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TOWARDS AUTOMATED HIGH CONTENT SCREENING ON A 672-MICROWELL SLIDE Emilie Weibull 1* , Sara Lindström 2,3 , Anna Segerman 4 and Helene Andersson-Svahn 1, 2 1 Division of Nanobiotechnology, Royal Institute of Technology, Albanova University Center, SWEDEN, 2 Picovitro, SWEDEN, 3 Cell and Molecular Biology, Karolinska Institute, SWEDEN and 4 Department of Genetics and Pathology, Rudbeck Laboratory, SWEDEN ABSTRACT We present an automated high content screening method for single-cell analysis on a microwell slide. The 672 wells are screened in 73 minutes, i.e. a 10-fold increase, yielding a negative/positive response using an automated confocal software. This screening method is highly sophisticated, rapid and results in high-resolution imaging. The next step is to apply the method to a heterogeneous glioma cell line using a stem cell- and astrocyte marker to study the link between marker expression and proliferation rate on single cells. The resulting images are input files in a cell analyzing software to attain information about cell density, co-localization, cell size and cell count. KEYWORDS: High Content Screening, Automated Cell Analysis, Microwell Slide, Single-Cell INTRODUCTION In high-throughput single-cell analysis the following features are often required for an efficient analysis of large numbers of individual cells: 1) fast, controlled and simple cell seeding, 2) short- or long term culturing and 3) live-cell imaging with an end-point option to screen for fluorescence expression/labels. Existing systems often lack one or more of these features limiting the true advantage of single-cell analysis. We have earlier presented a slide with methods for step 1 and 2 [1]. However, step 3 has so far been manually performed, i.e. time- and work intensive [2]. Here, we pres- ent the missing 3 rd step: an automated high content screening method for single-cell analysis on the previously presented microwell slide. A confocal microscope with an automated software called MatrixScreener was used with possible 4-channel screen- ing of all 672 wells. The high-throughput single-cell method enables a rapid and comprehensive method for detecting e.g. cell heterogeneity. The name of the raw image is connected to its position on the slide and the specific channel. This metadata is then used for keeping track of the images in the cell analyzing software. THEORY The confocal software MatrixScreener is used to create a 14 x 48 matrix, where every matrix point symbolizes a well on the slide. The matrix points have constant and identical proportions along the x, y axis and the hypotenuse is 1500 µm. The home position is adjusted after installing the slide in the confocal microscope. The range of the autofocus is set to 150 µm (maximum range 500 µm) which is scanned in 30 steps (z-axis). It is possible to choose which wells to scan and whether autofocus should be applied before or during scanning of the slide. If done before, only certain wells needs to be autofocussed to create a morphology map of the slide. After scanning the slide, the images and metadata are transferred and processed by the software Cellprofiler [3]. For each image, automatic calculations of the number of cells per well correlated to marker expression in single cells or clones can then easily be obtained. Cellprofiler identifies cells’ primary structures (nuclei) and with the help of proxim- ity to the nucleus the intensities of e.g. the marked Endoplasmatic Reticulum (ER) or cytoplasm can be identified. Cell- profiler also keeps track of the images’ positions, and places all images (multiple channels) from a specific well in an im- age set. This enables co-localization studies of proteins between cell clones or cells in different wells i.e. cell heterogeneity. The software compares the identified areas between different channels in a set. EXPERIMENTAL The previously presented microwell slide holds 672 wells in a 14 x 48 format and has a format of a standard micro- scopic slide (figure 1a-b). The microwell slide was coated with fibronectin whereupon epithelial carcinoma cells were seeded on the slide [1]. Cell growing conditions were maintained by covering the slide with a thin polydimethylsiloxane membrane and cultured for 24h, followed by immunostaining [4] with a slightly modified protocol to match the minia- turized format [2]. To facilitate high content screening of the slide a confocal microscope with a screening software was used for auto- mated imaging. The slide was placed on a motorized table (x, y and z directions) on the microscope. Each well was im- aged separately whereby the images were put together to reassemble the slide (figure 1c), showing the negative/positive (n/p) response in 672 wells in a straightforward manner. The software enables multichannel screening and overlay possibilities to identify e.g. co-localized proteins. It is possible to screen for up to 4 markers simultaneously. An op- tional pre-screening autofocus function creates a morphology map used for fast n/p response. The autofocus function can also be run on all wells when detailed cellular information is essential. 978-0-9798064-3-8/μTAS 2010/$20©2010 CBMS 965 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 3 - 7 October 2010, Groningen, The Netherlands
