Applications in Biofluidics

Transcription

1 Applications in Biofluidics Last Class: 1. Introduction of μpiv 2. Considerations of Microscopy in μpiv 3. Depth of Correlation 4. Physics of Particles in Micro PIV 5. Measurement Errors 6. Special Processing Methods Today s Contents: 1. PIV Thermometry 2. Evanescent Wave μpiv 3. Confocal μpiv 4. Application: Locomotion of C. elegans 5. Application: Wall Shear Stress 6. Application: Immunoassay Reactors 1

2 PIV Thermometry Computer (Post processing) Gravity driven flow Cover plate 12 bit CCD Camera (1376 x 1040 pixels) Thermocouple Emitter 610 nm Aluminum well Filter Cube Patch heater Insulator Microscope Objective Exciter 532 nm Hg lamp 60 mm Test piece Thermocouple 2

3 Principle Auto correlation Cross correlation Cross correlation 2 ( a) s o d 2, a 2 e / 2 ( b) s o d 2, c 2 e / 2 ( c) s o, c 2( d e 8M D t) / 3

4 Parabolic Profile C 30.2 C 40.6 C 50.0 C 60.0 C 70.9 C 0.2 R a d i a l L o c a t i o n Peak Width Increase (pix) Illustrates the effect of velocity gradient on the cross correlation function. 4

5 Temperature Calibration Temperature deduced using PIV o ( C) Measured temperature ( o C) Temperatures deduced using PIVT method plotted against measured temperatures. The average difference between the predicted and measured temperatures was ±2.1 C. 5

6 Evanescent Wave μpiv Snell s law For incident angles greater than the critical angle, total internal reflection occurs. 6

7 Evanescent Wave Images A comparison of (a) direct flood illumination versus (b) TIRF illumination of 200 nm diameter fluorescence polystyrene particles in water with a single 5 ns pulse from a Nd:YAG laser through a 100X objective with a NA =

8 Evanescent Wave μpiv 8

9 Confocal μpiv Ability to control depth of field. Elimination or reduction of background information away from the focal plane. Capability to collect serial optical sections from thick specimens. 9

10 Confocal Microscope (1) 10

11 Confocal Microscope (2) Nipkow disk allows point measurements to area measurements. 11

12 Confocal Microscope (3) fluorescent image confocal image 12

13 3D Reconstruction of Flow Velocity vector fields at different z positions of (a) pure water and (b) a 10% suspension of blood cells. 13

14 Flow Profiles Velocity vector fields at different z positions of (a) pure water and (b) a 10% suspension of blood cells. 14

15 Examples of Biofluidic Applications based on PIV 15

16 Locomotion of C. elegans Considerable progress has been made in the understanding of the motility of swimming micro organisms at low Reynolds number Re. The motivation in studying low Re locomotion lays in the potential impact on technological applications. The development of individual micro and nano scale artificial swimmers has rapidly increased, driven by applications such as targeted drug delivery and robotic surgery. Red Blood Cells Spermatozoa Self organizing Bacteria 16

17 What is C. elegans? ( Sex: Hermaphrodite or Male Food: E. coli (withscienceforlife.wordpress.com) 17

18 Swimming Gait The kinematics of swimming C. elegans at low Re in a viscous M9 buffer solution. 18

19 Resistive Force Theory 19

20 Velocity Fields Velocity fields and flow behavior a swimming C.elegans. The nematode shown is tracked in the buffer solution. 20

21 Velocity Fields v.s. Viscosities Effects of fluid viscosity (μ) on the kinematics of swimming C. elegans. Measurements for three kinematic metrics are shown. The annotations denote swimming speed (U), beating amplitude (A), and frequency (f), and power (P). 21

22 Velocity Fields: Force and Power Schematic illustration of the modeled nematode body shape for force and power calculation. 22

23 Concluding Remarks The analytical values of CN/CT provide reasonably accurate predictions of the nematode swimming speed. The experimental values are close to estimates using resistive force theory. The magnitudes of our measured forces are supported by recent experimental measurements of force delivered by crawling C. elegans using force sensing micropillars, with <F prop >~ 2nN(underestimated). The nematode C. elegans is a low Reynolds number swimmer. 23

