Abstract
In this paper, we present a novel cell counting method accomplished using a single-cell array fabricated on an image sensor, complementary metal oxide semiconductor sensor. The single-cell array was constructed using a microcavity array, which can trap up to 7,500 single cells on microcavities periodically arranged on a plane metallic substrate via the application of a negative pressure. The proposed method for cell counting is based on shadow imaging, which uses a light diffraction pattern generated by the microcavity array and trapped cells. Under illumination, the cell-occupied microcavities are visualized as shadow patterns in an image recorded by the complementary metal oxide semiconductor sensor due to light attenuation. The cell count is determined by enumerating the uniform shadow patterns created from one-on-one relationships with single cells trapped on the microcavities in digital format. In the experiment, all cell counting processes including entrapment of non-labeled HeLa cells from suspensions on the array and image acquisition of a wide-field-of-view of 30 mm2 in 1/60 seconds were implemented in a single integrated device. As a result, the results from the digital cell counting had a linear relationship with those obtained from microscopic observation (r2 = 0.99). This platform could be used at extremely low cell concentrations, i.e., 25–15,000 cells/mL. Our proposed system provides a simple and rapid miniaturized cell counting device for routine laboratory use.
Highlights
Today, cell counting is one of the most commonly performed routine laboratory tests in the field of cell biology
Materials The complementary metal oxide semiconductor (CMOS) sensor composed of 204861536 pixels in an area of 6.55 mm64.92 mm was used for imaging (DFK61BUC02; Imaging Source Europe GmbH; Bremen, Germany)
In contrast to the raw image, the area-size histogram generated from the masked image had a narrow peak at around 350 pixels corresponding to the area size of the uniform block consisting of 19619 pixels (Fig. 5f). These results indicate that it was possible to perform automated counting of individual cells based on shadow patterns extracted from a CMOS sensor image
Summary
Cell counting is one of the most commonly performed routine laboratory tests in the field of cell biology. Various types of desktop-sized automated cell counters including impedance-based [1,2] and image-based counters [3,4] have been developed and commercialized for routine laboratory use. These cell counters have been designed to reduce both operator error and the labor required for manual cell counting. In an imagebased cell counter, cell concentration is calculated from several microscopic images obtained by automated microscopy. Single cells are morphologically distinguished from debris or cluster from the images and the cell concentrations are calculated from the number of single cells identified in microscopic area. The ability to count small number of cells is becoming increasingly necessary to expand the utility in laboratories especially when using limited amounts of biological samples or preparing of cell standards for counting rare cells (e.g. circulating tumor cells or hematopoietic stem cells) [5]
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