contributing images is not homogeneous. 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. and transmitted securely. However, with each added replicate comes an increased demand on time needed to maintain the culture, perform experiments, and analyze results. The trick was to direct the software to look for a very large object. 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. Garbe JC, Bhattacharya S, Merchant B, Bassett E, Swisshelm K, Feiler HS, et al. On left, an image of a 96-well produced by EVOS. LabX is a marketplace with new, surplus, and used equipment for sale from a variety of vendors. Mean SD (n = 12). The complete medium we use for these cells is supplemented with fetal bovine serum (FBS). All rights reserved. Natick, Massachusetts: The MathWorks Inc. Modelling breast cancer requires identification and correction of a critical cell lineage-dependent transduction bias. 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. This was eventually accomplished by calculating an illumination function for the image set, which we then applied to each image to correct the uneven lighting. Vanderbilt University is committed to principles of equal opportunity and affirmative action. We have since overcome these obstacles and have created a rigorous cell counting pipeline for analyzing images captured by the EVOS scan function. For the purpose of developing our imaging pipeline, we used MCF-7 and MDA-MB-231 breast cancer cell lines labeled with histone-2-beta green-fluorescent-protein (H2B-GFP) [6, 8]. 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. Through this exercise, we encountered multiple problems that severely affected results (Table 1)not all of which we predicted from the outset. To remove these particulates, we it was crucial we filter the FBS-supplemented FluoroBrite medium through a 0.2um filtration unit. 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. 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). We therefore perform many co-culture assays in 96 well dishes and image them using our EVOS system. PMC legacy view One solution we considered was to create cropping masks unique to each well position. 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). Growth was assessed weekly by imaging the plates using the Scan function of the EVOS FL Auto imaging system. The second processing group performs a log contrast transformation of each pixel, which is essential to increase the cell nuclei signal relative to background. Some of these related to how we cultured the cells, such as high background (Fig 2), debris and noise (Fig 3); whereas others were unique to the imaging process, such as Illumination and focus inconsistencies (Figs (Figs44 & 5)all of which interfered with our ability to accurately identify and count cells. Inset is a magnification of the area outlined within the white box. 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). 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. 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, when we created these masks, we found the positions of wells across different plates was also not consistent. There are five objectives available for use: 100X Plan FL 1.28NA Oil, Coverslip Corrected. All images were assembled into a single folder, and the pipeline was applied to generate growth curves for each cell type. The powerful yet easy-to-use EVOS FL Cell Imaging System provides the flexibility to fit most epifluorescence microscopy applications. Hyland Scientifics goal is to provide the scientific community with high quality, pre-owned scientific equipment at an affordable price. An unexpected benefit of this approach is that it also removed condensation artifacts that would sometimes be misidentified as cell (these were bright areas due to condensation on the plates cover). Trusted diagnostic technologies and emerging analytical techniques form the backbone of this important industry. Versions of all the analysis CellProfiler3 pipelines and Matlab code are available for download from: BreastCancerLab.com, http://breastcancerlab.com/evos-cell-counting-pipeline/. Assessing cell counts by imaging has distinct advantages over these other methods. 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. Get Quote from Thermo Fisher Scientific for EVOS FL Cell Imaging System. However, without an automated tool to count cells in acquired images, it becomes the most tedious method of them all. From cell culture to complex protein analysis and multichannel fluorescence imaging, EVOS imaging systems help you perform a variety of routine and specialty applications. CellProfiler 3.0: Next-generation image processing for biology. These composite images have a soft grid pattern due to the lighting artifact (this can be observed in Figs Figs1,1, ,22 and and4).4). 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. These scans create coherent images of each well, but analyzing these images manually (or semi-manually) was a long and tedious process. The data underlying the results presented in the study are available on Github: https://github.com/WCHines/EVOS-Pipeline. There is some slight variance in the thickness of the plates, so perfectly focused images were not expected. Learn More. From fluorescence and electron microscopes, to gel imagers and more, shop LabX for a wide range of products and accessories to fit your needs. 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. During development of the pipeline, we encountered many obstacles. 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. Web Standards | 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. Shop for the latest new and used equipment designed to meet the challenges of this emerging and promising field. To facilitate automated counting, we needed to first optimize experimental conditions to create high quality images of uniform intensity and high contrast. The high number of replicates afforded by this approach reveals the phenotypic variation between wells, as well as between both cell types. Once identified, a mask of the well was created and applied to the greyscale image to remove everything outside of the well. To prevent attenuation of the cell nuclei signal that occurs during the illumination correction preprocessing step, a log contrast transform is applied (in Matlab) after masking the image. The field of neuroscience continues to offer groundbreaking insight into the most complex biological systems. 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. Shop for forensic lab equipment and get the latest trends on new products in forensic science. Applying these masks was removing significant portions of the well and often left edges that interfered with analysis. The site is secure. However, there are instances where using random fields for quantification may be confounding. Here, we present these problems, as well as solutions to each. 8600 Rockville Pike We thank members of our laboratory for their critical reading of this manuscript and useful discussions. These included: high background, illumination and stitching artifacts, low contrast, noise, focus inconsistencies, and image distortionall of which negatively impacted processing efficiency. The current paper represents a series of considerations and suggestions for the use with the EVOS platform and high-throughput imaging and common issues that may be encountered. 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. 1999-2022 Biocompare. HeLa cells expressing Mitochondria RFP were plated on a 6-well Falcon plate. We have overlaid a gray dashed line to indicate the edge of each image. 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. These software programs are powerful tools, but they do have many parameters that can be applied and/or adjusted. Shop now. Shop now for new and used products for materials research and testing applications. 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. LabX keeps pace with this industry by offering a wide range of instruments and solutions for the most demanding questions. 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 thus needed a strategy to attenuate the grid. The final step outlines each cell nuclei and generates a merged image of the outlines(purple outlines) and H2B-GFP fluorescent nuclei (green). 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. This system provides top-of-the-line imaging capabilities for cell-based immaging, such as live-cell analysis, image tiling, and Z-stacking. This was compounded when an imaging field contained few or no cells, causing the machine to search repeatedly for the correct focal plane. We chose to use CellProfiler imaging software because it is open-source, intuitive, and feature rich. 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. We found the log transform in Matlab performed the best (S1 Fig steps 78). 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. 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. The EVOS imaging system contains a scanning algorithm that screens culture plates and creates individual tiled images of each well. Unfortunately, adding FBS to the medium did introduce insoluble particulates that interfered with image processing (Fig 3). Site Development: Digital Strategies (Division of Communications) We submit the current pipeline in hopes that it will assist others with similar applications and goals. We describe an analytical pipeline for quantifying cells within stitched images generated by the EVOS imaging systema method that requires little user input and can be easily adopted for high throughput screening assays. We do not have an onstage incubator for the microscope although it will do time-lapse imaging. 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. Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America. We use cookies on this site to ensure the best service possible. From monoclonal and polyclonal antibodies to reagents, buffers and sera all spanning a wide range of applications.
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