# Experimental Study on Multi-Cell Counting Using an Inertial Microfluidic Device
###### Abstract
proposed a microparticle counting method using a metal mesh device (MMD) to trap particles above a certain size [12]. The MMD sensor effectively collects and detects particulate matter (PM) through size fractionation, but it primarily captures larger particles. As a result, smaller particles may not be detected efficiently, which highlights a limitation in the method's ability to monitor all PM sizes [13]. [PERSON] et al. explored various optical detection methods, highlighting the use of techniques like fluorescence and absorption while also addressing the challenges of integrating multiple functionalities on a single microfluidic platform [14]. Optical and electrical detection methods both offer significant advantages in microfluidic cell analysis. Electrical detection methods, such as electrochemistry and Coulter counting, provide high sensitivity without the need for labeling [15], while optical methods like spectroscopy and photometric approaches offer label-free detection with simpler equipment, suitable for point-of-care testing [16]. [PERSON] et al. introduced an acoustic flow cytometer that combines microfluidics with high-frequency ultrasound for non-invasive cell size measurement [17]. Proper focusing in microfluidic channels is crucial for accurate cell analysis, achieved through methods such as sheath flow, dielectrophoretic, acoustic, inertial, and hydrodynamic focusing [18]. Hydrodynamic focusing, in particular, allows for the precise control of particle positioning within microfluidic channels, facilitated by factors like channel geometry, flow rates, and Reynolds number [19]. The fabrication of microfluidic devices is accomplished through techniques such as photolithography, laser ablation [20], 3D printing, and soft lithography [21]. Soft lithography, as explored by [PERSON] et al., is a widely used method known for its simplicity and effectiveness in creating microscale features. Traditional cell counting techniques, such as hemocytometers and flow cytometry [22], face significant challenges that hinder their effectiveness in various applications. Hemocytometers are labor-intensive, time consuming, and require skilled personnel, leading to variability in results. Furthermore, they are not well-suited for high-throughput scenarios, making them inefficient for processing large sample volumes [23]. Despite the recent advancements in microfluidic technologies for particle manipulation, there is a gap in the development of a reliable, image-based microfluidic platform capable of counting multiple sized cells at clinically relevant ratios. Most prior research has focused on high-end technologies for single-cell analysis or utilized complex instrumentation unsuitable for limited resource communities. In this study, we present an image-based technique using an inertial microfluidic device for the multi-size microparticle counting of normal and diseased cell conditions. The microchannel dimensions and flow rates were selected based on the literature review, and full factorial analysis was conducted to enhance the particle counting accuracy based on the principles of inertial microfluidics. This device offers a comparable counting accuracy to conventional techniques like hemocytometers, suitable for conditions such as anemia, infections, and autoimmune disorders. The experiments aimed to identify the optimal device parameters, such as geometry of 150 \(\upmu\)m and the flow rate at 1 \(\upmu\)L/min, varying the cell size ratios that maximize counting accuracy while also validating the results against traditional hemocytometer measurements. The proposed integrated inertial microfluidic system streamlines and automates the entire process of counting microparticles, making it more accessible and affordable at the point-of-care.