On-chip multiple particle velocity and size measurement using single-shot two-wavelength differential image analysis

Mitsuru Sentoku 1,*, Shuya Sawa 1, and Kenji Yasuda 1,2

1 Department of Physics, School of Advanced Science and Engineering, Waseda University, Tokyo, 169-8555, Japan; 2 Department of Pure and Applied Physics, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, 169-8555, Japan

* Corresponding author: mitsen1019@fuji.waseda.jp1

Graphical Abstract

2

Title: On-chip multiple particle velocity and size measurement using single-shot two-wavelength differential image analysis

4994 µs590 nm

2500 µs530 nmRestoration

L2

590 nm 530 nm

L1

t1 t2

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v = L1�L2t1�t2

Multi-view module

Wavelength

Bright-field

Binarized

AbstractPrecise and quick measurement of samples' flow velocities is essential for cellsorting timing control and reconstruction of acquired image-analyzed data. We havedeveloped a simple technique for single-shot measurement of flow velocities ofparticles simultaneously in a microfluidic pathway. Microparticles were injectedthrough an imaging flow cytometer and two wavelengths of light with differentirradiation times were irradiated to the particles simultaneously. The mixture of twowavelengths transmitted lights was divided into two wavelengths, and the images ofthe same microparticles for each wavelength were acquired in a single capture. Thespeed was calculated from the difference in the particles' elongation in an acquiredimage which arose when applying two wavelengths of light with differentirradiation times. The distribution of polystyrene beads' velocity was parabolic andhighest at the center of the flow channel, consistent with the expected velocitydistribution of the laminar flow. Exploiting the calculated velocity, we restored theaccurate shapes and cross-sectional areas of particles in the images, demonstratingthe capability of this simple method for improvement of imaging flow cytometryand cell sorter for diagnostic screening of circulating tumor cells.

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Keywords: Imaging flow cytometer; precise velocity measurement; particle shape reconstruction; multi-view imaging

Introduction

4

Please see previously published paper, Odaka et al.,Micromachines, 2019, for more detail

• CTC forming clusters have been observed inside bloodstream.

CTC cluster. Bar, 10 μm

• CTC clusters are 23～50 times more likely to cause metastasise.g., A.Fabisiewicz, Medical Oncology, 2016

Advancement in image recognition and analysis technologies in the field of imaging cell sorting,

Difficulty overcoming the precise measurement ofmicroparticle flow speed for correct target collection

Machine learning based sorting Droplet microfluidics

e.g., M. Sesan, et al.,Sci. Rep., 2020e.g., N. Nitta, et al., Cell., 2018

Introduction

5

Chip design

Image recognition＋Applied voltageSorting by shape (area)

Development of a universal detection method of CTC clusters

System design

Fig. 1. Schematic of imaging cell sorter system.

cf. M., Odaka, et al. Micromachines, 2019 cf. H., Kim, et al. PLOS, 2014

Solutionl Development of a velocity measurement technology requiring

only single-capture of the object.l Estimation of the elongation and restoration via image

processing

Objective

Development of a universal detection method of CTC clusters

Task• Movement of particles while the shutter is open• shape is not consistent → not the accurate size measurement

Essential to know the flow velocity

Causes elongation

after t seconds

Difficulty identifying the particles

Results

7

Image acquisition flow cytometer with simultaneous two-wavelength differential imaging

(ii) LED(530 nm)

(iv) Microchip

(v) Objectivelens (x20)

(vi) Mirror

(ix) Controller

(vii) Multi-viewmodule

(vii) CCD camera

(i) Halogen lamp(590 nm)

(iii) Dichroic mirror

Sheath flow

Sampleflow

Sheath

flow

Cell

System set up

Sheath flow to focus the dispersed particles

Microchip design

Fig. 2. Schematic of imaging acquisition flow cytometer

Proof of concept

Principle

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(b)(a)

2500 µs4994 µsRestorationStationary Moving

Irradiation time: 2500 µs4994 µs 2500 µs4994 µs

(i) (ii) (iii)

530 nm590 nmWavelength: 530 nm590 nm 530 nm590 nm

0

500

1000

1500

0 500 1000 1500

Mea

sure

d ve

loci

ty [1

0-3

m/s

]

Setting velocity [10-3 m/s]

Photograph(480 px × 640 px)

Mirror A

Dichroicmirror A

Dichroicmirror B

CCDelement

Mirror B

2 3⁄ frame size window

590 !" 530 !"

image

Mirror C

Separates the image into two wavelength

12 µm single polystyrene beads Pre-set velocity vs Measured velocity

Linearly correlated

Multi-view unit

Bright-field

Binarized

Stationary Flowing Restoration

Image of a sample flowing inside microchip

Fig. Schematic of the multi-view unit

Fig. 3. Original and binarized images of polystyrene beads

590 nm

530 nm

Results

Results

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0123456789

0 10 20 30 40 50

n [%

]

X position [μm]

0

2

4

6

8

10

12

14

0 10 20 30 40 50 60 70

n [%

]

X position [μm]

(a) (c)

N = 91

(b)

(f)

N = 142

0

5

10

15

20

25

30

35

40

0 0.5 1 1.5 2 2.5 3

n [%

]

Velocity [10-3 m/s ]

(e)

Data acquisition position 149.3 μm

73.9 μm

0

10

20

30

40

50

60

70

0 0.5 1 1.5 2

n [ %

]

Velocity [10-3 m/s]

(d)

Data acquisition position 2

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60 70

Velo

city

[10-3

m/s

]

X position [μm]

(g)

00.20.40.60.8

11.21.41.61.8

0 10 20 30 40 50

Velo

city

[10-3

μm/μ

s]

X position [μm]

Ø Accurate flow velocity measurement is a prerequisite for determining the precise cell sorting timing to shift a target at the sorting point.

