Presentation for PhD colloquium before submitting the PhD thesis at Indian institute of science, Bangalore.

1. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Optical tweezers and its use in studying red blood cells –
healthy and infected.
Presentation for Thesis Colloquium
Apurba Paul
भारतीय िव�ान सं�थान
Department of Physics
Indian Institute of Science, Bangalore
June 16, 2016
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2. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Outline
1 Introduction
2 Materials and Methods
3 fc change due to malaria infection
4 The Bystander effect
5 fc change due to drug treatment
6 Clinical sample
7 Conclusion
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3. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Outline
1 Introduction
2 Materials and Methods
3 fc change due to malaria infection
4 The Bystander effect
5 fc change due to drug treatment
6 Clinical sample
7 Conclusion
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4. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Optical tweezers
What is Optical Tweezers?
It is an instrument which uses a tightly focused laser beam to trap a
microscopic particle in three dimensions.
Advantages
Manipulating single cells without mechanical contact.
Applying and measuring forces of the order of piconewtons.
Applications
Studying single cells and single molecules.
Molecular motors and kinesin motors.
Unzipping DNA or RNA.
Studying propagating waves in a 3D array of traps created by
hologram or SLM.
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5. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Optical tweezers
What is Optical Tweezers?
It is an instrument which uses a tightly focused laser beam to trap a
microscopic particle in three dimensions.
Advantages
Manipulating single cells without mechanical contact.
Applying and measuring forces of the order of piconewtons.
Applications
Studying single cells and single molecules.
Molecular motors and kinesin motors.
Unzipping DNA or RNA.
Studying propagating waves in a 3D array of traps created by
hologram or SLM.
A Paul (IISc) Colloquium June 16, 2016 4 / 41

6. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Optical tweezers
What is Optical Tweezers?
It is an instrument which uses a tightly focused laser beam to trap a
microscopic particle in three dimensions.
Advantages
Manipulating single cells without mechanical contact.
Applying and measuring forces of the order of piconewtons.
Applications
Studying single cells and single molecules.
Molecular motors and kinesin motors.
Unzipping DNA or RNA.
Studying propagating waves in a 3D array of traps created by
hologram or SLM.
A Paul (IISc) Colloquium June 16, 2016 4 / 41

7. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Theory of trapping
Different regime
Mie regime: when particle diameter is larger than wavelength, i.e.
d λ
Rayleigh regime: when particle diameter is smaller than
wavelength, i.e. d λ
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8. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Mie regime
When λ d
Momentum change of photon.
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9. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Mie regime
When λ d
Trapping by refraction
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10. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Malaria
Malaria Facts
Malaria is one of the severe human diseases, and a public health
problem in India.
19.8 crore confirmed cases and 5.84 lakh death have been reported
worldwide in 2013.
In India 8.8 lakh cases and 440 deaths reported in 2013.
Countries from tropical and sub-tropical regions are affected by
malaria.
It is caused by Plasmodium parasite.
There are several species but P. falciparum and P. vivax are most
common causes of human malaria.
Malaria Report 2014, World Health Organization, December 2015.
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11. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Pathogenesis
RBC in blood vessels
Knobs appear on the
RBC surface.
RBC becomes sticky
and adheres to other
surfaces.
Causes sequestering
and rosetting.
Blocks capillaries and
venules, interrupting
oxygen flow.
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12. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Diagnosis
Microscopic examination (thick and thin smear test).
Most reliable method of diagnosis, but takes long time and requires
a trained technician.
Rapid diagnostic test (antigen test).
Fastest diagnostic method, takes only 15 – 20 minutes, less
sensitive, requires a minimum of 20 parasites per µl.
Molecular method.
Highly sensitive, can detect 1 parasite per µl. Highly expensive
reagents and instrument are required.
Fluorescence method.
Better sensitivity than traditional microscopy, but can give false
positive results due to auto fluorescence substances in the blood.
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13. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Outline
1 Introduction
2 Materials and Methods
3 fc change due to malaria infection
4 The Bystander effect
5 fc change due to drug treatment
6 Clinical sample
7 Conclusion
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14. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Experimental Setup
Schematic Diagram
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15. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Picture of Experimental Setup
A
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16. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Picture of Experimental Setup
B
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17. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Trap stiffness
Definition
The trap stiffness is the force required to displace the trapped
particle by unit length from the trap center.
Measuring trap stiffness
There are several methods to measure trap stiffness
Escape force method.
Drag force method.
Equipartition method.
Step response method.
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18. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Trap stiffness
Definition
The trap stiffness is the force required to displace the trapped
particle by unit length from the trap center.
Measuring trap stiffness
There are several methods to measure trap stiffness
Escape force method.
Drag force method.
Equipartition method.
Step response method.
Power spectrum method.
A Paul (IISc) Colloquium June 16, 2016 15 / 41

19. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Trap stiffness
Definition
The trap stiffness is the force required to displace the trapped
particle by unit length from the trap center.
Measuring trap stiffness
There are several methods to measure trap stiffness
Escape force method.
Drag force method.
Equipartition method.
Step response method.
Power spectrum method.
A Paul (IISc) Colloquium June 16, 2016 15 / 41

20. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Power spectrum method
The power spectrum of the
thermal fluctuation of bead is
Lorentzian, and is given by
P(f) = kBT
2π2γ
1
f2
c +f2 .
Here fc is called corner
frequency.
Trap stiffness k is given by
k = 2πγfc.
We do Lorentzian fit of power
spectrum to get fc.
k and hence fc depends on a
variety of physical properties of
the trapped particle; laser power;
and the medium.
Power spectrum
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21. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
RBC sample preparation
Blood sample was first centrifuged at 2000 rpm for
15 minutes.
RBCs settled down at the bottom.
RBCs were then washed in PBS.
RBCs were then suspended in 1× PBS.
Individual RBCs were then trapped in optical
tweezers.
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22. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Outline
1 Introduction
2 Materials and Methods
3 fc change due to malaria infection
4 The Bystander effect
5 fc change due to drug treatment
6 Clinical sample
7 Conclusion
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23. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Change in fc due to malaria
Physical properties of RBC changes due to
malaria.
Since fc depends on physical property of
trapped object, it may change due to malaria.
The following study was to verify if we can
observe any change.
RBCs were separated from whole blood by
centrifugation, and then suspended in PBS for
measurement.
fc of 50 RBCs were measured for each sample.
Mean and standard deviation of fc were then
compared.
RBC before after trapping
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24. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
fc vs laser power of bead
fc of polystyrene bead should
vary linearly with laser power.
First experiment was to verify
it.
Figure shows fc of bead varies
linearly and going through
origin. fc vs power of trapping laser
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25. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
fc vs laser power of RBC
fc of RBC also changes
linearly with laser power
Interestingly infected RBC
shows higher slope than
normal RBC.
fc vs laser power for RBC
40
30
20
10
0
CornerFrequency(Hz)
4003002001000
Laser Power (mW)
Normal RBC
Infected RBC
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26. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Histogram
fc of nRBC and iRBC
Mean fc σ
nRBC 26.9 Hz 1.95 Hz
iRBC 32.0 Hz 3.3 Hz
Showing histogram of
fc at 300 mW laser
power.
Mean fc increases by
20%
Standard deviation
increases by 200%
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27. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Stage Specific
fc for different stage and time
¯fc (Hz) σ (Hz)
Normal RBC 29.4 ± 0.08 1.3 ± 0.08
Ring 36.5 ± 0.3 4.0 ± 0.3
Trophozoite 36.1 ± 0.02 3.6 ± 0.2
Mean fc does not
change with stage of
infection.
Mean fc does not
change with duration
of infection.
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28. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Outline
1 Introduction
2 Materials and Methods
3 fc change due to malaria infection
4 The Bystander effect
5 fc change due to drug treatment
6 Clinical sample
7 Conclusion
A Paul (IISc) Colloquium June 16, 2016 24 / 41

29. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
The bystander effect
Parasitemia count of cultured sample is 5–10%.
In previous experiment no attempt was made to
select hosting RBCs.
Not all measured RBCs from infected sample were
hosting the parasite.
But we have seen increase in ¯fc for all.
RBCs not hosting parasite were also getting affected.
We called it as bystander effect.
In the following experiments we have studied
bystander effect in detail.
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30. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
The bystander effect
Experimental detail
In the first experiment:
We have separated hosting and non-hosting malaria infected
RBCs.
Then fc was measured for both of them along with nRBCs.
¯fc and σ was calculated.
In the second experiment:
Healthy RBCs were collected from O-positive donors.
Healthy RBCs were incubated in spent medium.
RBCs were taken out from spent medium every 6 hr for a
total of 48 hr.
Mean and standard deviation of fc distributions were
calculated for each of them.
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31. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Purified RBCs
fc nRBC and non-hosting mRBC
8
6
4
2
0
4035302520
RBC type ¯fc (Hz) σ (Hz)
nRBC 25.6 ± 0.3 1.8
Hosting mRBC 31.3 ± 0.5 2.6
Non–hosting mRBC 30.0 ± 0.4 2.2
RBC type z-score p-value H◦ status
Normal & hosting −9.01 ∼ 10−15
Rejected
Normal & non-hosting −7.73 ∼ 10−15
Rejected
Hosting & non-hosting 1.91 0.28 Accepted
Hosting and non-hosting
RBC are showing same
change.
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32. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
nRBC incubated in spent media
¯fc with incubation time
32
30
28
26
5040302010
¯fc starts increasing
around 12 hr of
incubation.
At around 30 hr ¯fc
become almost equal
to iRBC.
After that it remains
constant.
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33. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Outline
1 Introduction
2 Materials and Methods
3 fc change due to malaria infection
4 The Bystander effect
5 fc change due to drug treatment
6 Clinical sample
7 Conclusion
A Paul (IISc) Colloquium June 16, 2016 29 / 41

34. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Introduction
We have seen that some released substance may be
responsible for bystander effect.
Experiment was done to identify released substance.
We treated RBC with different inhibitors (db-cAMP,
cd73, Diamide).
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35. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Materials and Methods
Inhibitors were prepared by dissolving it in sterile
DMSO and stored at -20°C.
Inhibitors then added to the RBC at 5 µM
concentration for 30 minutes.
RBCs were then taken out and washed and then
suspended in 1× PBS.
This was then taken for the measurement.
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36. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Results
fc for drug treated RBCs
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37. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Results
p-value for RBC ± inhibitors
Type 1 Type 2 P-value
nRBC+Diamide nRBC−Diamide 0.05
iRBC+Diamide iRBC−Diamide 0.05
nRBC+db-cAMP nRBC−db-cAMP 4.0 × 10−9
iRBC+db-cAMP iRBC−db-cAMP 0.047
nRBC+cd73 Inhibitor nRBC−cd73 Inhibitor 1.70 × 10−6
iRBC+cd73 Inhibitor iRBC−cd73 Inhibitor 5.58 × 10−5
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38. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Outline
1 Introduction
2 Materials and Methods
3 fc change due to malaria infection
4 The Bystander effect
5 fc change due to drug treatment
6 Clinical sample
7 Conclusion
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39. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Corner frequency
fc for clinical sample
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40. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Folding time
Folding time for nRBC and iRBC
nRBC iRBC
Folding time 0.80 s 1.33 s
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41. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Un-folding time
Un-folding time for nRBC and iRBC
nRBC iRBC
Un-folding time 26.67 s 11.67 s
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42. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Outline
1 Introduction
2 Materials and Methods
3 fc change due to malaria infection
4 The Bystander effect
5 fc change due to drug treatment
6 Clinical sample
7 Conclusion
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43. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Conclusion
We have demonstrated that fc increases due to malaria.
We have also seen that RBCs which are not hosting the parasite but
are from an infected sample also show changes – the bystander effect.
Normal RBCs incubated in spent medium also show similar changes
after 12 hr of incubation.
We have also identified the released substances responsible for
bystander effect by drug treatment.
Clinical samples (both falciparum and vivax) also show similar
changes and bystander effect.
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44. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Acknowledgment
First of all I would like to thank my supervisor Prof Vasant
Natarajan.
I also would like to thank Prof Utpal S Tatu.
I thank Raghuveer, all my lab seniors, and current lab mates.
I thank everyone from physics office and work shop.
I thank CSIR for fellowship.
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45. भारतीय िव�ान सं�थान
Introduction Materials and Methods fc change Bystander effect drug treated Clinical sample Conclusion Acknowledgment
Thank you for your kind attention.
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46. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Outline
8 Introduction
9 Materials & methods
10 fc change
11 The bystander effect
12 Drug treated RBC
13 Clinical sample
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47. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Mie regime
3D view
Geometry of single beam
gradient in ray optics
approach.
Center of the bead is at
the axis of beam.
Calculate forces for single
ray.
Sum of all forces will give
us total force.
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48. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Mie regime
Ray inside bead
When ray enters into bead
there will be multiple
reflection and refraction.
Power of the n’th emergent
beam will be PTRn, where
n = 0, 1, 2, ....
Corresponding angle will be
α + nβ.
ˆz is direction of propagation
of beam.
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49. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Mie regime
When λ d
Scattering force
Fz = Fs =
n2P
c
1 + R cos 2θ −
T2
[cos(2θ − 2θr) + R cos 2θ]
1 + R2 + 2R cos 2θr
Gradient force
Fy = Fg =
n2P
c
R sin 2θ −
T2
[sin(2θ − 2θr) + R sin 2θ]
1 + R2 + 2R cos 2θr
We will get stable trap only if Fg > Fs.
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50. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Rayleigh regime
When d λ.
Diffraction of the beam can not be ignored, so ray optics can’t be
used.
Electromagnetic theory or wave optics theory of light is used.
Bead can be considered as a dielectric in this situation.
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51. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Rayleigh regime
Scattering force
F scat(r) =
Cpr S(r, t) T
c/n2
= ˆz
n2
c
CprI(r)
Where Cpr is scattering cross section and is given by
Cpr =
8
3
π (ka)4
a2 m2 − 1
m2 + 2
Using this We get, Scattering force
F scat(r) = ˆz
n2
c
8
3
π(ka)4
a2 m2
− 1
m2 + 2
2
2P
πw2
0
× exp
2(˜x + ˜y)
1 + (2˜z)2
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52. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Rayleigh regime
When λ d
Gradient force is
F grad(r) =
α
2n2 0c
I(r)
Scattering force is
F scat(r) = ˆz
n2
c
8
3
π(ka)4
a2
n1
n2
2
− 1
n1
n2
2
+ 2
2
2P
πw2
0
× exp
2(˜x + ˜y)
1 + (2˜z)2
Positive polarizability particle will be attracted towards higher intensity
Gradient force should be greater than scattering force.
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53. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Life cycle
Life cycle of P. falciparum
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54. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Blood stage
Blood stage of P. falciparum
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55. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Blood stage
Blood stage of P. falciparum
Blood stage can be separated
in three stages,
Ring stage (6 – 18 hour)
Trophozoite stage (18 – 30
hour)
Schizont stage (30 – 48
hour)
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56. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Symptoms
Malaria syndromes typically consists
Fever, Shivering, Cough, respiratory distress, Joint pain, Headache,
Watery diarrhea, Vomiting and Convulsions
Sever malaria can cause
Coma and Cerebral oedema
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57. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Outline
8 Introduction
