Single channel layer, single sheath-flow inlet microfluidic flow cytometer with three-dimensional hydrodynamic focusing

Abstract

Flow cytometry is a technique capable of optically characterizing biological particles in a high-throughput manner. In flow cytometry, three dimensional (3D) hydrodynamic focusing is critical for accurate and consistent measurements. Due to the advantages of microfluidic techniques, a number of microfluidic flow cytometers with 3D hydrodynamic focusing have been developed in recent decades. However, the existing devices consist of multiple layers of microfluidic channels and tedious fluidic interconnections. As a result, these devices often require complicated fabrication and professional operation. Consequently, the development of a robust and reliable microfluidic flow cytometer for practical biological applications is desired. This paper develops a microfluidic device with a single channel layer and single sheath-flow inlet capable of achieving 3D hydrodynamic focusing for flow cytometry. The sheath-flow stream is introduced perpendicular to the microfluidic channel to encircle the sample flow. In this paper, the flow fields are simulated using a computational fluidic dynamic (CFD) software, and the results show that the 3D hydrodynamic focusing can be successfully formed in the designed microfluidic device under proper flow conditions. The developed device is further characterized experimentally. First, confocal microscopy is exploited to investigate the flow fields. The resultant Z-stack confocal images show the cross-sectional view of 3D hydrodynamic with flow conditions that agree with the simulated ones. Furthermore, the flow cytometric detections of fluorescence beads are performed using the developed device with various flow rate combinations. The measurement results demonstrate that the device can achieve great detection performances, which are comparable to the conventional flow cytometer. In addition, the enumeration of fluorescence-labelled cells is also performed to show its practicality for biological applications. Consequently, the microfluidic flow cytometer developed in this paper provides a practical platform that can be used for routine analysis in biological laboratories. Additionally, the 3D hydrodynamic focusing channel design can also be applied to various applications that can advance the lab on a chip research.

A Contact-Imaging Based Microfluidic Cytometer with Machine-Learning for Single-Frame Super-Resolution Processing

“…The recent advance of microfluidics-based lab-on-a-chip technology has introduced the possibility for the miniaturized microflow cytometer for potable flow cytometry [9]–[11], [14]–[15]. With the integration of complementary metal oxide semiconductor (CMOS) image sensor chip underneath the microfluidic channel, microfluidics-based lensless imaging systems [16]–[22] can be developed for portable contact-imaging [23] based microflow cytometer.…”

Pulsed laser activated cell sorter (PLACS) for high-throughput fluorescent mammalian cell sorting

“…Several sheathless 3D cell-focusing mechanisms have been demonstrated so far. These include focusing using acoustic pressure 9, 10 , optical 11,12 , magnetic 13 , and dielectrophoretic forces 12, 14 , hydrophoresis 15,16 and inertial focusing [17][18][19][20] . Among these, inertial focusing is a promising sheathless mechanism for integration with PLACS since both can operate at high speed (> 1 m sec -1 ).…”

Monolithic cell counter based on 3D hydrodynamic focusing in microfluidic channels

“…Furthermore many of the device presented above use three or even more inlets to implement the 3D focusing, while few inlets, enhancing the simplicity of the operations, are to be preferred. In addition, they usually provide asymmetric focusing in the two directions, with the focused sample showing an elliptical cross-section 8 and in several cases flowing close to one edge of the microchannel and not in its center 13 . A different fabrication technique with important advantages with respect to the standard lithographic approaches is femtosecond laser micromachining (FLM) 14 followed by chemical etching.…”