Phase imaging microscopy for the diagnostics of plasma-cell interaction
Yolanda Ohene, Ilya Marinov, Lucie de Laulanié, Corinne Dupuy, Benoit Wattelier, and Svetlana Starikovskaia
Citation: Applied Physics Letters 106, 233703 (2015); doi: 10.1063/1.4922525
View online: http://dx.doi.org/10.1063/1.4922525
View Table of Contents: http://scitation.aip.org/content/aip/journal/apl/106/23?ver=pdfcov
Published by the AIP Publishing
Articles you may be interested in
Probing the coupled adhesion and deformation characteristics of suspension cells
Appl. Phys. Lett. 105, 073703 (2014); 10.1063/1.4893734
Total three-dimensional imaging of phase objects using defocusing microscopy: Application to red blood cells
Appl. Phys. Lett. 104, 251107 (2014); 10.1063/1.4884420
Single beam Fourier transform digital holographic quantitative phase microscopy
Appl. Phys. Lett. 104, 103705 (2014); 10.1063/1.4868533
Quantitative phase imaging of human red blood cells using phase-shifting white light interference microscopy
with colour fringe analysis
Appl. Phys. Lett. 101, 203701 (2012); 10.1063/1.4767519
Waveguide evanescent field fluorescence microscopy: Thin film fluorescence intensities and its application in cell
biology
Appl. Phys. Lett. 92, 233503 (2008); 10.1063/1.2937840
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: 37.97.96.30
On: Mon, 15 Jun 2015 07:31:17
APPLIED PHYSICS LETTERS 106, 233703 (2015)
Phase imaging microscopy for the diagnostics of plasma-cell interaction
,2,a) Corinne Dupuy,3
Yolanda Ohene,1,a) Ilya Marinov,1,3,a) Lucie de Laulanie
2
1
Benoit Wattelier, and Svetlana Starikovskaia
1
Laboratoire de Physique des Plasmas UMR 7648 (CNRS, Ecole Polytechnique, Universit
e Pierre et Marie
Curie, Universit
e Paris Sud, Observatoire de Paris), route de Saclay, Palaiseau, France
2
PHASICS S.A., B^
atiment Explorer, Espace Technologique de Saint Aubin, 91190 Saint Aubin, France
3
Laboratory of Genetic Stability and Oncogenesis, (UMR8200), National Center for Scientific Research
(CNRS), Universit
e Paris-Sud, Institut Gustave Roussy, Villejuif, France
(Received 6 April 2015; accepted 3 June 2015; published online 12 June 2015)
Phase images of biological specimens were obtained by the method of Quadriwave Lateral
Shearing Interferometry (QWLSI). The QWLSI technique produces, at high resolution, phase
images of the cells having been exposed to a plasma treatment and enables the quantitative
analysis of the changes in the surface area of the cells over time. Morphological changes in
the HTori normal thyroid cells were demonstrated using this method. There was a comparison
of the cell behaviour between control cells, cells treated by plasma of a nanosecond dielectric
barrier discharge, including cells pre-treated by catalase, and cells treated with an equivalent
amount of H2O2. The major changes in the cell membrane morphology were observed at only
5 min after the plasma treatment. The primary role of reactive oxygen species (ROS) in this
degradation is suggested. Deformation and condensation of the cell nucleus were observed
2–3 h after the treatment and are supposedly related to apoptosis induction. The coupling of
the phase QWLSI with immunofluorescence imaging would give a deeper insight into the
C 2015 AIP Publishing LLC.
mechanisms of plasma induced cell death. V
[http://dx.doi.org/10.1063/1.4922525]
Plasma application in biology and medicine is a new
extensively developing field exploring interaction of nonequilibrium plasma with living cells or tissue.1–4 Different
factors, related to the plasma action, may be biologically relevant. They are the production of reactive oxygen and nitrogen species (ROS and RNS); atoms and radicals;
electronically excited states; electric field; and UV A, B, and
C emission. Recent research demonstrated the primary role
of ROS5 and particularly hydrogen peroxide as the mediator
of the cell death.6,7 Recently, it was suggested that the electric field and charged particles can improve ROS uptake and
diffusion in gelatin model.8 The difficulty in assessing the
role of each of the plasma agents is mainly due to the strong
difference between the typical time scale of plasma formation and the time delay prior to first observable response in
living cells. The effect therefore seems to be governed by
some intermediates.9 The aim of this paper is to test a high
contrast phase imaging microscopy for visualization of early
morphological changes occurring in cells under plasma
treatment.