Even after the hydrodynamic focusing, the variation in flow velocity distribution remained present.

Data acquisition position

Flow velocity distribution in microfluidic pathway

Number of particles vs X position Velocity of particles vs X position Number of particles vs velocity

Lower

Upper

Focused Focused

Laminar flow like distribution

Fig. 4. Flow velocity distribution of particles for upper and lower stream

Results

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0

0.5

1

1.5

2

2.5

0 10 20 30 40 50Ve

loci

ty [1

0-3

m/s

]X position [μm]

(a) (b)

1

23

45

1

2

3

4

5

1

23

4

5

Wavelength :

Irradiation time :

590 nm

4994 μs

530 nm

2500 μs

Simultaneous flow velocity measurement of multiple particles

• Two-wavelength images of five particles were obtained • The elongation was different depending on the flow velocities of

each particles. • The velocity was dependent on the location of microchannel.

Bar, 15 µm

Five particles flowing simultaneously Measurements of the five observed particle velocities

Quadratic approximation of laminar flowFig. ５。5. ow velocity measurement of multiple particles and its X positions

Results

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Irradiation time : 4994 μs 2500 μsWavelength: 590 nm 530 nm 590 nm 590 nm

4994 μs 2500 μs590 nm 530 nm

Conventional restoration method versus the proposed method

Stationary particle

Elongation caused by flow velocity

Conventional method vs this method

Compressed particle image

Properly restored image

Moving particle

• Conventional method compresses the particle to restore the image• The proposed method lifts the elongated particle up according to the

calculated velocity

Fig. 6. Shape comparison between conventional and our restoration method

Results

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90

110

130

150

170

190

210

0 5 10 15 20 25 30 35 40 45 50

Area

[μm

2]

X position [μm]

90

110

130

150

170

190

210

0 5 10 15 20 25 30 35 40 45 50

Area

[μm

2]

X position [μm]

90

110

130

150

170

190

210

0 5 10 15 20 25 30 35 40 45 50

Area

[μm2 ]

X position [μm]

Area of the restored particles using the method

• The size of the polystyrene beads is more consistent at various X positions of the microchannel

Conventional simple image reduction methodDistribution of the averaged area of the particles at different X positions

Without using any restoration method

Fig. 7. Graph of area of the particles at X positions

Discussion

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(a) (b)

0

2000

4000

6000

8000

0 0.5 1 1.5 2 2.5

Max

irra

diat

ion

time

[μs]

Velocity [10-3 m/s]

5.50

6.00

6.50

7.00

0 0.5 1 1.5 2 2.5

Max

den

sity

[107

cells

/ m

L]

Velocity [10-3 m/s]

Limitation of the method for imaging flow cytometry measurement

• Resolution and preciseness of the measurement is reliant on the hardware capability

• The difference in irradiation times of the two wavelengths acquisition must be greater than the curve (left graph) for elongation to appear

• If multiple particles appears within 80x80 pixel image, accurate particle recognition cannot be performed Ø must adjust the density and the velocity of the flowing particles

(less than the linear plot)

Fig. 8. Relation between velocity and max irradiation time (left) and max density (right)

Discussion

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590 nm 590 nm530 nm 530 nm

(a) (b)

Wavelength:Irradiation time: 4994 μs 4994 μs2500 μs 2500 μs

Application to flowing Hela cells

• The area of the captured cell image after binarization is 293.5 µm2

• The area size of the Hela cell is 170.6 µm2 after restoration.

Bar, 15 µm

Velocity was determined to be 1.41×10!" m/s

Fig. 9. Images of the flowing Hela cell before and after restoration

RestorationOriginal

True size acquired

1) Developed a system for measurement of flow velocities of each samples and reconstruction of size information from single image acquisition

2) Contribute to precise target recognition and target collection timing for diagnostic screening, such as circulating tumor.

In detail, please visit our publication of this issue:Sawa, S.; Sentoku, M.; Yasuda, K. On-Chip Multiple Particle Velocity and Size Measurement Using Single-Shot Two-Wavelength Differential Image Analysis. Micromachines 2020, 11, 1011.

Conclusions

Acknowledgments

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This research was funded by research and development projects of the

Industrial Science and Technology Program, the New Energy and Industrial

Technology Development Organization (NEDO, P08030), JSPS KAKENHI

Grant Number JP17H02757, JST CREST program, and Waseda University

Grant for Special Research Projects(2016S-093, 2017B-205, 2017K-239,

2018K-265, 2018B-186, 2019C-559), the Leading Graduate Program in

Science and Engineering for Waseda University from MEXT, Japan.

We would like to thank A. Hattori, M. Odaka, and all the members ofYasuda laboratory for their kind support and collaborations.