Accurate holographic cytometry using three-dimensional hydrodynamic focusing

Abstract

We present a microfluidic holographic cytometry technique using three-dimensional (3D) hydrodynamic focusing for accurate visualization, classification, and quantification of the cells and particles from a mixture. Our approach uses high-resolution, single-shot digital holographic microscopy to image moving cells and particles in a specially-designed microfluidic device that orders the cells and particles in a single file close to the bottom wall of the channel. Our 3D-focusing microfluidic device allows high-magnification holographic imaging without the need for computationally-expensive numerical refocusing used by the existing holographic cytometry techniques. Our microfluidic device also prevents the clustering of cells and can be fabricated at a low-cost using micromilling. To demonstrate the efficacy of our method, we consider a challenging case of classification from a mixture of unstained red blood cells and polystyrene particles, which are otherwise indistinguishable in brightfield and phase-contrast microscopy. Through experiments with cell-particle mixtures with varying proportions, we show that our holographic cytometry technique can precisely count and classify the cells and particles based on their reconstructed phase values. Our holographic cytometry technique has the potential for label-free classification and quantification of infected cells for applications such as disease diagnostics, cancer research, and genomics.

Squeezed state in the hydrodynamic focusing regime for Escherichia coli bacteria detection

“…18–22 In addition, hydrodynamic focusing also improves clogging problems by enclosing the core sample solution with the fluid cladding wall, serving as a critical fluidic functionality that runs in flow cytometers to compress and transport sample solutions. 23,24…”

“…[18][19][20][21][22] In addition, hydrodynamic focusing also improves clogging problems by enclosing the core sample solution with the fluid cladding wall, serving as a critical fluidic functionality that runs in flow cytometers to compress and transport sample solutions. 23,24 With the mature development of microfluidic technology, various microfluidic cytometers were demonstrated, aiming to replace bulky flow cytometer machines with lab-on-a-chip devices to adapt to point-of-care settings. [25][26][27][28] One of the most common designs is 2-dimensional (2D) hydrodynamic focusing with three microchannels serving as the inlets that are combined to a single focusing microchannel as the outlet.…”