Phase images of biological specimens were obtained by
the method of Quadriwave Lateral Shearing Interferometry
(QWLSI, Phasics10). This technique measures the optical
path difference (OPD) induced by the refractive index distribution in the cell. The optical path difference is the accumulation, due to the changes in a local refractive index, of the
phase delay along the cell thickness. For a typical mammalian cell, the maximum phase shift over the whole cell is on
a)
Y. Ohene, I. Marinov, and L. de Laulanie contributed equally to this work.
0003-6951/2015/106(23)/233703/5/$30.00
the order of a few hundreds of nanometers. The different organelles usually introduce the OPDs of tens of nanometers,
whereas vesicles or fibers can induce the OPDs of 1 nm or
lower. In this study, the local OPD measurement is achieved
by using a wavefront sensor, SID4Bio, consisting of a modified Hartmann mask (MHM) and a CCD camera, added to
the lateral port of a non-modified inverted bright field microscope. Such a wave front sensor measures the phase u ,
which is related to the argument of the complex electromagnetic field E(x,y):
Eðx; yÞ ¼ aðx; yÞeiuðx;yÞ ;
(1)
where a(x,y) is the field amplitude.
This phase is often related to the so-called optical path
difference W
uðx; yÞ ¼ k0 W:
(2)
Here, k0 ¼ 2p=k0 , is the wave vector and k0 the light
wavelength.
The optical path difference (OPD) is accumulated during propagation through the sample due to the variation of
light velocity propagating through the objects with varying
refractive index. To measure the wave front, the incident
light is diffracted by a complex amplitude diffraction grating
(MHM,11 Modified Hartmann Mask) into four replicas.
These replicas interfere on a CCD detector, which records
their interferogram. The acquired intensity exhibits a
deformed sinusoidal pattern. The deformation amplitude is
proportional to the gradients along x and y
106, 233703-1
C 2015 AIP Publishing LLC
V
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: 37.97.96.30
On: Mon, 15 Jun 2015 07:31:17
233703-2
Ohene et al.
Appl. Phys. Lett. 106, 233703 (2015)
2p
2p @W
xþ z
I ðr; zÞ ¼ I0 1 þ cos
p
p @x
2p
2p @W
yþ z
þ cos
p
p @y
"
!
1
2p
2p
@W
þ cos
ð x þ yÞ þ z
2
p
p @ ð x þ yÞ
!#)
2p
2p
@W
;
þ cos
ð x yÞ þ z
p
p @ ð x yÞ
(3)
where Io is a incident intensity, p is the grating period, and z
is the distance from the grating to the detector. To recover
the pattern deformation, the intensity signal is deconvolved
in the Fourier domain around the 1/p carrier frequencies. In
addition, the DC Fourier domain is used to reconstruct the
intensity image. The phase gradients are finally numerically
integrated, leading to the optical path difference. In this paper, we will show OPD images resulting from this treatment.
Technical details can be found elsewhere.10,12
The discharge configuration when at least one of the
electrodes is covered with dielectric is called dielectric barrier discharge (DBD). The DBDs are widely known and used
for generation of active species,13 but they were only
recently applied for the treatment of biological specimen.5,14
For cell treatment in plastic Petri dishes, we used nanosecond DBD (described elsewhere15) plasma. Briefly, short high
voltage (HV) pulses of 9 kV (pulse amplitude is doubled on
the electrode) and 30 ns duration were generated by a commercial pulser (FPG10, FID GmbH) and transmitted to the
discharge cell via 25-m long coaxial cable. The overall
energy deposited in the plasma for one HV pulse was about
18 mJ. The plasma was produced in 2 mm air gap between
the open flat high-voltage electrode and the medium in the
plastic petri dish. The ground electrode was placed below the
petri dish. For all plasma treatments, 10 000 pulses at a frequency 300 Hz were applied.
Inverted bright field TE2000 Nikon microscope
equipped with a PHASICS SID4BIO wave front sensor was
used to obtain the OPD images of reference cells and plasma
treated cells. Two Nikon microscope objectives were used: a
40 (NA ¼ 0.6) and an immersion oil 100 (NA ¼ 1.3)
combined with an additional 1.5 tube lens that enabled a
total magnification of 150.
