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Virus infectivity detection by effective refractive index using optofluidic imaging

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P.Y. Liu
P.Y. Liu
P.Y. Liu
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L.K. Chin
L.K. Chin
L.K. Chin
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Wee Ser at Nanyang Technological University
Wee Ser
Teck Choon Ayi at DSO National Laboratories
Teck Choon Ayi
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Abstract

This paper presents an optofluidic imaging system to detect influenza virus infection via co-culture of Madin Darby Canine Kidney (MDCK) cells. Influenza flu virus is a serious threat that can cause contagious infections in people in epidemic proportions. Hence, it is crucial to accurately detect and understand the morphological changes that occur in the cells when infected by the influenza virus. Recently, researchers are investigating the biophysical properties of cells and correlating them to biomedical conditions. For example, a decrease in refractive index (RI) is observed in bacterial infected cells [1]. In this paper, an optofluidic imaging system is developed to observe the change of RI in virus infected cells based on scattering signature.
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VIRUS INFECTIVITY DETECTION BY EFFECTIVE REFRACTIVE
INDEX USING OPTOFLUIDIC IMAGING
P. Y. Liu1, 2, L. K. Chin2†, W. Ser2, T. C. Ayi3, P. H. Yap3, T. Bourouina4
and Y. Leprince-Wang1†
¹ Université Paris-Est, UPEM, F-77454 Marne-la-Vallée, France
2School of Electrical and Electronic Engineering, Nanyang Technological University
Singapore 639798
3Defence Medical & Environmental Institute, DSO National Laboratories, Singapore 117510
4Université Paris-Est, ESYCOM, ESIEE Paris, F-93162 Marne-la-Vallée, France
ABSTRACT
This paper presents an optofluidic imaging system to detect influenza virus infection via co-culture of
Madin Darby Canine Kidney (MDCK) cells. Influenza flu virus is a serious threat that can cause
contagious infections in people in
epidemic proportions. Hence, it is
crucial to accurately detect and
understand the morphological changes
that occur in the cells when infected by
the influenza virus. Recently,
researchers are investigating the
biophysical properties of cells and
correlating them to biomedical
conditions. For example, a decrease in
refractive index (RI) is observed in
bacterial infected cells [1]. In this paper,
an optofluidic imaging system is
developed to observe the change of RI
in virus infected cells based on
scattering signature.
KEYWORDS: Optofluidics, Virus
infectivity, Scattering signature
INTRODUCTION
Figure 1 shows the biological
process of influenza virus infection
model. The influenza virus enters the
surface membrane cells of the lung and
throat by endocytosis. The virus RNA,
accessory proteins and RNA
polymerase are released into the
cytoplasm of the cells. A virus complex
is formed and carried into the cell
nucleus. The virus RNA replicates itself
in the cell nucleus and creates new
influenza virus particles. When the
RNA particles are increased in the
infected cells as compared to uninfected
cells, this leads to the change in the
Membrane
fusion
Invasion
Endosom
Viral genome
mRNA
Viral protein
Husking
Nucleus
Transcription
- replication
Figure 1: (a) Biological model of influenza virus infection
and (b) its effect on the scattering signature.
Uninfected cell
Infected cell
4
5
6
7
8
0246810
Normalized intensity
Scattering angle (o)
Normal
Infected
(a)
(b)
refractive index in the nucleus and
can be used as an indicator for
detection. Optofluidic
refractometers have been
developed extensively [2-4], and
in this paper, the change of
refractive index is determined by
observing the scattering signature
of infected and normal cells.
WORKING PRINCIPLES
Scattering signature has been
exploited to determine the state of
biological samples, for example the
infection of bacteriophage on
Escherichia coli [5]. When MDCK
cells are infected by influenza
virus, the multiplication of RNA
particles increases the effective
refractive index of the cells. This
change can be reflected by
measuring the scattering image of
the MDCK cells at different time
points after infection. In the
experiments, different plates of MDCK cells are cultured and infected by influenza virus. At each time point
of 1 hr, a plate of MDCK cells are trypsinized and injected to the optofluidic chip to capture the bright field
and scattering images of the MDCK cells. Microfluidics facilitate the alignment of the MDCK cells with the
incident laser for capturing of the scattering image as shown in Figure 2. The images are subsequently
analyzed to determine the size of the MDCK cells (bright field) and the distance of 1st scattering peak from
the center (scattering).
