Accepted Manuscript

Title: Review and Perspectives on Microfluidic Flow

Cytometers

Authors: Ruey-Jen Yang, Lung-Ming Fu, Hui-Hsiung Hou

PII: S0925-4005(18)30581-1
DOI: https://doi.org/10.1016/j.snb.2018.03.091
Reference: SNB 24376

To appear in: Sensors and Actuators B

Received date: 23-11-2017

Revised date: 22-2-2018
Accepted date: 15-3-2018

Please cite this article as: Ruey-Jen Yang, Lung-Ming Fu, Hui-Hsiung Hou, Review
and Perspectives on Microfluidic Flow Cytometers, Sensors and Actuators B:
Chemical https://doi.org/10.1016/j.snb.2018.03.091

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A Revised Paper Submitted to

Sensors and Actuators B: Chemical

Review and Perspectives on Microfluidic Flow Cytometers

by

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Ruey-Jen Yang1, Lung-Ming Fu2,3*, Hui-Hsiung Hou1

1
Department of Engineering Science,

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National Cheng Kung University, Tainan, 70101, Taiwan
2
Graduate Institute of Materials Engineering,

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3
Department of Biomechatronics Engineering,
National Pingtung University of Science and Technology, Pingtung 912, Taiwan

Corresponding author: Prof. Lung-Ming Fu

e-mail: loudyfu@mail.npust.edu.tw
Tel: +886-8-7703202-7553 U
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Tel Fax: +886-7740552
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Highlights
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 Microflow cytometers are one of the most powerful approaches for cells /
particles analysis.
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 The review focuses on proposals for microfluidic focusing techniques and

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detection / analysis methods.

 A comprehensive review of the main applications of microflow cytometers over

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the past ten years.

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ABSTRACT

Modern microflow cytometers are sophisticated instruments capable of measuring

multiple physical characteristics of a single cell simultaneously as the cells flow in

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suspension through a measuring device. Cytometers are the tool of choice for the

high-speed acquisition and analysis of large single cell populations. However,

traditional devices lack the ability to provide intracellular spatial information. In the

past few decades, various flow cytometer systems with the ability to combine cell /

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particle detection and the acquisition of two or three-dimensional spatial information

have been proposed. However, these devices suffer a number of drawbacks

Accordingly, the problem of developing more sophisticated microflow cytometers

based on electrical impedance, optical detection and image analysis methods has

received significant attention in the literature. This review describes some of the

major advances made in the microfluidic cytometry field over the past ten years. The

review focuses specifically on recent proposals for enhanced microfluidic focusing

techniques and detection / analysis methods, respectively. Overall, the review

provides a useful insight into the microflow cytometer technology field for both new

and existing users.

Keywords: microfluidic; microflow; cytometer; cytometry.

1. Introduction

Flow cytometry is widely used throughout the life science and clinical diagnosis

fields for the characterization and analysis of cells. Traditional cytometers exploit the

forward or side-scattering properties of particles and / or the fluorescence intensities

of attached probes, and typically enable cells to be detected. The traditional flow

cytometers are often bulky and mechanically complicated and required specialists to

operate the devices. The conventional flow cytometers can hardly detect particles

smaller than 0.5 micron in diameter via light scattering measurement. With the rapid

advancement of micro-electro-mechanical systems (MEMS) technology in recent

decades, microfluidic chips or Lab-on-a-Chip (LOC) devices have emerged as a

powerful technique for the rapid in situ detection of small amounts of samples in the

chemistry, biology, food, medicine and environmental monitoring fields [1-9].

Microchip-based flow cytometers are an extremely important class of LOC device,

and have received extensive attention in the literature [10,11]. Many functioning

devices have been reported; with those intended for point-of-care (POC) applications
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being particularly common [12-15]. Compared to their conventional counterparts,

microfluidic cytometers have a greatly reduced reagent / sample volume consumption

and a far lower cost. The typical sample volume is in the range of 10-9 and 10-18 liters.

The volume of high cost reagents is therefore greatly reduced. The microfluidic flow

cytometers are portable and disposable. In addition, through their integration with

other LOC devices, they provide the opportunity for parallel processing and the

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automation of complete chemical processes with fewer handling steps and associated

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errors and costs [16]. Notably, modern MEMS techniques make possible the

realization of microfluidic cytometers capable of detecting and analyzing single cells

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smaller than 0.5 micron in diameter with a high sensitivity [17-19].

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In traditional flow cytometers [20-22], the cells are suspended in solution and

injected into the fluidic system, whereupon they are hydrodynamically focused by a
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sheath flow such that they travel through the center of the fluidic channel at a uniform
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velocity. Upon arrival at the interrogation zone, the cells are individually interrogated
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by a focused laser beam passing perpendicularly across the channel. For each cell, the
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cytometer system collects both forward light scatter (FSC) and orthogonal (side) light

scatter (SSC) intensity information. The FSC information is used to characterize the
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particle size and refractive index, while the SSC information is used to investigate the

internal structure of the cell.

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Single cell analysis is of great importance in performing the biological assay of

highly heterogeneous cell populations, such as human blood [23,24]. Microflow

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cytometers provide the means to investigate not only whole cells, but also cellular
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components, such as organelles, nuclei, DNA, RNA, chromosomes, cytokines,

hormones and proteins [23]. Furthermore, microfluidic cytometers provide an

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efficient technique for detecting the membrane, cytoplasmic and nuclear antigen

properties of biological samples [24]. As a result, they play a critical role in many

biological and medical applications.

This review provides a historical perspective on the development of microfluidic

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cytometers. Particular emphasis is placed on recent advances in the microfluidic

focusing systems and detection methods used in such devices. The related literature is

systematically reviewed and discussed, and potential real-world applications

described where appropriate. The review concludes with a brief perspective on further

likely developments within the field over the coming years and decades.

2. Microfluidic focusing systems for flow cytometers

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In conventional microfluidic cytometers, the potential for two or more particles

to enter the optical interrogation region simultaneously is minimized by focusing the

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sample flow into a very narrow stream by means of a surrounding sheath flow stream.

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Importantly, microfluidic focusing not only confines the particles to a single-file

stream, but also ensures a uniform particle velocity by removing the contact between
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the sample flow and the flow cell walls, thereby eliminating the parabolic flow profile
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that would otherwise exist. Such an effect is extremely beneficial since variations in
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the particle velocity not only undermine the reliability of the detected signals, but can
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also cause synchronization problems downstream for cell sorting. As a result, the

development of effective microfluidic focusing modules is crucial in ensuring the

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performance of microflow cytometers. The fundamentals of the focusing flow system

can be referred to our earlier publications [25-27]. As shown in Table 1, many

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focusing methods based on two-dimensional (2-D), three-dimensional (3-D),

dielectrophoresis, acoustic, and inertial techniques have been proposed. The details of
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these proposed methods are described in the following sections.

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2.1 Two-dimensional microfluidic focusing systems

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2-D microfluidic focusing techniques are among the most commonly used for

particle confinement and have been applied in many microfluidic cytometer

applications [28-41]. 2-D focusing methods have the advantage of simplicity.

Consequently, the associated microchannel geometries can be easily mass produced at

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low cost using standard fabrication techniques such as replica molding and soft

lithography. However, in 2-D focusing methods, the sample stream can only be

focused in one plane (typically, the horizontal plane).

Akagi et al. [29] developed a microflow cytomer based on a simple 2-D flow

focusing module for the multivariate real-time analysis of live cells. It was shown that

the 2-D focusing effect, while relatively simple, was sufficiently precise to facilitate

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the cellular DNA content measurement of live tumor cells using DRAQ5 DNA probe.

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Moreover, the feasibility of the proposed system for the dose-response profiling of

anti-cancer agents on human hematopoietic cancer cells was also demonstrated.

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Skommer et al. [30] presented a microflow cytometer with a simple 2-D

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hydrodynamic focusing technique for the multi-parameter analysis of apoptosis in

relation to the cell cycle position. The device enabled up to six parameters to be
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detected by means of spatially separated solid-state 473 nm (10 mW) and 640 nm (20
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mW) lasers driven by X-Y stages. The practical applicability of the proposed system
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was demonstrated by analyzing the caspase activation and dissipation of the
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mitochondrial inner membrane potential (Δψm loss) of live tumor cells in relation to

their DNA content. Akagi et al. [31] presented a planar-flow chip-based cytometer for
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the multi-parameter analysis of programmed dead tumor cells, in which the cells were

focused into a single-file stream using an air-over-liquid system (Figs. 1(a) and 1(b)).
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The results confirmed that the proposed cytometer provided the means to track the

pharmacological dose-response profiling of anti-cancer agents using sample volumes

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as low as 10 µl (Fig. 1(c)). Erickson and Jiminez [32] developed a 2-D hydrodynamic
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focusing microfluidic cytometer for measuring the forward light scatter, chlorophyll

fluorescence induction and lipophilic stain fluorescence of algal cells at a rate of 100
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cells/s. The cytometer was used to evaluate the photoenergy conversion of unstressed

(nutrient replete) and stressed (nutrient limited) P. tricornutum cells. It was shown that

the stressed cells yielded a lower photoenergy conversion and a higher Nile Red (NR)

fluorescence than the unstressed cells.