The human thyroid epithelial cell line (HTori-3) was grown
in phenol red-free RPMI 1640 (Invitrogen, Inc.), supplemented
with 1% (v/v) antibiotics/antimycotics (Invitrogen), 2 mmol/L
of L-glutamine (Invitrogen), and 10% (v/v) FCS (PAA
Laboratories). In all experiments (details shown in Table I),
1.5 105 cells were plated in 35 mm petri dish two days prior
to treatment. At the time of treatment, the cells reached
60%–70% confluence. The cells were washed twice with warm
Phosphate Buffer solution (PBS) supplemented with CaCl2 and
MgCl2 and exposed to nsDBD plasma in 1.5 ml of PBS.
Production of hydrogen peroxide was measured by fluorescence
intensity of resorufin at 595 nm, produced in reaction of cellimpermeable 10-acetyl-3,7-dihydroxyphenoxazine (Ampliflu
Red reagent, SigmaAldrich) with H2O2 in the presence of peroxidase (HRP). For H2O2 treatment, the equivalent to plasma
produced H2O2 concentration of 100 lM was obtained by dilution of a standard 30% H2O2 solution (Merck) in PBS with
CaCl2 and MgCl2. For experiments with catalase (specific enzymatic H2O2 scavenger) (Merck), 400 U/ml were added 15 min
before the treatment. Cells were incubated from 5 min up to 1 h
in plasma treated PBS or hydrogen peroxide solution, and then,
the cells washed twice with warm PBS and supplied with a fresh
RPMI 10% SVF medium. The phase contrast images were
taken directly in petri dish on live cells T0 ¼ 1 h after the treatment or on the cells fixed with a 4% paraformaldehyde (EMS)
between microscope glass slide and cover slip when higher
magnification was needed. In the last case, the samples were
treated beforehand and could be kept at 4 C over several days.
First, microscopic analysis was made on live cells 1 h after the treatment. Figure 1(a) shows that the control cells
generally have a larger, more extended appearance compared
to the treated cells. The cell membrane is relatively smooth,
with some variation in shape and size from cell to cell. The
cells, which have been treated with H2O2 (Figure 1(b)), demonstrate a slight deformation to the cell membrane; a slight
decrease in the cell size can be seen. Significant cell shrinkage and formation of protrusions on the cell membrane are
observed after the plasma treatment (Figure 1(c)). In this
case, the cell sizes are considerably smaller than in the control or H2O2 treated samples.
The images at 150 magnification for longer time scale
are given in Figure 2. They present two examples of a representative cell in each of the conditions. The image of the
control condition after 1 h highlights some of the key
FIG. 1. Examples of the phase images,
with a numerical high pass filter for
detail enhancement, of the Htori thyroid cells, at magnification 60 and
NA ¼ 0.6, in each treatment condition.
A scale of optical thickness (in nm) is
indicated
near
each
frame.
Visualization in the petri dish. The reference time is 1 h after the treatment.
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: 37.97.96.30
On: Mon, 15 Jun 2015 07:31:17
233703-3
Ohene et al.
Appl. Phys. Lett. 106, 233703 (2015)
FIG. 2. Phase images with a numerical
high pass filter for detail enhancement,
at 150 magnification with NA ¼ 1.3,
of each of the conditions of the cells at
different times after the treatment.
features of the thyroid cells. The nucleolus can be seen as
dark circle zone. The cell membrane is well defined, having
a slightly greater optical thickness compared to the cytosol.
Some regions of the membrane are easier to observe than
other, often these are the regions, where the membrane has
folded onto itself. The H2O2 treated cells are similar in
appearance to the control cells with only minor changes
observed.
Treatment by nanosecond DBD plasma causes severe
changes in the cells. It is evident that 1 h after the plasma
treatment, the cell membrane had deformed to have small
spine-like protrusions. Over time, the cytosol seems to be
expulsed from the cell, leaving the spine structure of stress
filaments, this can be found in Figure 2(c). Formation of
membrane protrusion and cell shrinkage after plasma treatment can be caused by significant membrane permeabilization. At the same time, the cell nucleus undergoes a strong
FIG. 3. A plot of the average surface area and the standard deviation, measured at 40 magnification and NA ¼ 0.6, of the cells in each of the treatment
conditions (C for control cells, P for plasma treatment, and H for H2O2
treatment).
deformations and condensation, which is a hallmark of apoptosis induction.
To quantify the changes in the morphology of the cells,
30 cells at 40 magnification, NA ¼ 0.6 for each condition,
were isolated and the surface area was computed (Figure 3).
The results of the measurements reveal that there is a notable
difference between the surface area of plasma treated cells
and the other conditions. In spite of the changes in morphology described above, the surface area of the control cells is
comparable to that of the H2O2 treated cells. The surface
area of the plasma treated cells is notably lower for all the
experimental conditions.