EXPERIMENTAL RESULTS AND DISCUSSIONS
Figure 3 shows an example of the scattering image of normal and infected MDCK cells. MDCK cells
with the incident laser for capturing of the scattering image as shown in Figure 2. The images are
subsequently analyzed to
determine the size of the
MDCK cells (bright field) and
the distance of 1st scattering
peak from the center
(scattering). Figure 4 shows
the statistical measurements of
normal and infected MDCK
cells at 0 hr and 5 hrs after
infection. For normal MDCK
cells, the positions of 1st peak
are relatively similar.
However, the positions of 1st
peak are significantly
decreased in infected cells
after 5-hr infection. This is
(a)
(b)
Figure 3: An example of the scattering image of (a) normal, and (b)
infected MDCK cell.
Figure 2: Schematic illustration of the optofluidic imaging of
trypsinized MDCK cells flowing in microchannel.
Laser light
Focusing lens
Microchip
Scattering signature
Microscopic image
Image and signal
processing unit
CCD sensor
CCD sensor
Microscopic light
Focusing lens
Beam splitter
correlated with the fact that infected cells have increased refractive index, and subsequently narrower
scattering bands.
CONCLUSIONS
In conclusion, a different approach to detecting virus infection has been presented. The results show
that refractive index changes due to the changes in the nucleus of the cells when MDCK cells are infected
by the influenza virus. It is imperative that a direct detection method can be conceived to monitor the
changes in the lung and throat to allow early detection and treatment of the influenza flu virus.
ACKNOWLEDGEMENT
The authors would like to acknowledge the financial support from Environmental and Water Industry
(EWI) Development Council of Singapore (Grant No. 1102-IRIS-05-02).
REFERENCES
[1] A. E. Ekpenyong, S. M. Manm, S. Achouri, C. E. Bryant, J. Guck and K. J. Chalut, “Bacterial
infection of macrophages induces decrease in refractive index,” J. Biophotonics, 6, 393, 2013.
[2] L. K. Chin, A. Q. Liu, X. M. Zhang, C. S. Lim, J. H. Ng, J. Z. Hao and S. Takahashi, “Differential
single living cell refractometry using grating resonant cavity with optical trap,” Appl. Phys. Lett., 91,
243901, 2007.
[3] L. K. Chin, A. Q. Liu, C. S. Lim, C. L. Lin, T. C. Ayi, and P. H. Yap, “An optofluidic volume
refractometer using Fabry–Pérot resonator with tunable liquid microlenses,” Biomicrofluidics, 4,
024107, 2010.
[4] W. Z. Song, A. Q. Liu, S. Swaminathan, C. S. Lim, P. H. Yap, and T. C. Ayi, “Determination of
single living cell’s dry/water mass using optofluidic chip,” Appl. Phys. Lett., 91, 223902, 2007.
[5] J. Q. Yu, W. Huang, L. K. Chin, L. Lei, Z. P. Lin, W. Ser, H. Chen, T. C. Ayi, P. H. Yap, C. H. Chen
and A. Q. Liu, “Droplet optofluidic imaging for λ-bacteriophage detection via co-culture with host
cell Escherichia coli,” Lab on a Chip, DOI: 10.1039/c4lc0042k.
CONTACT
† L. K. Chin; phone: +65-6790 6532; lkchin@ntu.edu.sg
Y. Leprince-Wang; yamin.Leprince@u-pem.fr
0
5
10
15
20
25
1000 1200 1400 1600 1800
Cell radius (µm)
1st Peak
Infected
Control
0
5
10
15
20
25
30
1000 1200 1400 1600 1800
Cell radius (µm)
1st Peak
Infected
Control
Figure 4: Significant shift in the position of 1
st
peak is observed after 5 hrs of virus infection.
0 hr
5 hrs
... Classification-based methods apply shape features, geometric features, color features, texture features, and statistical features for EM detection, such as in [51][52][53][54][55][56][57][58][59][60][61][62][63][64][65]. In [51,52,55], shape features were selected as vital information for EM detection, including contour features, area features, squareness, angular, roundness, etc. ...
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