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 Choi et al. [33] presented a flow cytometer system for the sensitive, accurate and

rapid detection of pathogenic bacteria. In the proposed device, the samples were

pushed hydrodynamically toward pyrolytic graphite edge (PGE) electrodes by a

virtual wall solution with extremely low ion conductivity (Fig. 2(a)). The

experimental and simulation results showed that the effective channel width of the

device could be controlled by adjusting the flow rate of the wall system. Consequently,

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the device had the ability not only to detect micron-sized particles and bacteria, but

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also to distinguish between them. Huang et al. [34] developed an integrated

microfluidic system for sorting motile human sperm consisting of laminar

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stream-based microchannels, a 2-D hydrodynamic side-focusing module, and a flow

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cytometer (Fig. 2(b)). Briefly, the semen samples were stained by SYBR-14 (green

fluorescence) and PI (red fluorescence) dye and injected into the microfluidic chip.
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The viability and motility of the sperms within the sample were then assessed based
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upon differences in the interactions between dead and live sperm and the laminar flow
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within the channel. The viability results of sample injected from two-outlet and
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four-outlet chip were 95.2% and 92.2% that presented the higher quality of sorting

sperm. The results confirmed that the microfluidic system provided an effective and
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low-cost solution for selecting the most appropriate sperms for IVF (in vitro

fertilization) treatment. Table 2 presents a brief overview of other 2-D microfluidic

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focusing systems presented in the literature.

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2.2 Three-dimensional microfluidic focusing systems

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3-D microfluidic focusing systems focus the sample flow in both the horizontal

direction and the vertical direction, and consequently improve the accuracy and
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reliability of the detected signals. However, due to the planar property of chip-based

microfluidic devices, achieving on-chip 3-D focusing is more difficult than 2-D

focusing. Traditionally, 3-D hydrodynamic focusing was achieved using

multiple-layer chip designs, in which the sample stream is confined to the central flow

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path by means of sheath flows both above and below the sample and to its left and

right (Fig. 3(a)) [42-48]. However, in recent years, many new techniques for 3-D

microfluidic focusing have been proposed, including horizontal focusing + secondary

flow on the vertical, horizontal focusing + groove-generated sheath effect on the

vertical, horizontal focusing + micro-weir effect on the vertical, horizontal focusing +

dielectrophoresis on the vertical, and structure effect-induced Dean flow (Figs.

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3(b)-3(e)) [49-65]. 3-D microfluidic focusing offers the potential to develop novel

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sensors, cytometers and sample-processing modules with a superior performance.

Consequently, the use of fluid dynamics and specialized microchannel designs to

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achieve 3-D microfluidic focusing has attracted significant attention in the literature.

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Fu et al. [46] developed a 3-D focusing microflow cytometer based on

hydrodynamic forces and a micro-weir structure applied in the vertical direction. The
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experimental results showed that the proposed device successfully separated 5-μm
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and 10-μm diameter polystyrene beads in the vertical direction. It was further shown
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that the separation of the beads led to their sequential flow through the interrogation
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region of the device, and thus improved the performance of the detection process. The

same group [47] later presented a 3-D focusing sheathless microflow cytometer in
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which the sample stream was progressively focused along the centerline of the

channel by the Saffman shear lift force developed by a series of micro-weirs of

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gradually reducing height. The device was shown to achieve a focusing performance

of approximately 99.76% and 99.57% for polystyrene beads with diameters of 5 µm

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and 10 µm, respectively.

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Lee et al. [52] proposed a 3-D focusing microflow cytometer in which the sample

stream was focused initially in the horizontal plane by two sheath flows and then in
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the vertical plane by a series of staggered micro-weirs with a gradually reducing

height (Fig. 3(c)). The performance of the proposed device was evaluated by counting

a mixed sample of 7 and 15 µm fluorescent polystyrene beads. The results revealed

that the device was capable of achieving a detection throughput of at least 60

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particles/s. Frankowski et al. [63] developed two microfluidic chips for blood cell

analysis applications, in which the 3-D hydrodynamic focusing effect was achieved

using two different approaches, namely two-stage cascade focusing and spin focusing

(vortex), respectively. In the first technique, the sample stream was confined to the

central region of the channel using a cascaded arrangement of channels with different

heights. By contrast, in the second technique, focusing was performed by means of a

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spinning sheath flow (Fig. 3(f)). The potential of the proposed cytometer was

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demonstrated by detecting immunologically labeled CD3 positive and CD4 positive

T-lymphocytes in blood. Table 2 summarizes the other 3-D microfluidic focusing

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systems presented in the literature.

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2.3 Dielectrophoresis microfluidic focusing systems

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Dielectrophoresis (DEP) is one of the most widely used techniques for the
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microfluidic manipulation of particles, cells, viruses, DNAs and other objects [66-70].
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DEP is also extensively used for trapping, patterning, focusing, separation,
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transportation and deposition purposes in a variety of bio-sensing applications [71-75].

The DEP force depends on the particle size, the dielectric properties of the particles
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involved, and the medium in which the particles are transported (Fig. 4(a)). Moreover,

in the case of an AC field, the DEP force varies as a function of the frequency.
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Depending on the dielectric properties of the medium and particle, respectively, the

DEP response can be switched from negative to positive, where the frequency at
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which this transition occurs is known as the crossover frequency.

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Lin et al. [66] proposed a 3-D focusing effect based on a combination of

hydrodynamic forces and a negative DEP force applied in the vertical direction. The
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feasibility of the proposed device for practical applications was demonstrated using

both micro polystyrene beads with diameters of 10 and 20 µm and diluted human

RBCs. The experimental results confirmed that the DEP force improved the

uniformity of the detected signal amplitude and therefore enhanced the accuracy and

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reliability of the detection results. Electrode-based dielectrophoretic focusing

approaches were traditionally implemented using negative DEP forces [75,76].

However, in a more recent study, Wang et al. [77, 78] presented a method for the

separation of beads and cells using two vertical interdigitated electrode arrays

embedded in the sidewalls of the microchannel (Fig. 4(b)). The microelectrode arrays

not only extended the region of the DEP effect, but also induced a non-uniform

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electric field in the direction of the channel width. By adjusting the frequency and

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amplitude of the AC signals applied to the electrodes, objects with different DEP

properties were driven to different positions across the channel width and

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subsequently directed to different collection outlets. The potential of the proposed

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device was demonstrated by separating human kidney cells (HEK293) from N115

mouse neuroblastoma cells as well as polystyrene microbeads from modified HEK293

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cells. Chu et al. [79] presented a 3-D particle focusing microchannel using a positive
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DEP effect and a dielectric structure placed between two symmetric planar electrodes.
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As shown in Fig. 4(c), particles experiencing positive DEP (through tuning the AC
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voltage frequency) are forced to travel along the top edge of the dielectric structure

and then focused to the centerline of the microchannel in the pressure-driven laminar
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wake of the structure. The experimental results obtained using 2 mm diameter

polystyrene beads showed that a focusing efficiency of 90% was achieved within 2
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mm given an applied voltage of 15 Vp-p and a flow rate of up to 0.01 ml/min. Choi et

al. [80] later optimized the geometry of the dielectric structure through numerical
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modeling.
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Shaker et al. [81] proposed an impedance-based microflow cytometer for single

cell morphology discrimination in which a negative DEP force generated by liquid

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electrodes was used to focus the cells at the center of the channel and impedance

measurements obtained in the longitudinal and transverse directions were used to

derive an index describing the particle anisotropy (Fig. 5(a)). Nikolic-Jaric et al. [82]

developed a DEP cytometer based on a differential electrode array (Fig. 5(b)). As

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each cell flowed over the array, the DEP force varied in accordance with the

biological properties and physiological state of the cell. Consequently, a measurable

change in the capacitance occurred, from which the apoptosis state of the cell could

then be inferred. Thomson group [83-87] recently developed a series of DEP-focusing

cytometers based on various detection techniques, including conductivity, sensitivity

and dielectric. Table 2 summarizes some of the other proposals presented in the

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literature for DEP-based microfluidic focusing cytometers.