24 Atherosclerosis : Wall Shear Stress The susceptibility of vascular branches to atherosclerosis is believed due in part to the unusual fluid dynamic environments that the vessel wall experiences in the regions. Fluid mechanical studies have shown that atherosclerosis may occur at branching points where the geometry is complex, a large Reynolds number and a lower than average wall shear stress. In general, the complex flow pattern is associated with a spatially nonuniform shear stress and wall curvature. The rate of change of shear stress and shear rate have been shown to be important, as well as local turbulence and unsteady flow. In addition local disturbed flow patterns, recirculation zones, long particle residence times have been suggested to play significant roles in the onset and development of atherosclerosis. 24

25 Velocity Profile in a Symmetric bifurcation 25

26 Principle of Wall Shear Stress 26

27 Experimental Setup 27

28 Measurement Results (1) (a): stagnant flow area, (b): below center of the recirculation eddy (c): reattachment flow area (d): fully developed flow area. 28

29 Measurement Results (2) Effect of pre treatment of ECs with TNFa on THP 1 cell adhesion under disturbed flow. ECs were maintained in static condition without (A) or with TNFa (125 U/ml) treatment (B) for 4 h, and then exposed to VSF with THP 1 cell suspension at a concentration of 5105 cells/ml. 29

30 Concluding Remarks The μpiv studies on the motions of flowing THP 1 cells under disturbed flow have demonstrated the existence of a higher near wall cell concentration and a longer residence time in the regions near the step and the reattachment point. The flowing THP 1 cells in the vicinity of the reattachment point possess a greater normal velocity component towards the wall, which may enhance the convective transport of these cells from the bulk flow to the ECs. The preferential adhesion near the step and the reattachment point under VSF may result from the combination of multiple factors, including cell sedimentation, near wall concentration, residence time, normal velocity component towards the wall, and tangential velocity component along the wall. 30

31 Immunoassays Reactors The availability of genetic information for many organisms has created a need for bioanalytical technologies that are capable of simultaneously determining the concentration of the thousands of proteins that make up cells. Microarrays are now routinely used for simultaneously characterize the concentration of thousands of oligonucleotides at a sensitivity that exceeds 1 nm. The left figure presents a conventional optical micrograph of an array of plastic reactors of three volumes that are supported on a 60 μm thick alumina membrane with a minimum pore size of 20 nm. 31

32 Experimental Setup The advantages of this technique are that (1) it requires only a single receptor, (2) the rate of reaction allows the assay to be completed in 35 min, (3) the total volume of analysis is less than a nanoliter, and (4) the sensitivity can reach a single molecule per particle. 32

33 Principle of Diffusometry TEM image of a red fluorescent microparticle that has reacted with the M13 virus. Specifically, the model assumes that the diffusion coefficient of tagged particles is given by where ξ virus increases linearly with an increase in virus concentration. A least squares fit yields the following correlation 33

34 Measurement Results (1) A large number of images are processed to yield particle tracks. The terminated paths indicate loss of particle from the viewing volume. Diffusion coefficients of antibody functionalized particles and unmodified particles as a function of virus concentration. 34

35 Measurement Results (2) Simulation results Variation of particle size is the dominant source of variation in the measured diffusion coefficient of the microparticles. 690±40 nm 690±0 nm Percentage change in diffusion coefficient of different sized particles as a function of the hydrodynamic radius of the pathogen. 35

36 Concluding Remarks High sensitivity: the particle diffusometry technique had single virus binding per particlesensitivity, which amounts to about 500 viruses/nl. There appears to be three practical limitations to implementing this technology. First, in this study large particles have been used which limited the analyte to large viruses. However, if 40 nm particles were used it may be possible to detect analytes as small as 1 nm. Second, the reliability of the particle diffusometry technique was increased by using multicolor particle tracking, which allowed uncoated microparticles to be used to detect changes in environmental parameters, such as temperature and viscosity. This leaves the variation in particle size as the primary source of noise in the measurement. Third, simulations suggest that at least 6000 data points are needed to obtain statistically convergent measurements for uniform sized particles. It may be difficult to track individual particles for 6000 frames in practice. 36