1. Small forces – Big results Abhijit Sarkar Assistant Professor Physics Department The Catholic University of America Washington D.C. sarkar@cua.edu http://www.vsl.cua.edu/sarkar

2. Basic Principle m F Aspiration Pipette N S z x y Not to scale Top View of Flow Cell Bead diameter ~3µm Bead-to-Magnet Distance 0µm to 3500µm

3. Sample Cell with Microspray Pipette Aspiration Pipette m z x y Not to scale 1mm Protein Microspray Needle

4. CCD 3D Electronic Micromanipulator PC with Labview 3D Hydraulic Micromanipulator Objective 40X, 0.65NA Light Source Condenser Micropipettes Chamber Stage DNA Syringe Pump

5. Lambda DNA 50 kbp Anti-DIG DIG Avidin Biotin Putting Handles on DNA

6. L 0 = 16.4µm, b (persistence length) = 50nm, K 0 = 1000pN Force-Extension Measurement for Single DNA Molecule

7. Brownian Fluctuation Method f xx Stiff Pipette Calculate gradient over image Diffraction around bead produces a central peak and series of concentric circles of decreasing gradient intensity Detecting these concentric circles can then be used to obtain a most-likely bead center Allows sub-pixel determination of bead center (accuracy on order of 10nm) Force & Extension Measurements

8. Stokes Law Force Calibration m m m NS F Not to Scale Stokes Drag Force Law mmm

9. Grab frame and crop Convert to 8-bit grayscale with bright beads Apply filter to eliminate background Search for peaks Need to follow beads over time – which bead is which? Bead Velocity Measurements

10. 000290. 149 327. 783 359. 569 396. 433 429. 967 504. 652 538. 831 068.0 46 99.7 81 0072.4 74 304. 048 372. 414 400. 173 436. 243 476. 998 000203. 603 227. 477 140. 174 197. 821 246. 761 335. 727 405. 934 436. 722 472. 668 506. 635 00179. 084 229. 898 254. 253 183. 858 233. 694 280. 864 366. 781 520. 132 577. 111 607. 567 649. 801 00206. 634 319. 356 346. 114 206. 963 285. 330 327. 463 477. 100 561. 412 00000295. 288 378. 064 401. 465 242. 976 393. 688 497. 401 534. 759 000000000 341. 122 458. 892 553. 425 597. 310 000000000 422. 436 528. 481 00000000000 477. 711 541. 328 00000000000 510. 350 000000000000 Crocker algorithm – proved inefficient at high force, when bead displacement approaches bead separation Constraint of minimizing negative y-velocity Constraint of minimizing x- velocity deviation Iterative ranking of bead assignments consistent with these constraints – usually unique back-check to identity assignments Bead Tracking Method

11. Nucleosome Formation & Rupture Under Tension Single DNA Molecule f Nucleosome Formation N  l ~ N x 50 nm f Nucleosome Rupture Nf*  l =  G Predicted f* ~ 1.5 pN f*f*

12. Nucleosome Formation and Rupture

13. Nucleosomes Under Moderate Load f ~ 6.5pN

14. Start with extension as function of time Edge preservation is critical Red is raw data - Blue is Bilaterally filtered data Finding Nucleosome Pop-off Events Microns Time