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2.4 Microfluidic acoustic focusing systems

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Acoustic actuation is capable of generating large forces on particles in a

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microfluidic system. Under the effects of acoustic actuation, the particles move

toward either the pressure nodes or the antinodes, depending on the density and
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compressibility of the particle and the medium within which they are carried.
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Furthermore, by actuating a series of interdigital transducers on a piezoelectric
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material at their resonant frequency, the particles can be forced to travel across the
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surface, thereby creating a sample transport effect [88-91].

The literature contains many proposals for microfluidic actuation using either
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traveling surface acoustic waves (TSAWs) [92-94] or standing surface acoustic waves

(SSAWs) [95-98]. SAW-based devices have several important advantages for

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nanoparticle manipulation, including a wide operating frequency range (e.g.,

∼10–1000 MHz, corresponding to acoustic wavelengths from ∼4–400 μm) and good
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biocompatibility [99,100]. Moreover, the actuation force propagates within the fluid
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itself, and hence the clogging and poor yields associated with physical separation

methods such as nanopore filters are avoided. Recent studies have shown that acoustic
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fields not only provide an effective means of achieving sample transport at the

microscale, but can also be used to perform efficient sample focusing. For example,

Collins et al. [101-103] used a set of interdigital transducers to produce narrow

acoustic beams with a width of just ∼10–20 μm (Fig. 6(a)). Nawaz et al. [104]

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demonstrated an acoustofluidic fluorescence activated cell sorting (FACS) device (Fig.

6(b)) in which the cells were focused three-dimensionally using a “microfluidic

drifting” method and then sorted using short bursts of SSAWs. It was shown that the

proposed device not only achieved a sorting efficiency of more than 92% for a mixed

sample of labeled and unlabeled HeLa cells, but also maintained a post-sort cell

viability of more than 99%.

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Acoustic focusing microfluidic cytometers have been successfully applied for

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the counting and sorting of many different particles, cells and viruses [105-112]. Chen

et al. [107] presented a SSAW-based microfluidic cytometer in which the analytes

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were concentrated by a 3-D focusing field produced by dual IDTs and detected in the

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downstream region using a laser induced fluorescence (LIF) technique (Fig. 7(a)).

The results obtained using calibration beads showed that the proposed device
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achieved a coefficient of variation less than 10% at a throughput of ~1000 particles/s.
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Grenvall et al. [108] presented an acoustic focusing microfluidic chip for the
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sheathless positioning of cells and particles and subsequent sorting and counting using
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a planar electrode Coulter-type impedance spectrometer (Fig. 7(b)). The proposed

device was used to analyze both single and mixed size bead suspensions as well as
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diluted whole blood samples. The results were shown to be in good agreement with

those obtained using a conventional benchtop Coulter counter. Zmijan et al. [110]
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proposed an imaging flow cytometer based on the use of ultrasonic standing waves to

focus the particles onto the image plane of a high-resolution CMOS camera (Fig. 7(c)).
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The device achieved a throughput of 208,000 beads/s given a camera frame rate of 80
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fps. Moreover, throughputs of 52,350 and 60,400 cells/s were obtained for ATDC5

(pre-chondrocytic) cells and primary leukemia cells, respectively. Table 2 summarizes

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the other proposals for microfluidic acoustic focusing cytometers.

2.5 Microfluidic inertial focusing systems

Inertial focusing methods utilize cross-stream particle motion to focus the

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particles into one or multiple streams. Inertial focusing can be induced in many

different types of channel, including straight microchannels [113-115], curved

microchannels [49-51, 116-119], and specific-to-type (e.g., groove-structure)

microchannels [56-59,120-123]. In general, the particles / cells in a channel flow

experience both a shear gradient force and a wall effect force, and particle / cell

focusing occurs when a balance between these two forces occurs. For straight

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microchannels, the symmetrical cross-section results in four equilibrium regions (Fig.

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8(a)). In curved channels, the inertia effect produces two counter-rotating vortices

perpendicular to the primary flow direction (Fig. 8(b)). These vortices generate a drag

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force, known as the Dean force, which acts on the particles / cells and drives them to

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points of equilibrium outside the vortex structure. Finally, in groove-structure

microchannels, the vortex flow structure induced in the cross-section of the

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microchannel exerts a drag force on the particles / cells, and a focusing effect occurs
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at the point where the shear gradient force, wall effect force and drag force reach
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equilibrium with one another (Fig. 8(c)).
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Many inertial focusing microfluidic cytometers have been presented for the

counting and sorting of particles, cells, viruses, and other objects [124-129]. In a
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recent study, Oakey et al. [124] fabricated a staged inertial microfluidic device

comprising both curved and straight microchannels to confine particles into a single
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streamline without the need for a sheath flow. It was shown that the resolution of the

proposed device was comparable to that of a commercial cytometer with

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hydrodynamic focusing. Moreover, the effectiveness of the device was found to

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improve with an increasing flow rate; thereby improving its potential for high

throughput analysis applications. Tang et al. [125] presented an impedance microflow

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cytometer consisting of an inertial focusing module implemented in an asymmetrical

curved serpentine channel (Fig. 9(a)) with liquid electrodes. The feasibility of the

proposed cytometer was demonstrated by investigating the size distributions of breast

tumor cells (MCF-7) and white blood cells (WBCs) in human whole blood samples.

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Wang et al. [129] developed a microflow cytometer based on a glass capillary, in

which the cells / particles were focused into a single position through the Dean

vortices produced by wrapping the capillary tube into a helical structure with three

loops (Fig. 9(b)). The device was shown to be capable of counting 10 μm microbeads

with a high throughput of 13000 beads/s and fluorescently labeled WBCs in diluted

whole blood samples. Table 2 shows some of the other proposals for microfluidic

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inertial focusing cytometers presented in the literature.

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3. Microfluidic detection systems for flow cytometers

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he detection performance of microflow cytometers relies on a precise

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streamwise positioning of the particles such that the particles flow through the

interrogation region in a single-line fashion. As described above, microflow

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cytometers use a variety of focusing techniques, including hydrodynamic sheath flows,
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DEP, acoustic waves and inertial forces. The focused particles are traditionally
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detected and analyzed using optical scattering-based methods [130-135] (Fig. 10(a)).
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However, non-optical techniques such as impedance detection (Fig. 10(b)) [136-140]

and digital image processing (Fig. 10(c)) [141-145] have become increasingly
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common in recent years. The main features of the various detection methods are

shown in Table 3.
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3.1 Optical detection systems

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Traditional microflow cytometers invariably collect forward scatter (FSC) and

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side scatter (SSC) information, together with at least a few colors of fluorescence

[146-151]. The FSC and SSC data provide insights into the particle size and internal
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granularity, while the fluorescence information enables the use of

immunophenotyping to discriminate between different cell populations. Xun et al.

[148] presented a microflow cytometer based on a polydimethylsiloxane (PDMS)

microfluidic chip with a SSC scatter and fluorescence detection capability. In the

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proposed device, the particles (white blood cells) were focused using a sheathless

geometric confinement effect and were detected using a fluorescence-based method.

The coincidence error of the focusing mechanism was shown to be less than 0.069%.

Moreover, the coefficients of variation (CVs) of the SSC and fluorescein

isothiocyanate (FITC) channels were found to be 8.37% and 2.46%, respectively. Guo

et al. [151] developed an optofluidic cytometer comprising microfluidic focusing and

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optical detection components integrated on a glass / PDMS chip. The biological

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information of the cells was investigated by measuring the FSC, SSC and FL

emissions as the cells passed through the detection region. In particular, the FSC

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signal measured at a small angle (0.5o~ 20o) was used to characterize the cell size and

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viability, while the SSC signal measured at a large angle (15o~150o) was used to

evaluate the cellular granularity. Finally, the fluorescence signal (measured at the
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same angle as the SSC signal) was used to determine the specific biological properties
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of the cell. The experimental counting results obtained for samples consisting of 5 μm
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fluorescent particles and HeLa cells mixed in ratios of 1:1, 2:1 and 4:1, respectively,
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were shown to be in good agreement with those obtained using a commercial

cytometer.
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For microflow cytometers, a sensitive and accurate analysis of the fluorescence

signals requires calibration of both the intensity response and the spectral response of
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the instrument [52-53,152-162]. Furthermore, the detection of multiple fluorophores

using a single instrument requires the use of a compensation process to deconvolve

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the individual contributions of the various fluorophores in each detector. Li group

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[152] developed a microfluidic cytometer based on electrokinetically-induced

pressure-driven flow and a miniaturized dual-wavelength fluorescent detection system

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for counting two different fluorescent particles simultaneously (Fig. 11(a)). In the

proposed device, two excitation lights were provided by a single bi-color blue/red

LED from one side of the microchannel and the two emission lights were captured by

two photo-detectors placed above and below the chip, respectively. The device was

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shown to achieve a throughput of around 20~40 particles/s for a mixed sample

containing 7.0 μm Dragon Green fluorescent beads and 7.0 μm Flash Red fluorescent

beads. In a later study, the same group utilized a similar fluorescent detection platform

to analyze three species of microalgae cells (Isochrysis galbana, Dunaliella salina

and Tetraselmis chui) [153]. The device not only achieved a limit of detection of just

3 μm, but also provided the means to distinguish between dead and living cells;

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thereby excluding interference from other particles and dead cells.