Based on the results, there was a specific interest to
understand when the changes in plasma treated cells become
detectable. Figure 4 shows phase images of the cells, which
have been treated by the plasma after a time period of 5, 10,
and 30 min, respectively. It is clear that already 5 min after
the treatment, the protrusions on the membrane begin to
appear. These structures, which form on the membrane,
seem to be unique to the plasma treatment and are not
observed after H2O2 treatment.
The role of plasma produced hydrogen peroxide in cell
death was addressed by using catalase enzymatic scavenger.
The images of the cells pre-treated by catalase before the
plasma treatment can be found in Figure 5. The cells were
imaged after a time period of 5 min, 1 h, and 2 h after the
treatment. It is clearly seen that the appearance of the cells is
very similar to the control cells after the same time periods. It
means that catalase can efficiently protect the cells (Figure 5)
from the changes caused by plasma action. On the other
hand, the equivalent to plasma produced concentration of
hydrogen peroxide (Figures 1(b) and 2(b)) leads to significantly less morphological changes in cells comparing to
plasma action.
The fact that catalase protects the cells means that the
direct effect of electric field and UV light in our conditions
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: 37.97.96.30
On: Mon, 15 Jun 2015 07:31:17
233703-4
Ohene et al.
Appl. Phys. Lett. 106, 233703 (2015)
FIG. 4. Phase images with a numerical
high pass filter for detail enhancement,
at 150 magnification and NA ¼ 1.3,
of Htori cells at the indicated time period subsequent to the plasma treatment. A scale of optical thickness (in
nm) is indicated near each frame.
FIG. 5. Phase images with a numerical
high pass filter for detail enhancement,
at 150 magnification and NA ¼ 1.3,
for plasma treated cells, which have
been pre-treated with the enzyme catalase to protect the cells from the ROS.
A scale of optical thickness (in nm) is
indicated near each frame.
TABLE I. Summary of the experimental conditions for each of the experiments.
T0, visualization in Petri dish
T0, visualization on slides
Control cells
H2O2 treatment, 100 lM
Discharge treatment, 10 000 ns pulses at 300 Hz
Catalase pretreatmenta 400 unit/ml
1h
1 h and 6 h
1h
1 h, 2 h, 4 h, and 6 h
1h
5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h
…
5 min, 1 h, and 2 h
a
With further discharge treatment, 10 000 ns pulses at 300 Hz
play a minor role comparing to ROS action in spite of high
electric field in nsDBD. As it is shown above, the cell membranes undergo strong deformations rapidly after the plasma
treatment. This can be due to the partial loss of cell membrane integrity. It should be noted that no pH changes has
been observed during the plasma treatment. Although hydrogen peroxide was shown to be one of the major plasma produced ROS,5,7 given its relatively low reactivity, other
species able to react with cell membrane as OH, O2, HOO,
and OONO18 should be considered. Recently, superoxide
anion, hydroxyl, and hydroperoxyl radical concentrations
were measured in plasma jet treated PBS using EPR spin
traps.17 Hydroxyl radical can directly interact with membrane molecules leading to breaking of covalent C-O, C-C,
and C-N bonds;18 however, its low life-time in the media
(1 ps) almost prevents OH diffusion. Peroxynitrite radical
can be produced in reaction of superoxide anion with nitric
oxide,16 and it is able to permeate cell membrane by lipid
peroxidation mechanism.19 Moreover, OONO can be scavenged by catalase’s heme group,20 although its life-time is
not well established. Hydroperoxyl can also efficiently initiate lipid peroxidation, while O2 due to its electrical charge
and fast dismutation does not seem to interact directly with
cell membrane.21,22 Further experimental study of plasma
produced reactive species that can interact with cell membrane inducing the killing of the cell is necessary.
Peroxynitrite and hydroperoxyl seem to be good candidates
and will be studied in future work.
The capabilities of the phase imaging technique,
Quadriwave Lateral Shearing Interferometry as a diagnostic
for the plasma action on living cells have been demonstrated.
The QWLSI produces phase images of the cells and enables
the quantitative analysis of the changes in the surface area of
the cells over time. The minimum optical path difference
registered by QWLSI technique in axial direction is below
1 nm (RMS). Thanks to high contrast of OPD images, the
cell membrane is clearly visible and its deformations due to
cell modification are easily detected.
The morphological changes in the HTori normal thyroid
cells were demonstrated on the time scale 5 mins–6 h after
the treatment. A comparison of the cell behaviour between
control cells, H2O2 treatment, and a treatment by plasma of a
nanosecond dielectric barrier discharge shows that the
plasma treatment resulted in significant cell membrane damage starting already a few minutes after the treatment.
