1. TMV Diffusion in Complex Solutions: When Science Goes Wrong Randy Cush* & Paul Russo Louisiana State University Indiana University Purdue University Indianapolis October 30, 2002

2. Generic Talk Outline Thank hosts for wonderful day Tell joke or story Explain what we’re trying to do Explain what we did Explain why that is so cool Destroy evil-doers Thank friends Explain why that is totally wrong Fix it Move on

3. See, e.g., Lodge & Muthukumar, J. Phys. Chem. 1996, 100, 13275-13292

4. DLS for Molecular Rheology of Complex Fluids: Prospects & Problems + + + Wide-ranging autocorrelators > 10 decades of time in one measurement! – – – Contrast stinks: everything scatters, esp. in aqueous systems or most supercritical fluids, where refractive index matching cannot hide the matrix. Studied a lot Barely studied

5. Our Hypothesis Solutions containing rodlike diffusers may provide evidence for entanglement-like phenomena in solution…or at least prove interesting, fun and challenging while helping us develop micro-rheology tools. Evidence would be: * sudden drop-offs in mobility with concentration * failure to follow continuum mechanics

6. Yeah, challenging…. Early de Gennes paper on rod/coil diffusion: 19 citations Same era de Gennes paper on coil/coil reptation: 479 citations We may expect some problems! Why it’s worth it: composite precursor fluids, dissolution rate, phase separation rate, relation to GPC, CGE of rods, intracellular transport.

7. Desirable rod properties Stiff—no bending Monodisperse (all the same size) Non-aggregating Water-soluble We don’t have to make ‘em! Let mother nature do the work: plants make viruses (unwillingly) with most of these qualities.

8. Seedlings  Sick Plants  And close-up of mosaic pattern. DIY farming--keeping the “A” in LSU A&M

9. TMV Characterization Sedimentation, Electron Microscopy and DLS Most TMV is intact. Some TMV is fragmented –(weaker, faster mode in CONTIN) Intact TMV is easy to identify –(stronger, slower mode in CONTIN)

10. Better Views http://www.uct.ac.za/depts/mmi/stannard/linda.html

11. An ear of corn has about as many kernels as TMV has protein subunits (ca. 2130). The protein subunits enfold a spiral-wound strand of RNA which will encode the next generation. TMV is more extended than an ear of corn. A Minnesota Farmboy’s Corny View of TMV

12. Tobacco from the Carolinas to Connecticut

13. If tobacco goes away… “Traditionally in Kentucky great mounds of brush are piled and burned in February to prepare a bed for tobacco seedlings. I remember spending most of the day hauling and piling brush. My dad would start the fire in late afternoon and we would sit up most of a cold February or March night stoking the fire, watching the stars, and roasting hot dogs or marshmallows over the bonfire. Many times neighbors would stop by and sit with us for a spell around the fire, talking into the night.” From: http://www.webcom.com/duane/farm2.html

14. Rotation & Diffusion of TMV in Polymer Solutions ++ + + Fabulous new autocorrelators for scattering  10 decades of time in one measurement! – – – Contrast for scattering stinks: everything scatters, esp. in aqueous systems where refractive index matching cannot hide matrix. Matrix Polymer Solvent TMV Probe

15. Solution: Use Polarizers to Hide Matrix

16. Dynamic Light Scattering  Hv = q 2 D trans + 6D rot LASERV H  PMT Hv Geometry (Depolarized) Uv Geometry (Polarized) V   Uv = q 2 D trans PMT LASER  q2q2 6D rot  q2q2 D trans

17. Strategy Find polymer that should not “entangle” Find a rodlike probe that is visible in DDLS Measure its diffusion in solutions of each polymer separately Random coil Polysaccharide Invisible in HvDLS Highly-branched Polysaccharide Invisible in HvDLS Rigid rod Virus Visible in HvDLS Dextran Ficoll TMV Find polymer that should (???) “entangle” BARELY

18. As expected…

19. All measurements made at low TMV concentrations—no self-entanglement

20. Hv correlation functions for 14.5% dextran and 28% ficoll with and without added 0.5 mg/mL TMV The dilute TMV easily “outscatters” either matrix Matrix is invisible

21. Hey, it works!

22. I didn’t think—I experimented. ---Wilhelm Conrad Roentgen

23. Early results—very slight errors rotationtranslation Macromolecules 1997,30, 4920-6.

24. Stokes-Einstein Plots: if SE works, these would be flat. Instead, apparent deviations in different directions for D rot and D trans

25. Macromolecules 1997,30, 4920-6. At the sudden transition: L/  c.m. ~ 13 and L/  ~ 120 L   cm

26. rotation translation END OF PUBLISHED DATA We believed that the transition represented topological constraints. It was suggested that more systems be studied. BEGIN FICOLL When we did Ficoll, many more points were added!