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Shi et al. [160] proposed a sheathless microflow cytometer for performing

four-part differential leukocyte counts (lymphocyte, monocyte, neutrophil and

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eosinophil) by means of a fluorescent dye assay. The leukocytes were selectively

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stained using three different dyes (propidium iodide (PI), fluorescein isothiocyanate

(FITC) and basic orange 21 (BO21)), and were illuminated by a 488 nm laser through
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a condenser lens. The fluorescent emissions were collected by a lens and passed
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through a 514 nm long pass filter to remove the residual excitation light. A 593 nm
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dichroic mirror was then used to separate the collected fluorescence emissions into
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two beams. By combining the fluorescent assay with a sheathless microflow

technique, the proposed system achieved a minimal sample volume of just 5 µL.
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Furthermore, the total leukocyte count showed an error of less than 90% compared to

that obtained using a commercial cytometer. Frankowski et al. [147,161] presented a

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cytometer for the differentiation of fluorescently labeled blood cells based on multiple

frequency AC impedance and an integrated fluorescent light scattering analysis

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technique (Fig. 11(b)). It was shown that the cytometer was capable not only of
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differentiating WBCs in haemolyzed blood samples, but also of distinguishing among

platelets, erythrocytes, granulocytes, monocytes and lymphocytes in whole human

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blood in a single measurement process.

In addition to the stand-off optical detection systems described above, the

literature also contains many proposals for detection systems based on the integration

of other micro-optical components, such as filters based on dye-doped substrates,

15
liquid micro-lenses with tunable focal length and transmission properties, and optical

waveguides [38-39,41-45,57-60,163-170]. Watts et al. [163] proposed a photonic

integrated microfluidic device for performing on-chip optical excitation and forward

scattering collection in a planar format. The results showed that the device was

capable of counting 5 μm polystyrene beads at a rate of 24 events/s. Furthermore,

compared to the results obtained from the free-space collection of SSC signals, the

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on-chip forward-scattered results demonstrated a false positive rate of just 0.4%, a

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missed events rate of 6.8%, and a coincident rate of 96.3%. Xie et al. [164] developed

a novel cytometer in which label-free single cell analysis was performed by

R
illuminating single static cells on a glass slide using a scanning optical fiber projected

SC
through a small numerical aperture (NA) microscope objective lens. It was shown that

a lens with a low resolution of around 1.30 μm was sufficient to perform the high
U
resolution analysis of yeast cells with distributed sizes. The practical viability of the
N
proposed cytometer was further demonstrated using standard microspheres with mean
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diameters of 3.87 and 4.19 µm, respectively.
M

Martini et al. [165] presented a simple approach for performing optical detection

using a patterned mask integrated into the flow channel wall. In the proposed device,
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the particles flowing through the channel were excited by an external light source

passed through a light guide and the resulting emission pattern was recorded as the
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particles flowed past the mask; resulting in a temporal modulation of the emission

signal. The known modulation (determined by the geometry of the mask pattern) was
E

then used to selectively de-convolve the particle-based fluorescence from the

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background signal. It was shown that the use of different colored masks enabled the

detection of multiple fluorophores. Fan et al. [167] proposed a 3-D microfluidic

A

device comprising 32 detection channels, 64 sheath flow channels, and a high

refractive index microball lens array for high throughput multicolor fluorescence

detection. The performance of the proposed device was evaluated using a mixed

sample of 10 µm green and 8 µm red polystyrene beads and a mixture of fluorescence

16
labeled Ramos and HeLa cells. The results revealed that the device was capable of

achieving a total throughput of 358,400 cells/s across all 32 channels. Sony recently

launched a commercial hyperspectral microflow cytometer which improves the

enumeration efficiency by detecting all of the emitted sample fluorescence at

wavelengths ranging from 500 to 800 nm [168]. In the proposed system, the sample

emission is dispersed into multiple separate bands by means of ten consecutive prisms

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and the bands are then focused onto specific channels of a 32-channel photomultiplier

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tube (PMT) by a microlens array assembly. The system not only provides the ability

to remove autofluorescence from the sample (thereby improving the detection

R
accuracy), but can also function as a regular polychromatic flow cytometer by

SC
combining the channels in the PMT. Kasuga et al. [169] developed a non-damaging

microfluidic chip-based device for cell sorting and mass-spectrometry

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microproteomics analysis. The cell sorting device was operated by using a
N
commercial On-chip sort system (On-chip Biotechnologies, Tokyo, Japan), which has
A
6 fluorescence detectors and 2 scatters (forward and side). The device achieved a
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reasonable microproteomics performance for samples consisting of as little as 100

cells. In addition, the performance achieved using a larger sample size of 1000 cells
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rivaled that obtained using 1 g of whole cell lysate.

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3.2 Electrical impedance detection systems

Impedance-based microflow cytometers are an emerging tool for the

E

high-throughput analysis of the dielectric properties of cells and internal cellular

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components. As shown in Figs. 12(a), (b) and (c), such cytometers are based on the

use of coplanar electrodes [137-140, 171-175], parallel electrodes, [176-180] and

A

constriction channels [181-185], respectively. Irrespective of the design, detection is

performed using an excitation electrode and multiple sensing electrodes embedded

within the microfluidic channel.

Bisegna et al. [186-188] presented a method for correcting the measured particle

17
position in impedance-based flow cytometers using a multiple coplanar electrode

design and a signal processing technique based on a metric encoding particle. Given

an optimal flow rate and sample concentration, the theoretical and experimental

throughputs were determined to be 166 and 144 events/s, respectively. Javanmard et

al. [189-191] developed a portable system for personalized blood cell counting

consisting of a microfluidic impedance cytometer and portable analog readout

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electronics. The analog outputs were processed by an analog-to-digital converter

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(ADC) and then transmitted via Bluetooth to a user-accessible mobile application.

The electrode sensing platform was found to be capable of detecting changes in

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impedance as small as 0.032%; allowing for the detection of 3 μm diameter particles

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in a 300 μm wide channel. Moreover, the sensitivity of the proposed cytometer

system was shown to be comparable to that of a high-end benchtop impedance

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spectrometer. Xie et al. [192] proposed a sheathless impedance flow cytometer for
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label-free cell classification and viability testing purposes. The proposed device had
A
the advantages of a high SNR and a low coincidence ratio. Furthermore, when applied
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to HeLa cell screening, the throughput was as high as 172 cells/s. Finally, the results

obtained for the ratios of apoptotic, necrotic and live cells in a drug-treated cell
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sample containing more than 10,000 cells were found to be in good agreement with

those obtained using a traditional flow cytometer.

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Parallel-electrode impedance-based cytometers were first reported by Gawad et

al. [193], and consist of one or more pairs of electrodes placed in the top and bottom
E

or sidewalls of the microchamber (Fig. 12(b)). Mansor et al. [194] proposed a simple
CC

impedance microflow cytometer for the detection of yeast cell concentrations by

means of a dual tungsten microneedle placed at the half-height position of the

A

microchannel. It was shown that the device had the ability to detect various cell types

with a size ranging from 5 to 25 µm. Haandbak et al. [195, 196] developed an

impedance microflow cytometer for the label-free dielectric characterization of single

cells at frequencies as high as 500 MHz. The performance of the proposed device was

18
evaluated by discriminating wild-type yeast from mutant cells based on a difference in

their dielectric properties at a frequency of approximately 250 MHz. The mutant cells

were found to exhibit a higher opacity magnitude than the wild-type cells as a result

of a difference in the size and distribution of the vacuoles. In addition, the results

extracted from an FEM model for the dielectric properties of the yeast cells were

shown to be in good agreement with those presented in the literature. In a later study

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[197], the same group proposed a platform combining a microfluidic impedance

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cytometer with a high-speed camera to allow the physical morphology of single yeast

cells of the S. cerevisiae species to be assessed in parallel with their dielectric

R
properties. The experimental images and impedance measurements were used to

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identify specific signatures in the impedance data associated with particular

morphological characteristics. The validity of the proposed approach was

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demonstrated by discriminating between single and budding yeast cells.
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The first microfluidic impedance flow cytometer incorporating a constriction
A
channel was proposed by Chen et al. [198]. In the proposed device, Ag/AgCl
M

electrodes were placed at the inlet and outlet, respectively, and single cells were

aspirated through the constriction channel while their elongations and

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single-frequency impedance profiles were measured simultaneously (Fig. 12(c)) The

device was used to discriminate between two different bone cells (osteoblasts and
PT

osteocytes) using a constriction channel with a size of 6 µm x 6 µm and a frequency

of 100 kHz. The results revealed that of the two cells, the osteoblasts had a greater
E

elongation length, a longer transit time and a higher impedance amplitude ratio (i.e., a
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greater difference between the highest impedance amplitude recorded and the

background impedance amplitude in the absence of any cells).