Nucleus deformation was observed several hours after the
treatment. To reveal the type of cell death, immunofluorescence will be combined with QWLSI in the future work. The
role of short-living species in membrane lipid peroxidation is
suggested and will be examined in more detail in future
experiments.
The work of LPP and IGR scientific teams was partially
supported by the PEPS MelaNan Project (CNRS). Work of I.
Marinov was supported by PostDoc Project from Plas@Par,
and work of Y. Ohene was supported by Master Plas@Par
Program, both with financial support managed by ANR as
part of the Program “Investments for the Future” (ANR-11IDEX-0004-02).
1
G. Fridman, G. Friedman, A. Gutsol, A. B. Shekhter, V. N. Vasilets, and
A. Fridman, Plasma Process. Polym. 5, 503 (2008).
2
K.-D. Weltmann, M. Polak, K. Masur, T. von Woedtke, J. Winter, and S.
Reuter, Contrib. Plasma Phys. 52, 644 (2012).
3
J. Schlegel, J. K€
oritzer, and V. Boxhammer, Clin. Plasma Med. 1, 2
(2013).
4
D. B. Graves, Clin. Plasma Med. 2, 38 (2014).
5
M. Vandamme, E. Robert, S. Lerondel, V. Sarron, D. Ries, S. Dozias, J.
Sobilo, D. Gosset, C. Kieda, B. Legrain, J.-M. Pouvesle, and A. Le Pape,
Int. J. Cancer 130, 2185 (2012).
6
I. Marinov, Ph.D. thesis, University Paris Sud, France (2013).
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: 37.97.96.30
On: Mon, 15 Jun 2015 07:31:17
233703-5
7
Ohene et al.
S. Bekeschus, J. Kolata, C. Winterbourn, A. Kramer, R. Turner, K. D.
Weltmann, B. Br€oker, and K. Masur, Free Radical Res. 48, 542 (2014).
8
E. J. Szili, J. W. Bradley, and R. D. Short, J. Phys. D. Appl. Phys. 47,
152002 (2014).
9
M. Hoentsch, T. von Woedtke, K.-D. Weltmann, and J. Barbara Nebe,
J. Phys. D. Appl. Phys. 45, 025206 (2012).
10
P. Bon, G. Maucort, B. Wattellier, and S. Monneret, Opt. Express 17(15),
13080 (2009).
11
J. Primot and N. Guerineau, Appl. Opt. 39(31), 5715 (2000).
12
P. Bon, S. Lecart, E. Fort, and S. Lev^eque-Fort, Biophys. J. 106(8), 1588
(2014).
13
U. Kogelschatz, Plasma Chem. Plasma Process. 23(1), 1 (2003).
14
G. Fridman, A. Shereshevsky, M. M. Jost, A. D. Brooks, A. Fridman, A. Gutsol,
V. Vasilets, and G. Friedman, Plasma Chem. Plasma Process. 27, 163 (2007).
Appl. Phys. Lett. 106, 233703 (2015)
15
A. Duval, I. Marinov, G. Bousquet, G. Gapihan, M. Svetlana, A.
Rousseau, and A. Janin, PLoS One 8, 1 (2013).
D. B. Graves, J. Phys. D. Appl. Phys. 45, 263001 (2012).
17
H. Tresp, M. U. Hammer, J. Winter, K.-D. Weltmann, and S. Reuter,
J. Phys. D. Appl. Phys. 46, 435401 (2013).
18
M. Yusupov, A. Bogaerts, S. Huygh, R. Snoeckx, A. C. T. van Duin, and
E. C. Neyts, J. Phys. Chem. C 117, 5993 (2013).
19
S. Marla, J. Lee, and J. Groves, J Proc. Natl. Acad. Sci. U.S.A. 94, 14243
(1997).
20
L. Gebicka and J. Didik, J. Inorg. Biochem. 103, 1375 (2009).
21
J. Aikens and T. A. Dix, J. Biol. Chem. 266, 15091 (1991), available at
http://www.jbc.org/content/266/23/15091.
22
F. Liu, P. Sun, N. Bai, Y. Tian, H. Zhou, S. Wei, Y. Zhou, J. Zhang, W.
Zhu, K. Becker, and J. Fang, Plasma Process. Polym. 7, 231 (2010).
16
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: 37.97.96.30
On: Mon, 15 Jun 2015 07:31:17