Biological Applications of Optical Tweezers Explained

1. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Biological Applications of Optical Tweezers A Presentation for Comprehensive Exam Apurba Paul Department of Physics Indian Institute of Science, Bangalore 30 June, 2010

2. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Outline of Talk 1 Introduction 2 Experimental Setup 3 Experiment, Results and Discussion 4 Future Plan

3. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Optical Tweezers Introduction to Optical Tweezers Optical Tweezers Instrument used to trap micron size particle using tightly focused laser beam is called optical tweezers Application Optical tweezers has a wide application in the ﬁeld of physics and biology Studying and manipulating single cells Manipulating single molecules like DNA Measuring forces of order piconewton

4. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Optical Tweezers Working Principle There are two regimes to explain the trapping of the particle by optical tweezers Rayleigh Regime: When d λ Mie or Ray Optics Regime: When d λ

7. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Optical Tweezers Rayleigh Regime When d λ Trapped due to Electric Field Intensity Gradient Energy at Electric ﬁeld is U = −P · E = −αE · E = −αE2 = −αI Minimum at focus if α is positive. Trapping Due to Intensity Gradient

8. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Optical Tweezers Mie Regime When d λ can be explain using ray or geometrical optics and the change of momentum of photon due to refraction. Trapping due to momentum change

9. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Optical Tweezers Trap Stiffness And Its Measurement Trap Stiffness: The stiffness depends on several parameters Wavelength and Power of Laser Refractive index of the Bead and Medium Size and Shape of the particle Numerical aperture and magnification of the Objective Koen et. al., IEEE Journal of Selected Topics in Quantum Electronics, Vol. 2, No. 4, December 1996

14. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Optical Tweezers Trap Stiﬀness And Its Measurement Measurement There are several methods for stiﬀness measurement Escape Force Method Drag Force Method Equipartition Method Step Response Method Power Spectrum Method Koen et. al., IEEE Journal of Selected Topics in Quantum Electronics, Vol. 2, No. 4, December 1996

20. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Optical Tweezers Power Spectrum method and Corner Frequency Bead does Brownian fluctuation inside the trap. The power spectrum of the fluctuation is a Lorentzian. Given By Sx (f ) = kbT (γπ2) (f 2 c + f 2) γ = Stokes viscous drag coefficient. fc = Corner Frequency The corner frequency is related with stiffness by relation ktrap = 6π2ηdfc Figure: Brownian fluctuation 3.5 3.4 3.3 3.2 3.1 3.0 2.9 ---------Position(arbitraryunit)------> 6s543210 ---------Time(s)------->

21. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Optical Tweezers Power Spectrum method and Corner Frequency Bead does Brownian fluctuation inside the trap. The power spectrum of the fluctuation is a Lorentzian. Given By Sx (f ) = kbT (γπ2) (f 2 c + f 2) γ = Stokes viscous drag coefficient. fc = Corner Frequency The corner frequency is related with stiffness by relation ktrap = 6π2ηdfc Figure: Brownian fluctuation 3.5 3.4 3.3 3.2 3.1 3.0 2.9 ---------Position(arbitraryunit)------> 6s543210 ---------Time(s)-------> Figure: Power Spectrum 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 ---------Amplitude--------> 1 10 100 1000 -------Frequency (Hz)------->

22. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Optical Tweezers Power Spectrum method and Corner Frequency Bead does Brownian fluctuation inside the trap. The power spectrum of the fluctuation is a Lorentzian. Given By Sx (f ) = kbT (γπ2) (f 2 c + f 2) γ = Stokes viscous drag coefficient. fc = Corner Frequency The corner frequency is related with stiffness by relation ktrap = 6π2ηdfc Figure: Brownian fluctuation 3.5 3.4 3.3 3.2 3.1 3.0 2.9 ---------Position(arbitraryunit)------> 6s543210 ---------Time(s)-------> Figure: Power Spectrum 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 ---------Amplitude--------> 1 10 100 1000 -------Frequency (Hz)------->

23. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Malaria Introduction to Malaria Malaria is a mosquito borne disease Malaria transmitted by the bite of female Anopheles Per year 200-300 million cases of infection are found, and 1-2 million people die due to malaria. There are ﬁve species of Malaria Plasmodium falciparum Plasmodium vivax Plasmodium ovale Plasmodium malariae Plasmodium knowlesi Aﬀects liver and damages the Red Blood Cell (RBC).

24. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Malaria Life Cycle of Plasmodium While taking blood meal from infected human, female Anopheles gets infected and the protozoa grows inside its salivary gland to form sporozoite. sporozoite enter blood stream from infected mosquito and migrate to liver. Here they multiply to merozoite and enter blood stream again. Merozoite infect RBCs and develop ring stage, tropozoite and schizont stage and multiply asexually. Then they come back to blood stream in merozoite stage Life Cycle of Plasmodium

25. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Malaria Diagnosis of Malaria Symptoms of Malaria are very non specific so, specific laboratory based diagnosis techniques to detect malaria are required. There are several Diagnostic tools Microscopic examination of blood films Antigen Test or Malaria Rapid Diagnostic Test Molecular Test Miller et. al. Nature, Vol 415, 2002

29. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Schematic Experimental Setup Beam Splitter Cube Nd:YAG 1064 nm Laser Power 300mw He:Ne 632 nm Laser Power 5mw PC QPD Eyepiece λ/2 wave plate 1.44 NA 100X Oil Immersion Objective Sample IR Block M4 M3 M1 M2 L1 L2 A B C D x=(A+C)-(B+D) y=(A+B)-(C+D) Quadrant Photo Detector(QPD)

30. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Photo Experimental Setup

31. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Photo Experimental Setup

32. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Checking Checking QPD Kept QPD at dark place and took the data. 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 ----Amplitude-----> 1 10 100 1000 ----Frequency (Hz)-----> Kept the He:Ne laser on and checked the power spectrum. 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 ----Amplitude-----> 1 10 100 1000 ----Frequency (Hz)----->

33. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Checking Checking Alignment We trapped single 3 µm polystyrene bead. We measured corner frequency at diﬀerent power from 250 mW to 350 mW Plot of the fc vs power is a linear as shown. 50 40 30 20 10 0 -----Cornerfrequency-----> 4003002001000 ----Power of Laser (mw)---->

34. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Sample Preparing RBC Sample We have taken RBC samples from the Prof. Utpal Tatu’s lab which are suspended in PBS to be trapped in optical tweezers Figure: Untrapped RBC Figure: Trapped RBC Photograph showing how an RBC reorient itself when it is trapped.

35. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Experiment & Result Power vs fc for Infected and Normal RBCs We trapped single RBC We measured fc for different power which increases linearly with laser power and it passes through origin. But the slope increases significantly for infected RBC than normal. Which shows clear difference between normal and Infected RBC 40 30 20 10 0 CornerFrequency 4003002001000 Laser Power (mw) Normal RBC, Slope = 0.0868 Infected RBC, Slope = 0.1032 Power dependence of fc . Here slope for normal RBC is 0.08 and for Infected RBC is 0.10

36. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Experiment & Result Distribution of Corner Frequency We have measured the corner frequency of the ensemble of 50 RBCs for both normal and infected RBCs and checked the histogram 8 6 4 2 0 NoofCounts 40353025 fc (Hz) Histogram for Normal RBC Mean fc =26.964 standerd deviation =1.95232 Mean fc = 26.9 Standard deviation (σ) = 1.95 4 3 2 1 0 Noofcounts 40353025 Corner frequency(Hz) Histogram for Infected RBC Mean fc =32.2 Standerd Deviation =5.29 Mean fc = 32.2 Standard deviation(σ) = 5.29 So mean fc is increasing almost by 20% and σ by 200%.

37. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Experiment & Result fc at diﬀerent stages of infection We measure fc for every stage of infection for every two hour. We see fc is independent of time and stages of infection

38. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Experiment & Result Result of this work has recently published in the following paper: Vishal Saraogi, P. Padmapriya, Apurba Paul, Utpal S. Tatu, Vasant Natarajan Change in spectrum of Brownian ﬂuctuations of optically trapped red blood cells due to malarial infection, Journal of Biomedical Optics, Vol 15 No 3 (2010)

39. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Experiment & Result Bystander Effect There are only 2-5% of RBCs actually infected by malaria. We have measured fc by selecting RBCs randomly from the sample. So most of the RBCs for which we have measured the fc are not actually hosting the parasite. But the above result shows an increase in corner frequency for all RBCs from infected sample. We call it as Bystander Effect. So it is very much likely that the medium is also affected by the infection.

40. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Experiment & Result fc for RBC samples that actually host the parasite and those that do not This experiment was done to check if the RBCs which do not host the parasite from the infected sample are actually aﬀected or not. We separate the actual host and non host and see the histogram of fc 5 4 3 2 1 0 Noofcounts 40353025 Frquency (Hz) Figure: Histogram for actual host RBCs, Mean fc =32.0 0.20 0.15 0.10 0.05 0.00 Noofcounts 40353025 Frequency (Hz) Figure: Histogram for non host RBCs, Mean fc =31.4

41. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Experiment & Result fc for normal RBC in the spent medium This experiment was to check if medium is aﬀected. All RBCs were taken out from infected blood and the serum was used. Normal RBCs were injected into it and incubated for 48 hrs. We then take out the RBCs and measure the fc. Mean fc is 31.3 Hz which is within 80% that of infected RBCs.

42. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Experiment & Result Time vs fc for Normal RBCs in the spent medium We incubated RBCs in the spent medium. Measure fc for every 6 hr of incubation. The variation of mean fc vs time. 32 31 30 29 28 27 Meancornerfrequency(Hz) 40302010 Time (Hr)

43. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Discussion Discussion In summary Corner frequency of power spectrum of the Brownian fluctuation of RBC is significantly increasing due Malarial infection. The change is independent of the stages of infection. Those RBCs which are actually not hosting the parasite also show increase of fc (bystander effect). We have shown that even the medium is affected. So we can say that some chemical is released in the medium due to infection, which is affecting the so called non infected RBCs from the infected pool.

44. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Future Plan We want to ﬁnd out what is causing this change in the medium. So far we did this experiment using P falciparum only. We want to do the same experiment using P vivax We did this experiment using cultured sample. We want to test it for actual patient sample where the parasite count is even less (1-2%) We want to extend the experiment to other diseases which aﬀect the cell like cancer, leukemia, thalasemia. We want to do some single molecule experiments using DNA

45. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Acknowledgement I want to thank my supervisor Prof. Vasant Natarajan for his constant support and guidance. I extend my sincere thanks to Prof. Utpal Tatu and Pallavi for providing us samples and for valuable advices. This work was supported by the Board of Research in Nuclear Sciences (DAE), the Life Sciences Research Board (DRDO), and the Department of Biotechnology, India

46. Introduction Experimental Setup Experiment, Results and Discussion Future Plan Acknowledgement Thank You

Biological Applications of Optical Tweezers Explained

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