A

Wang and Chen group [182,199] presented a constriction channel-based

impedance cytometer for quantifying the specific membrane capacitance (Cspecific

membrane) and cytoplasm conductivity (σcytoplasm) of cells by means of cell

elongation and impedance profile measurements obtained at frequencies of 1 kHz and

19
100 kHz. The device was used to characterize various cells, including kidney tumor

cells (786-O), non-small cell lung carcinoma cells (CRL-5803), and lung

adenocarcinoma epithelial cells (CCL-185). The same group later proposed a

microfluidic platform in which the clogging problem associated with

constriction-based impedance cytometers was resolved by tuning the deformation of a

PDMS membrane serving as one side of the microchannel by means of an applied

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pneumatic pressure [200]. Zhao et al. [201,183] used a constriction channel-based

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impedance cytometer to differentiate neural stem cells and characterize two different

tumor cells (A549 and H1299).

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Generally speaking, impedance microflow cytometers with a coplanar or parallel

SC
electrode design have a much higher throughput (~1000 cells/s) than those with a

constriction channel design (~100 cells/s). Developing microflow cytometers with a

U
throughput performance comparable to that of traditional flow cytometers represents a
N
major challenge since a tradeoff must be made between the throughput and the signal
A
quality. However, various studies have shown that the potential exists to increase the
M

throughput by performing data acquisition at a higher sampling rate, or by

implementing multiple detection channels.

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3.3 Image analysis detection systems

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Image analysis detection methods for microflow cytometers combine the

single-cell imaging capabilities of microscopes with the high-throughput capabilities

E

of conventional microflow cytometers. Optical imaging is arguably the most effective

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means of visualizing living cells with high spatiotemporal resolution. Accordingly,

state-of-the-art cellular assay techniques increasingly adopt optical imaging

A

techniques for the classification of different cell types / stages and the dissection of

the respective cellular functions. However, adding an imaging capability to microflow

cytometers reduces the throughout to ~1000 cells/s from the ~100,000 cells/s

achieved by gold-standard flow cytometers. In general, the detectors used in imaging

20
microflow cytometers fall into two different types: (1) camera-based imaging devices

[202-213], such as charge coupled devices (CCD) and complementary

metal-oxide-semiconductor (CMOS) devices; and (2) photodetector-based imaging

devices [214-225], such as those based on photomultiplier tubes (PMT) and avalanche

photodiodes (APDs).

In imaging microflow cytometer systems using CCD or CMOS detectors and a

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wide-field illumination technique, 2D cell images can be successfully produced

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provided that a sufficient number of photons are sensed within a given exposure time.

The conundrum in this case is that of increasing the speed of the imaging process.

R
Marr group [202-205] presented a high throughput microflow cytometer based on a

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high-speed CMOS camera imaging analysis system for investigating the dynamic

viscoelastic behavior of RBCs and their time-dependent mechanical response under

U
oscillating external forces (Fig. 13(a)). In the proposed device, the stretching stress
N
distribution on the cell was produced using a one-dimensionally focused laser beam
A
with a laser power oscillating in a frequency range of nearly three orders of magnitude.
M

It was shown that the phase shift between the stimulus and the cell response as a

function of the oscillation frequency was sufficiently high to distinguish between

ED

infected and uninfected erythrocytes (Fig. 13(a)). Moreover, the device was capable

of probing the cell viscoelasticity at rates of more than 20 cells/s; a throughput well
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below the equilibrium relaxation timescale.

Schonbrun et al. [206,207] presented an image-processing-based microflow

E

cytometer in which the traditional trade-off between the throughput and the exposure
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time was overcome by projecting 16 fields of view onto the CMOS camera

simultaneously; each with a magnification of 4 times and a submicron resolution

A

(Fig. 13(b)). The use of multiple imaging channels enabled the same throughput to be

maintained with a flow velocity reduced in proportion to the degree of parallelization

(i.e., the number of imaging channels used). The experimental results revealed that the

device was capable of imaging latex beads, RBCs and acute myeloid leukemia cells at

21
rates of 2000-20,000 cells/s. The same group later demonstrated a method for

eliminating motion blur in the fluorescence imaging of flowing cells using a

pseudo-random excitation pulse sequence followed by deconvolution of the measured

motion-blur image using a Wiener filter [208]. Clark [210] used a commercial

imaging flow cytometer (Amnis® IFC, EMD Millipore Corp, Germany) to observe

and quantify extracellular vesicles (EVs) by capturing the image and

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fluorescence-intensity data with a CCD camera. The author concluded that the

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Amnis® IFC not only provides an ideal platform for the highly sensitive detection of

EVs with a reduced sample volume and preparation time, but also gives the means to

R
distinguish true single particles from aggregates and cellular debris.

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Photodetector-based imaging methods provide a higher bandwidth and lower

dark noise than CCD/CMOS cameras in imaging microflow cytometer systems.

U
However, the readouts of single-pixel PMTs only present the number of photons
N
detected in the time domain, i.e., they provide no spatial information. Accordingly, in
A
some high-speed microscopy laser scanning cytometer techniques, PMTs are
M

combined with laser spot scanning methods to collect the entire light emitted or

scattered by the illuminated cells in the temporal domain. Cell images are then
ED

constructed by assembling the intensity signal in the time domain according to the

laser scanning position. Goda et al. [214] developed an ultrafast optical imaging flow
PT

analyzer technique designated as serial time-encoded amplified microscopy (STEAM)

for the blur-free imaging of cells flowing at high speed. In contrast to the light
E

emitting diode (LED), laser, and mercury lamp light sources used in conventional
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microflow cytometers, the proposed system used a mode-locked femtosecond pulse

fiber laser to generate near-infrared light with a wide spectral bandwidth (Fig. 14(a)).
A

Each pulse (representing one frame of the camera) was stretched in time and digitized

in real time using an ADC. By choosing a shutter speed in the picosecond range, the

camera effectively froze the motion of the cells, resulting in the acquisition of

blur-free images. When applied to the imaging of budding yeast cells and breast

22
cancer cells, the device exhibited an unprecedented throughput of 100,000 particles/s

with a false positive rate of just one in a million. Huang et al. [216] presented a

technique for achieving ultrafast imaging by replacing the spatial light modulators

(SLMs) used in conventional compressive sensing (CS) techniques with a spectral

resonance modulator (SRM) implemented using an etalon array.

Tsia group [217-219] proposed an asymmetric-detection time-stretch optical

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microscopy (ATOM) technique for the label-free, high-contrast imaging of single

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cells (human whole blood and leukemic monocytes) with an ultrahigh microfluidic

speed and a sub-cellular resolution (Fig. 14(b)). The experimental results showed that

R
the system was able to achieve live-cell imaging at flow speeds as high as ~10 m/s,

SC
corresponding to an imaging throughput of ~100,000 cells/s. Moreover, it was

reported that the system could be further enhanced through the integration with
U
field-programmable gate arrays (FPGAs) or graphical processing units (GPUs).
N
Han and Lo [220,221] applied a spatial–temporal transformation technique to
A
retrofit a conventional flow cytometer into an imaging microflow cytometer system.
M

The resulting system provided the ability to encode the time-domain signal waveform

with a specially designed spatial filter such that the waveform consisted of a sequence
ED

of patterns separated in the time domain. In particular, by inserting a spatial filter with

a known pattern in the image plane, the fluorescence, transmission and scattered light
PT

signals emitted from different parts of the cells passed through the different slits at

different times. The cell images acquired in various modes (e.g., fluorescence or
E

scattering) were then assembled using algorithms specific to the particular spatial
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filter used. Zhang et al. [225] presented a microflow cytometer based on a Brillouin

spectroscopy technique for the label-free, non-contact and noninvasive measurement

A

of the nuclear mechanical properties of cell populations at a throughput of around 200

cells per hour.

