Fabrication of digital cell counting device based on a CMOS sensor integrated with a microcavity array.

<p>(a, b) Schematic diagrams of shadow-imaging platform. (a) Only the light transmitted through the microcavities positioned above the CMOS sensor is received at the sensor surface. (b) When cells are trapped on the microcavities, the light is attenuated by the trapped cells. (c, d) Schematics of the expected CMOS sensor images acquired before (c) and after cell entrapment (d).</p

Digital Cell Counting Device Integrated with a Single-Cell Array

<div><p>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.</p></div

Calculated light intensity received at the sensor surface.

<p>(a) Schematic model of the shadow imaging. (b) Simulated profile of light intensity received at the sensor surface below the microcavity array. The black line indicates a profile with no cells present in the closely aligned microcavities. The red line indicates a profile with a single cell trapped on one of the closely aligned microcavities. <i>I₀</i> was defined as 1.0. The light through the trapped cell was assumed to be attenuated by 100%.</p

Evaluation of cell counting accuracy.

<p>CMOS sensor image (a) and fluorescent micrograph (b) of HeLa cells trapped on a microcavity array. Scale bar; 60 µm. (c) Cell count measured by CMOS sensor images compared with the count of HeLa cells stained with Hoechst 33342 in the fluorescent micrograph. Plot area in lower range (red box) is enlarged and shown on the top left side.</p

Effects of wavelength and distance between the microcavity array and sensor surface on shadow patterns.

<p>(a, c) Parts of the CMOS sensor images corresponding to selected rows of microcavities acquired under illumination at various wavelengths in constant distance: 1,095 µm (a) and at various distances in constant wavelength: 365 nm (c). Three of the seven microcavities in a row (a) and two of the eight microcavities in a row (c) were occupied by microparticles. (b, d) Cross-sectional variation in light intensity was measured in each image.</p

Digital cell counting device composed of a CMOS sensor and a microcavity array.

<p>(a) Photograph of the device. (b) Micrograph of the microcavity array surface. Scale bar; 60 µm. (c) Side-sectional view of the digital cell counting device.</p

Image processing for cell counting.

<p>(a, b) The raw CMOS sensor image (a) and image merged with a pattern mask to be divided into uniform blocks (b) were processed by threshold adjustment and size extraction. (c, d) The shadow patterns were extracted from the raw image (c) and the masked image (d). Figures in red within (c, d) indicate the serial number of the extracted patterns. (e, f) Size histograms of the extracted shadow patterns were also generated.</p