27. Huh? D rot still diving in Ficoll? rotation translation

28. Maybe we should think now.

29. The chiral dextran and ficoll alter polarization slightly before and after the scattering center. With a strongly depolarizing probe, this would not matter, but…  TMV = I Hv /I Uv ~ 0.003 While matrix scattering is minimal, polarized scattering from TMV itself leaks through a “twisted” Hv setup. Most damaging at low angles

30. Mixing in Polarized TMV Light Uv light from misalignTrue Hv light  q2q2 q2q2 D rot too low 6Drot q2q2  

31. Even at the highest concentrations, only a few degrees out of alignment.

32. Polarimeters are long for a reason

33. Slight, but important, improvement.

34. Improved D rot /D trans Ratio Plots

35. Improved Stokes-Einstein Plots Black = TMV Translation Blue = TMV Rotation

36. Check D trans by FPR a.k.a. FRAP

37. Hydrodynamic Ratio—Effect of Matrix M at High Matrix Concentration

38. Effect of Dextran Molecular Weight— High Dextran Concentration (~ 15%) TMV Translation TMV Rotation

39. Behavior of Dextran Matrix

40. Pathological Science?*  The magnitude of the effect is substantially independent of the intensity of the causative agent.  The effect is of a magnitude that remains close to the limits of detectability; or, many measurements are necessary because of the very low statistical significance of the results.  It makes claims of great accuracy.  It puts forth fantastic theories contrary to experience.  Criticisms are met by ad hoc excuses.  The ratio of supporters to critics rises up to somewhere near 50 percent and then falls gradually to oblivion. Langmuir Nobel 1932 *The science of things that aren’t so Lecture December 18, 1953 GE Labs

41. Studying “entangled” TMV by FPR Fluorescently labeled probe rod Unlabeled rods Solvent

42. L d LC formation  = 4 / A 2  5 / dL 2 Reduced # Density   dL 2 / 5 Doi-Edwards-Onsager Reference Volumes for Rods = number density = # of rods per unit volume

43. D expected to decrease by half, but at what concentration? DD D  X

44. Modulation FPR Device a’la Lanni & Ware * * * * AOM M M D RR DM OBJ S PMT PAOS TA/PVD L SCOPE Measuring Translational Diffusion

45. FPR Data for TMV Solution: very low dye content.

46. D tracer self of TMV vs. c TMV

47. TMV vs Helical, Semiflexible Polymer

48. Thanks! Randy Cush David Neau Ding Shih Holly Ricks Jonathan Strange Amanda Brown Zimei Bu Zuhal & Savas Kucukyavuz—METU Seth Fraden—Brandeis Dan DeKee—Tulane Nancy Thompson—Chapel Hill N$F

49. THE END

50. Misalignment from thick polarizer in “active” part of detector train, exacerbated by tiny cells used to squelch optical rotation & conserve TMV

51. L d LC formation  = 4/A 2  5/dL 2 Reduced # Density   dL 2 /5 Doi-Edwards-Onsager Reference Volumes for Rods = number density = # of rods per unit volume

52. Conditions for use as a Probe Is the TMV Probe Dilute? A TMV concentration of 0.5 mg/mL, well below the theoretical overlap concentration, was chosen. See Figure 2. Does dilute TMV overwhelm the matrix scattering? At 0.5 mg/mL the TMV easily “outscatters” both matrices. See Figure 3. Is the probe compatible with the matrix? -Solutions stable months after preparation -Angle dependent Hv SLS -Dtrans goes up, not down (Figures 6 & 8)

53. Effect of Dextran Concentration The dependence of D rot and D trans upon added dextran is shown in Figure 4. The quotient D rot /D trans is plotted against viscosity in Figure 5. By combining both transport coefficients, each inversely proportional to viscosity in dilute solution, we can remove the effect of solution viscosity. Figure 6 reveals like positive deviations from the Stokes-Einstein continuum expectation that diffusion be inversely proportional to viscosity (below 6.5%). Above 6.5% the deviations become greater for both D rot and D trans but in opposite directions

54. There once was a theorist from France who wondered how molecules dance. “They’re like snakes,” he observed, “As they follow a curve, the large ones Can hardly advance.” D ~ M -2 P.G. de Gennes Scaling Concepts in Polymer Physics Cornell University Press, 1979 de Gennes

55. Outline Characterize the TMV –Is it intact and behaving properly? Establish conditions for use of TMV as probe –Can the probe be dilute and still overwhelm the matrix scattering? –Will the probe stay mixed with the matrix solutions without aggregating? Show the effect of the dextran and ficoll matrices on TMV diffusion

57. Effect of Ficoll Concentration The dependence of D rot and D trans upon added dextran is shown in Figure 4. The quotient D rot /D trans is plotted against viscosity in Figure 7. Figure 8 shows slight like positive deviations from the Stokes-Einstein continuum expectation (below 11%). Above about 11% ficoll the deviation slowly becomes greater for D rot and slightly greater for D trans but in opposite directions Figure 9 compares TMV behavior in ficoll to that in dextran.

60. Too-Good-to-be-True Conclusion? Below 6.5% dextran the diffusion of the rodlike TMV probe is controlled mostly by viscosity. Above 6.5% dextran a sharp transition suggests topological constraint for TMV rotation while translation is not much affected. The transition is more gradual in ficoll. The TMV probe senses something different for linear vs. highly branched polymers in solution. Looks good for topological models!

61. Alternate Conclusion? The systems studied so far place (impossibly?) strict demands on geometric & polarization alignment. –Revised polarization placement –Difficult zero angle measurements requiring even more TMV New systems must be studied: –TMV is OK –Dextran/Ficoll must go! Depolarized probe diffusion has the potential, as yet unrealized, to assess strength of hydrodynamic vs. topological effects.

62. To Do Get Cush to estimate the total number of TMV’s he produced. Slopes of intrinsic viscosity plot don’t meet the 0.5 rule. What has Deutch & Pecora to do with Dtrans vs. M? What does regular viscosity have to say about M-dependence? At what concentration was that M- dependent stuff done?