4. Conclusion and Future Directions

23
 Microflow cytometers are one of the most powerful techniques for the rapid

analysis of cells and particles. Moreover, advances in Lab-on-a-Chip (LOC)

technology have facilitated the use of microfluidic devices (including microflow

cytometers) throughout academia, research, and commercial instrumentation. This

review has described the many sheath flow and sheathless approaches which have

been proposed for particle focusing in microflow cytometers. The various detection

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systems used in such platforms have also been introduced and discussed. For each

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method presented (both focusing and detection), the relative merits and performance

metrics have been described and explained. Overall, the review provides a useful

R
summary of the current state-of-the art for microflow cytometers, and is thus expected

SC
to be of significant interest to both experienced practitioners in the field and novices

entering the field for the first time.

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As shown in Table 1, all microfluidic focusing systems have their own particular
N
advantages and disadvantages. Thus, comparing the various systems directly is far
A
from straightforward. In practice, the choice of an optimum method depends on the
M

particular needs of the application in question, which typically involve simplicity,

integration, efficiency, throughput, reliability, cell effects, and others. In general, 2-D
ED

microfluidic focusing has the advantages of a simple fabrication process,

low-complexity driving and power requirements, convenient control, and

PT

straightforward integration. However, 2-D focusing systems have an inherently low

detection sensitivity. Fortunately, the sensitivity performance can be improved using

E

various techniques such as impedance system and image analysis, as shown in Table 3.
CC

For example, given fluorescence labeling of the sample, a sensitivity of optical system

can be achieved using impedance methods.

A

Sample processing is a crucial step in many biochemical and biomedical assays

since most biofluids are complex. As a result, it is necessary to isolate the target cells

in some way prior to analysis. Existing image analysis systems typically achieve a

counting efficiency of around 1,000~100,000 events/s. However, a trade-off exists

24
between the throughput and the signal quality. Consequently, microfluidic cytometers

combined with impedance detection systems (with a throughput of more than 1000

events/s) are regarded as a convenient and straightforward approach for performing

the high-throughput label-free characterization of sample cells and particles.

Despite the many advantages of microfluidic cytometers for disease diagnosis

and POC applications, many significant challenges remain to be overcome, including

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their extension to clinical contexts and rare cell diagnosis, and the need for a lower

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cost, reduced size and greater simplicity. Thus, future studies on microfluidic

cytometers are likely to focus on widening their applications and promoting their

R
integration ability. For example, Fu et al. [137] recently developed a

SC
lab-on-PCB-based micro-cytometer integrated impedance detection system for the

detection and enumeration of rare circulating tumor cells (CTCs). Similarly,

U
Kasukurti et al. [203] presented a microfluidic viscoelasticity cytometer (VC) capable
N
of performing real-time and continuous measurements using fluorescence activated
A
cell sorters (FACS) for label-free red blood cell (RBC) detection. In addition, Clift et
M

al. [226] proposed a multi-color microflow cytometer for resolving the specific cell

types of multi-cellular models without impacting the cell viability.

ED

Due to the high surface area-to-volume ratio of microfluidic channels, the

surface properties (e.g., hydrophilicity/hydrophobicity, electric charge, and roughness)

PT

have a significant impact on the performance of microfluidic flow cytometers. For

example, surface adhesion / adsorption / fouling may degrade the enumeration

E

accuracy for cells, bacteria, proteins and other organisms [227-230]. Consequently,
CC

further efforts are required to improve the performance of microflow cytometers

through the development of suitable surface modification technologies.

A

Although the microflow cytometer field has undergone dramatic advances over

the past few decades, further innovations are required to reduce the cost, size and

complexity of cytometry systems and to increase their sensitivity. Thus, the coming

years are expected to bring significant further enhancements of the microfluidic

25
cytometry field, including full automation of the acquisition processes, a further

reduction in the device size through the use of LOC approaches, and more efficient

flow control machinery to avoid motion blur and other undesired artifacts. The

resulting improvements are likely to play a key role in facilitating medical diagnostics

and biomedical research worldwide.

Acknowledgement

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The authors would like to thank the Ministry of Science and Technology of

Taiwan for the financial support of this study under Grant Nos. MOST 103-2320-B-

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020-001-MY3, MOST 103-2221-E-020-025-MY3, MOST 106-2314-B-020-002

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-MY3, MOST 106-2221-E-020-019-MY3, and MOST 107-2622-B-020-003-CC2.

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Biographies

Ruey-Jen Yang received the Ph.D. degree from the University of California at
A

Berkeley in 1982. From 1982 to 1993, he was a Research Scientist and Engineer at
Scientific Research Associates and Rockwell International. He moved back to Taiwan
in 1993 and now he is a Professor of Engineering Science Department at National
Cheng Kung University, Tainan, Taiwan, R.O.C. His research interests are microflow
physics, computational fluid dynamics, vortex dynamics, and bifurcation theory.

Lung-Ming Fu received M.S. and Ph.D. degrees in Engineering Science from

National Cheng Kung University (NCKU), Taiwan, in 1997 and 2001. He had his
42
postdoc training in Department of Engineering Science at NCKU during 2002-2003.
He is currently a professor in the Graduate Institute of Materials Engineering at
National Pingtung University of Science and Technology. His research interests are in
microfluidic systems, microfluidic paper-based devices and applications, MEMS
fabrication technologies, micro-sensor and computational fluid dynamics.

Hui-Hsiung Hou received the Ph.D. degrees in engineering science from National
Cheng KungUniversity (NCKU), Tainan, Taiwan, R.O.C., in 2013. He is currently
postdoc training with the Department of Engineering Science at NCKU. His graduate
and postdoc work was focused on analysis and application of microfluid systems.

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Figure Captions

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Fig. 1 (LM Fu)

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(a) (b)

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U
N
Concentration of
species at – 30 kPa
A

(c)
M

104 104 104 Live

Live Live
93.68% 73.23% 16.82%

103 103 103

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102 102 102

101 101 101

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APO-Early APO-Late APO-Early APO-Late APO-Early APO-Late

3.15% 1.68% 15.40% 9.54% 55.89% 25.98%
100
100 100 0
100 101 102 103 104 100 101 102 103 104 10 101 102 103 104
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Fig. 1 Principles of 2-D hydrodynamic focusing technique for confining cells to

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single-file flow. (a) microphotograph showing 2-D hydrodynamic focusing effect

inside chip-based cytometer; (b) CFD results for 3-D contours of species
concentration; and (c) multi-parameter analysis of programmed tumor cell death
A

using microflow cytometer. (Reprinted from Ref. [31] with permission of Elsevier.)

43
Fig. 2 (LM Fu)

(a) (b)

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IP
104
PI
Waste

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103 DOUBLE

FL3-H
Semen 102

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101
Media SYBR-14
100
100 101 102 103 104
FL1-H

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Fig. 2 (a) Schematic illustration of flow cytometry-based submicron-sized bacterial
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detection system using 2-D hydrodynamic side-focusing flow. (Reprinted from Ref.
A
[33] with permission of Royal Society of Chemistry.) (b) Schematic illustration of
M

motile human sperm sorting process using 2-D hydrodynamic side-focusing

microfluidic system [34].

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E PT
CC
A

44
Fig. 3 (LM Fu)

(a) (b) 1 B
1
(c)
A
2 2
C 3
3
4
4 Weir

3D view

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IP
Weir
Cross-section view

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SC
(d) (e)
Sheath
Core
Sheath U (f)
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drain sample
A
Sheath
fluid
grooves for
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optical fibers Detection channel

Spin
hydrodynamic
focusing
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Sheath flow Sample flow

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Fig. 3 Microfluidic 3-D focusing devices. (a) Horizontal and vertical direction focusing
device. (Reprinted from Ref. [48] with permission of Royal Society of Chemistry.) (b)
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Horizontal focusing and vertical secondary flow effect device. (Reprinted from Refs.
[49,50] with permission of Royal Society of Chemistry.) (c) Horizontal focusing and
CC

vertical micro-weir effect device. (Reprinted from Ref. [52] with permission of
Springer.) (d) Dean flow induced by structure-effect device. (Reprinted from Ref. [54]
with permission of Royal Society of Chemistry.) (e) Horizontal focusing and
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groove-generated sheath effect device. (Reprinted from Refs. [56] and [58] with
permission of Royal Society of Chemistry and Elsevier.) (f) Microflow cytometer with
integrated mirror and six grooves for 3-D vortex hydrodynamic focusing [63].