9 Materials & methods
10 fc change
11 The bystander effect
12 Drug treated RBC
13 Clinical sample
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58. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Motorized stage
Works on statics and dynamic
friction.
Has 30 nm resolution.
Can be controlled from
computer.
Schematic of Motorized stage
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59. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Quadrant Photodetector (QPD)
We use QPD for data
acquisition.
Data saved directly to PC.
Movement of the bead is
recorded by the movement of the
backscattered light on qpd.
Acquisition rate is 16 KHz and
number of data point is 100000.
QPD
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60. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Quadrant Photodetector (QPD)
Circuit diagram of QPD
Vx =
(VA + VD) − (VB + VC )
VA + VB + VC + VD
Vy =
(VA + VB) − (VC + VD)
VA + VB + VC + VD
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61. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Trap stiffness
Escape force method
Measures minimum force required to remove bead
from trap.
Force applied by viscus drag.
Force is given by Fd = 6πηav.
Sensitive position detector is not required.
Less accurate.
Measurement is done at the non linear region of trap.
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62. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Trap stiffness
Drag force method
Measure the displacement by applying
known force and then calculate stiffness.
Trap stiffness k is given by k = F
δx .
Force can be applied by moving medium
with respect to trap.
Displacement should be within Hookeian
region.
More accurate than escape force method.
Accurate and well calibrate position
detector required.
Precise knowledge of drag force required.
Drag force method
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63. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Trap stiffness
Equipartition method
Use equipartition theorem to measure
trap stiffness.
Formula 1
2k σ2
x = 1
2kBT.
High resolution and well calibrated
position detector is required.
Precise knowledge of viscosity is
needed.
Shape, size and optical properties of
particle is not so important.
Data acquisition electronics should
have sufficient bandwidth.
Position histogram
Countpercentage
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Position (arbitrary unit)
3.5 4
Hisogram
Gaussian fit
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64. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Trap stiffness
Step response method
If trap position is moved very fast, trapped
bead follows with time lag.
Equation: x(t) = x◦ 1 − exp k
γ t
Precise knowledge of γ is required.
Calibrated position detector is not required.
Analog bandwidth of the data acquisition
system should be high.
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65. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Trap stiffness
Power spectrum method
trapped bead executes Brownian fluctuation
constrained by harmonic potential.
Equation of motion is
m¨x(t) + γ ˙x(t) + kx(t) = (2kBTγ)1/2
η(t)
Where (2kBTγ)1/2
η(t) represents Brownian force.
Here η(t) = 0 and η(t)η(t ) = δ(t − t ).
We can ignore the first term as the measurement
interval is much higher than collision interval.
Taking fourier transform and squaring we will get
P(f) = kBT
2π2γ
1
f2
c +f2
fc is called corner frequency.
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66. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Power spectrum
Pros and cons
Require high resolution position detector tor record.
Calibration not required, any arbitrary scaling gives same
corner frequency.
Data acquisition with sufficiently high band width and low
noise needed.
Precise knowledge of viscosity needed to calculate k.
This method can be used test alignment as slight
misalignment deviates power spectrum from Lorentzian.
This is the easiest method and used in our experiment.
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67. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Null test
To test noise level of QPD.
(A): dark spectrum was
taken keeping QPD at dark.
Flat spectrum indicates less
noise. Peaks may indicate
line noise.
(B): light spectrum was
taken with all laser on
without trap.
Low frequency slope may
indicates pointing instability
and mechanical vibrations.
Dark & light spectrum
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68. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Calibration
QPD was calibrated to
know bead displacement
vs voltage relation.
Output voltage was
recorded by moving bead
for known distance.
Slope of the linear portion
is 0.6 mV/nm.
Calibration of QPD
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69. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Statistical analysis
Mean and standard error
We measured 5 fc for each RBC.
We measured fc of 25 to 50 RBC for each sample.
We plotted histogram and measured mean and
standard error.
Mean was calculated as:
µ =
1
N
N
i=1
xi ≡ ¯x
Standard error in mean was calculated as:
σµ =
σ
√
N
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70. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Statistical analysis
P-value and hypothesis testing
Hypothesis testing is statistical method to
verify if observed difference in distribution
is real or by chance.
There are two hypothesis, Null and
Alternative Hypothesis
P-value was calculated to test hypothesis.
Z-score was used to calculate p-value.
Z-score is given by
z =
µ1 − µ2
σ2
1
n1
+
σ2
2
n2
Z-score distribution
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71. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Sample preparation
In vitro culturing of P. falciparum
Using candle jar method.
At 37◦C temperature.
With 10% human O-positive serum and 2% hematocrit.
Synchronization
Synchronization of stage was done using 5% sorbitol.
We incubate infected RBC in sorbitol for 5 minute.
This will rupture the Trophozoite and Schizont stage cells and Ring
stage will remain unaffected.
Percol purification.a
RBC with different stage of infection was separated using percol density
gradient method.
Since the density of different stage is different, RBC with different
stage will be at interface of different percol concentration.
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72. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Outline
8 Introduction
9 Materials & methods
10 fc change
11 The bystander effect
12 Drug treated RBC
13 Clinical sample
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73. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Discussion
¯fc of RBC increases significantly with malaria
infection.
σ of fc distribution also increases.
¯fc does not changes with duration and stage of
infection.
Changes my be due to the change in physical
properties of RBC due to malaria infection.
A Paul (IISc) Colloquium June 16, 2016 30 / 41

74. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Outline
8 Introduction
9 Materials & methods
10 fc change
11 The bystander effect
12 Drug treated RBC
13 Clinical sample
A Paul (IISc) Colloquium June 16, 2016 31 / 41

75. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Discussion
We have seen nRBC get affected by spent media.
This confirms the “bystander effect”
This can be due to substances released to the media
by parasite.
Our next goal is to identify the “released substance”
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76. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Outline
8 Introduction
9 Materials & methods
10 fc change
11 The bystander effect
12 Drug treated RBC
13 Clinical sample
A Paul (IISc) Colloquium June 16, 2016 33 / 41

77. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Results
fc value of drug-treated RBCs
Sl.No. Inhibitors Types of RBCs Corner frequency (Hz)
1. Diamide
nRBC+diamide 22.75 ± 0.33
nRBC−diamide 21.67 ± 0.42
iRBC+diamide 27.45 ± 0.46
iRBC−diamide 28.61 ± 0.34
2. db-cAMP
nRRC+db-cAMP 24.67 ± 0.43
nRBC−db-cAMP 20.79 ± 0.41
iRBC+db-cAMP 29.35 ± 0.30
iRBC−db-cAMP 28.38 ± 0.37
3. CD73 inhibitor
nRBC+CD73 inhibitor 23.63 ± 0.41
nRBC−CD73 inhibitor 21.99 ± 0.33
iRBC+CD73 inhibitor 26.96 ± 0.40
iRBC−CD73 inhibitor 29.69 ± 0.51
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78. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Discussion
Normal RBC ± db-cAMP is showing maximum
change.
CD73 inhibitors on both nRBC and iRBC showing
prominent change.
These may be the released substance responsible for
bystander effect.
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79. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Outline
8 Introduction
9 Materials & methods
10 fc change
11 The bystander effect
12 Drug treated RBC
13 Clinical sample
A Paul (IISc) Colloquium June 16, 2016 36 / 41

80. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Introduction
All earlier works were done using cultured sample.
Sample taken from patient may differ from the
cultured sample.
So we have done this experiment to study clinical
sample.
Also culturing P. Vivax is difficult, since, it invades
mainly reticulocytes.
In India, occurence of of P. falciparum and P. vivax
are same, so we had opportunity to study P. vivax
also.
In this study we have measured folding and
un-folding time with fc.
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81. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Ethical clearance
Since the study contain human involvement we had
taken ethical clearance.
Clearance was taken from Indian Institute of Science
Ethics Committee (reference no: 25/2014)
Clearance was taken from Bangalore Medical Collage
and Research Institute Ethics Committee (reference
no: BMCRI/PS/04/2015–16)
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82. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Sample collection
Blood sample were collected from patients coming
of Bangalore Medical Collage and Research Institute.
First everything was explained to the patients and
then blood was collected.
Blood was collected in anticoagulant-containing
tube.
Blood were then kept in ice box and transported to
lab.
Blood were then prepared and used for experiment.
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83. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Discussion
Both P. vivax and P. falciparum shows about 20% increase in
corner frequency.
This shows the technique is effective for clinical sample also.
Folding time increases and un-folding time decreases due to
infection.
Though P. vivax does not invade RBCs, we have seen changes for
RBCs also.
This also strengthen the bystander effect hypothesis.
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84. भारतीय िव�ान सं�थान
Introduction Materials & methods fc change The bystander effect Drug treated RBC Clinical sample
Future outlook
We can test specificity of the changes observed.
We can directly measure changes in elasticity due to drug
treatment using dual trap.
We can use the same technique for other cells also.
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