45
Fig. 4 (LM Fu)

(a) (b)
Mixed cells Separation zone

Cell 1
Electrodes

100μm Cell 2

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Lock-in

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amplifier DAQ 100μm
Filters
1V/ μA
DEP electrodes #2
Drive signal Left
Detection outlet

R
electrodes
Fluid
flow
Center

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Particle
line
DEP focusing Detection
electrodes
15k Ω 15k Ω
Right
Shunt resistor Shunt resistor outlet
Differential
Amplifier

U DEP electrodes #1
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(c) Particle focusing of Particle focusing of
top section cross view
A
y

z Particle
x
M

x
ED
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Fig. 4 (a) Microfluidic cytometer employing dielectrophoretic focusing / centering of

cells and hydrodynamic focusing of sample stream. (Reprinted from Ref. [67] with
E

permission of Royal Society of Chemistry.) (b) Microelectrode arrays patterned along

microchannel sidewalls and force balance plot of cells in separation zone of
CC

microfluidic DEP device. (Reprinted from Refs. [77] and [78] with permission of John
Wiley and Sons and Royal Society of Chemistry.) (c) Schematic view of 3-D particle
focusing channel using positive DEP and dielectric structure between two planar
A

electrodes. (Reprinted from Refs. [79] and [80] with permission of Royal Society of
Chemistry and Springer.)

46
Fig. 5 (LM Fu)

(a) Microelectrodes Microchannel

(b)

Flow

V-
TOP VIEW Orientation

T
direction

IP
Flow
Focusing

R
40μm
line

SC
V+

Flow

U
N
A
Fig. 5 (a) Microflow cytometer for single cell shape-based discrimination (upper) with
details of electric field in DEP focusing/orientation section (middle) and M-phase cell
M

population flow (lower). Reprinted from Ref. [81] with permission of Royal Society of
Chemistry.) (b) Microelectrode array of DEP cytometry device showing configuration
ED

of electrode pads (upper and middle) and trajectory of small sphere flowing through
microfluidic channel with actuation by nDEP force (lower). (Reprinted from Ref. [82]
with permission of AIP Publishing LLC.)
E PT
CC
A

47
Fig. 6 (LM Fu)

(a) (b)

streaming
SAW
IDTs

T
streamlines
particle trajectory
acoustic filed

IP
Sample IDT
inlet
Sheath Laser
spot Target
inlet Ttrigger outlet

R
Outlet Waste
TDelay outlet

SC
channel boundary SSAW
IDT active
region

Standing SAW Cell

(Pressure)

SAW 100 μm
IDT

U IDT
N
Fig. 6 (a) Principle of SAW-based nanoparticle focusing, streaming field and acoustic
A
radiation forces generated by substrate vibrations produced by IDT. (Reprinted from
M

Ref. [103] with permission of Royal Society of Chemistry.) (b) Photograph and
schematic illustration of acoustic-fluidic FACS device indicating focusing region and
principle of cell/particle focusing at channel middle position under effects of SAW
ED

force. (Reprinted from Ref. [104] with permission of American Chemical Society.)
E PT
CC
A

48
Fig. 7 (LM Fu)

(a) (c)

Transducer
SSAW
Unfocusing
Objective
Transducer
Galvo mirror
Lens

Mirror

T
Camera Illumination optics
Syringe pump
Focusing

IP
White light source

(b) TFC: Filter cube

PFG: Programmable function generator

R
SC
U
N
A
Fig. 7 (a) Schematic illustration of SSAW-based microfluidic cytometer with
integrated LIF detection system. (Reprinted from Ref. [107] with permission of Royal
M

Society of Chemistry.) (b) Schematic illustration of experimental setup for microchip

impedance spectroscopy (MIS) flow cytometry (left) and working principle of 2-D
ED

acoustic particle focusing (right). (Reprinted from Ref. [108] with permission of Royal
Society of Chemistry.) (c) Schematic illustration of experimental setup for acoustic
focusing flow cytometry system with CMOS image detection [110].
E PT
CC
A

49
Fig. 8 (LM Fu)

(a) (b) (c)

input D C B A
segment I
flow 200 μm

segment II

inlet

cross-section
cross-section

T
particle in equilibrium
particles undergoing Dean
recirculation
Inner wall

IP
F2

Outer wall
F1 FD

FD

R
velocity contour
wall effect force Dean vortex flow cross-section
shear gradient force

SC
Fig. 8 (a) Principle of inertial force focusing in square channel, in which four
equilibrium positions exist where shear gradient force is equal to wall effect force.
(Reprinted from Ref. [113] with permission of Royal Society of Chemistry.) (b)
Photograph of microfluidic chip and schematic illustration showing two
U
N
counter-rotating Dean vortices orthogonal to main flow direction. (Reprinted from
Ref. [116] with permission of Springer.) (c) Photograph of grooved microchannel and
A
schematic illustration of resulting helical streamlines. (Reprinted from Ref. [123] with
permission of Elsevier.)
M
ED
E PT
CC
A

50
Fig. 9 (LM Fu)

(a) (b)

Helical
capillary
tube Inertial lift force
(3 loops) Dean drag force
Inlet Flow 2
1 3 4
Outlet
1 2 3 4

T
Outer wall
Inner wall

IP
Inlet Outlet Outlet (after 90 0)

R
SC
Fig. 9 (a) System configuration and working principle of liquid electrode impedance

U
microflow cytometer with inertial focusing effect in asymmetrical curved serpentine
N
channel. (Reprinted from Ref. [125] with permission of American Chemical Society.)
(b) Microfluidic helical capillary–based inertial focusing of micro-particles and cells
A
for high-throughput 3-D focusing flow cytometry (Reprinted from Ref. [129] with
permission of AIP Publishing.)
M
ED
E PT
CC
A

51
Fig. 10 (LM Fu)

(a) (b) (c)

HF21S Input
Input Ch1
Output HF2TA Ch2

Sample Gas
EI
Electrodes

PDMS
Glass

T
count
20

IP
Exciation at 500kHz 15
10
5
0
count
100 100 2.5
Hypoxia 10 20 30 40 50
2.0 Normoxia
Thresholding Noise reduction
Δ/Z (107 Ω)

R
10-1 10-1 1.5
EXT

10 μm
SSC

10 μm 1.0
10-2 7.26 μm 10-2 5 μm
0.5
5 μm 3 μm

SC
3 μm 0
10-3 10-3 -0.30 -0.25 -0.20 -0.15 -0.10 -0.05 Finish Binary mask
0.01 0.1 1 10 0.01 0.1 1 10 AO(rad)
FL FL

Fig. 10 (a) Schematic illustration of PV-cell based optofluidic cytometer for

simultaneous extinction, side scatter and fluorescence signal measurement [130]. (b)
U
N
Schematic illustration of experimental setup for electrical impedance microflow
cytometer (upper) and scatter plots of sickle RBCs under normoxia and hypoxia given
A
electrical excitation at 500 kHz (lower). (Reprinted from Ref. [140] with permission of
Elsevier.) (c) Schematic of webcam-based wide-field flow cytometer using imaging
M

analysis and computational analysis technique. (Reprinted from Ref. [144] with
permission of Royal Society of Chemistry.)
ED
E PT
CC
A

52
Fig. 11 (LM Fu)

(a) Bi-colour blue/red LED

(b)
Photodiode
Filter (I)

Microfluidic chip
Filter Filter PE
FITC
Data Filter SSC
acquisition

Photodiode
Power supply

T
FL3-H::FL3-Height

PD 1

IP
(II) (III)
LED
PD 2
(inside)

R
SC
PD: Photo-detectors
FL2-H::FL2-Height

Fig. 11 (a) Photographs and schematic illustrations of optical detection system and
experimental setup, and number counts for two fluorescent particles determined by
U
N
commercial flow cytometer. (Reprinted from Ref. [152] with permission of Royal
Society of Chemistry.) (b) (I) Measurement setup for combined flow cytometry
A
optical and vector impedance measurements. (II) Photograph of microfluidic
cytometer-glass chip featuring platinum electrodes with arrows indicating sheath and
M

sample flows. (III) Magnitude of impedance versus reactance at 10 MH for whole

blood sample. (Reprinted from Refs. [147] and [161] with permission of Royal Society
ED

of Chemistry and John Wiley and Sons.)

E PT
CC
A

53
Fig. 12 (LM Fu)

(a) (b) (c)

Glass AC signal
Electrode Function
generator
Lock-in amplifier
t0 t1 t2 t3 t4
Ag/AgCl Cell loading channel Ag/AgCl
Flow electrode electrode

PDMS
Differential I-V converters Electric Aspiration
amplifier
field lines channel
-P
Detection circuit
Glass

Differential signal (V)

I inner
Lock-in

T
amplification

C cell R cell
R channel

IP
C channel
Time (ms) R leak

R
Fig. 12 (a) Principle of impedance flow cytometry with coplanar electrodes.
(Reprinted from Ref. [171] with permission of Royal Society of Chemistry.) (b)

SC
Principle of impedance flow cytometry with parallel electrodes [176]. (c) Principle of
impedance flow cytometry with constriction channel design and equivalent circuit
model. (Reprinted from Ref. [181] with permission of Royal Society of Chemistry.)

U
N
A
M
ED
E PT
CC
A

54
Fig. 13 (LM Fu)

(a) (b)

T
R IP
(e)

SC
(d)

(c)

U (b)
N
A
Fig. 13 (a) Schematic illustration showing experimental setup for microflow
cytometer with CMOS image analysis detection method (upper) and measured
M

distributions of cell deformation for ~100 cells given various modulation frequencies
and cell types (lower). (Reprinted from Ref. [202] with permission of Elsevier). (b)
ED

Multiple field-of-view imaging microflow cytometer, image plane of 12 field-of-view

device for sample of 3.5 μm latex beads, and scatter plot of minor vs. major axis of
human RBC images. (Reprinted from Ref. [206] with permission of Royal Society of
Chemistry).
E PT
CC
A

55
Fig. 14 (LM Fu)

Illumination & Imaging Optics

(a) (b)
Microfluidic device
Sample Laser Mirror
beam Flowing
inlet
cells
Dispersive
Blood draw Preparation Loading Sheath Outlet Data analysis fiber Beam Beam Mirror
inlet Fiber
Diffraction splitter splitter
collimator
Broadband Objective lens Objective lens grating
pulse laser
In-line
Diffraction lenses lenses amplifier
grating Spectral
Oscilloscope
Photodetector Mirror Mirror shower
(digitizer)
Mirror
Photodetector
Collimator Grating
Real-time

T
Mirror Collimator Dispersive fiber oscilloscope Broadband Beam
pulsed laser splitter

Real-Time Optoelectronic Time-Stretch Image Processor Broadband Spectrally- Time-multiplex of Time-stretch with

IP
pulsed laser encoding asymmetrically detected pulses optical amplification
Microchannel
White blood cells
White
blood cell
Aggregated

R
platelets Platelet

Spectral shower
ID dispersed
rainbow Thin PDMS

SC
Output Input
Thick PDMS

Flow
direction

Fig. 14 (a) Schematic illustration of flow analyzer consisting of CMOS camera and
real-time optoelectronic time-stretch image processor, and conventional microflow U
N
cytometer scatter plots of white blood cells. (Reprinted from Ref [215] with
permission of Royal Society of Chemistry). (b) Schematic illustration of ATOM
A
microfluidic channel platform, interferometric time-stretch (iTS) microscope setup,
M

and corresponding temporal and spectral waveforms [218].

ED

Table 1 Overview of microfluidic focusing methods for microflow cytometers.

Focusing system Advantages Disadvantages
PT

2-D focusing Simple fabrication Vertical focusing independence

Simple driving power Low sensitivity
High throughput
E

3-D focusing High sensitivity Complex fabrication

CC

High throughput Complex driving power

Inertial focusing High throughput Complex fabrication
Simple driving power Specific geometry
Flow difficult control
A

Dieletrophoresis High sensitivity Complex fabrication

focusing Complex driving power
Low throughput
Acousitic focusing High sensitivity Complex fabrication of electrodes
High throughput Complex driving/detection power

56
Table 2 Summary of microfluidic focusing systems for microflow cytometers.
Ref. and First Year Focusing Driving force Detection type Throughput Experimental
author type samples
[27] Lee 2014 2-D Hydrodynamic Optical (PMT) N/R Calibration particles
[35] Hirai 2015 2-D Hydrodynamic Optical (PMT) 3.5 particles/s Bladder cancer cells,
prostate cancer cells
[36] Spencer 2014 2-D Hydrodynamic Impedance (parallel 132 cells/s CD4+ lymphocytes,
electrodes) 368 beads/s calibration beads
[37] Wang 2013 2-D Hydrodynamic Optical (APD) 0.04~0.13 Microalgae cells
cells/s
[38] Golden 2013 2-D Hydrodynamic Optical (fiber + 650±150 Escherichia coli
4PMTs) spheres/min 0157:H7

T
[39] Nawaz 2013 2-D Hydrodynamic Optical (fiber + 3754 events/s CD4+ lymphocytes,
3PMTs (FL,FSC, RBCs, WBCs

IP
SSC))
[40] Vercruysse 2015 2-D Hydrodynamic Image analysis N/R RBC-lysed blood
samples

R
[41] Fu 2004 2-D Electrokinetic Optical 6 cells/s Polystyrene latex
(fiber+APD) beads, RBCs

SC
[42] Rosenauer 2010 3-D Hydrodynamic Optical (fiber+ 600 beads/s Yeast cells,
(2H + 1V) amplified polystyrene particles
Si-photodetector)
[43] Kennedy 2011 3-D Hydrodynamic Optical (fiber + 215 beads/s Calibration

[45] Rosenauer 2011 3-D

(4H + fillister )
Hydrodynamic
(2H + 1V)
3PMTs)

U
Optical (fiber+
amplified
15 beads/s
microparticles
Polystyrene beads
N
Si-photodetector)
[53] Hou 2009 3-D Hydrodynamic Optical (APD) 20 beads/s Polystyrene beads
A
(2H+ micro-weir)
[57] Golden 2009 3-D Hydrodynamic Optical (fiber + N/R Escherichia coli
(2H+ groove) 4PMTs)
M

[58] Hashemi 2011 3-D Hydrodynamic Optical (fiber + N/R Synechococcus sp.,
(2H+ groove) 4PMTs) Nitzschia d.,
Thalassiosira p.
[60] Lin 2012 3-D Hydrodynamic Optical (fiber + 150 beads/s Leukaemia cells,
ED

(single sheath- PMT) beads

flow)
[64] Zhao 2016 3-D Hydrodynamic Optical (PMT) 1400 ~ 20000 Polystyrene beads
(2H + 3 layers beads/s
PT

structure)
[65] Shivhare 2016 3-D Hydrodynamic N/R N/R Polystyrene beads
(2H + 2V)
[67] Evander 2013 DEP Hydrodynamic Impedance (parallel N/R RBCs
E

electrodes)
[69] Sadeghian 2017 DEP Hydrodynamic + Image analysis N/R Polystyrene beads,
CC

Electrokinetic WBCs, k562 cells

[72] Song 2015 DEP Electrokinetic Image analysis N/R Human
mesenchymal stem
cells
[87] Mohammad 2017 DEP Electrokinetic Impedance N/R Chinese hamster
A

(capacitance sensor) ovary cells

[100] Kishor 2017 SAW Hydrodynamic + Impedance 0.7 ~ 20 Polystyrene beads
Electrokinetic (IDT sensor) beads/μL
[105] Graves 2012 SAW Hydrodynamic + Optical (PMT) 3000 events/s CD4+ T cells
Electrokinetic
[106] Piyasena 2012 SAW Hydrodynamic + Image analysis 50000 cells/s CD4+ T cells
Electrokinetic
[111] Xue 2017 SAW Hydrodynamic + Image analysis N/R Human
Electrokinetic mesenchymal stem
cells

57
[112] Kalb 2017 SAW Hydrodynamic + Image analysis 100K beads/s Microsphere,
Electrokinetic 1M cells/s truly rare blood cell
[114] Wang 2015 Inertial Hydrodynamic Optical (PMT) 2000 beads/s Polymer beads,
focusing 850 cells/s fibroblast cells
[116] Johnston 2014 Inertial Hydrodynamic Image analysis ~33000 Fluorescent
focusing particles/s microspheres
[117] Fan 2015 Inertial Hydrodynamic Image analysis < 39000 Fluorescent particles
focusing particles/s
[122] Chung 2013 Inertial Hydrodynamic Image analysis 36000 MCF7 and Jurkat
focusing particles/s cells, microbeads
[125] Tang 2017 Inertial Hydrodynamic Impedance (parallel 5000 cells/s MCF7 cells and
focusing electrodes) WBCs
[126] Hur 2010 Inertial Hydrodynamic Image analysis 106 cells/s Fluorescent particles
focusing RBCs

T
[128] Butement 2016 Inertial Hydrodynamic Optical +Image 2 bead/s/s Fluorescent beads
focusing

IP
N/R: not report; 2-D: two-dimensional; 3-D: three-dimensional; H: horizontal focusing;
V: vertical focusing; DEP: dielectrophoresis force; SAW: surface acoustic wave

R
SC
Table 3 Overview of detection methods for microflow cytometers.
Detecting system Typical detectors Summary
Optical system PMT
U
Throughput is more random, but don’t
N
APD exceed 1,000 events/s in generally.
Optical waveguide
A
Impedance system Coplanar electrodes Throughput in coplanar electrode design and
Parallel electrodes parallel electrode design are about 1,000
M

Constriction channel events/s and constriction channel design is

about 100 events/s.
Imaging analysis Camera-based device Throughput in camera-based device is about
ED

Photodetector-based device 1,000 ~ 10,000 events/s and

photodetector-based device is about 5,000 ~
100,000 events/s.
E PT
CC
A

58