3 Dimensional Magnetic Force Microscope (3DFM) Overall Funding. The Next Step in Biological Force Microscopy How to do force microscopy inside a cell?

Transcription

1 3 Dimensional Magnetic Force Microscope (3DFM) Russell M. Taylor II Slide 1 Overall Funding Sustained funding $1M+: NIH/NCRR (3+ yr), NIH/NIBIB (5-year), UNC CF Foundation Slide 2 The Next Step in Biological Force Microscopy How to do force microscopy inside a cell? * Puncture the cell membrane to image inside the cell? Slide 3

2 One Solution: Put the Probe Inside the Cell Problems: How to measure the probe s position? How to apply forces? Slide 4 One Method is the Optical Trap Problems: * Limited forces due to laser heating * Nonspecific forces Laser Biological Cell Scan in x,y,z Slide 5 Our Solution 3-D Force Microscope Magnetic Particle Solenoid pole tips substrate Cell optical tracking Magnetic fields apply forces to magnetic particle Particle position is monitored using optical tracking 1. Very specific forces 2. Little localized optical heating 3. Relatively high forces Slide 6

3 3DFM: Goals & Objectives Single Particle Tracking Magnetic particle manipulation Measurement of viscoelastic (mechanical) properties Slide 7 3DFM: Concept Video Slide 8 3DFM User Interface Concept Seeing, Feeling, and Manipulating in 3D Visual: Display context + detail in 3D Force: Simultaneous sense & control Replay: Re-analyze original data in context Control: From exploratory to controlled Escape from Flatland Graphics: Open-Source VTK Network: Our Public-Domain VRPN Slide 9

4 3DFM: System Overview Coarse tracking and context Optical microscope 3D translation stage centers bead 2-axis translation stage for X&Y Piezoelectric stack actuator for Z Force application to bead Four magnetic cores apply field Fine Tracking of bead in X,Y,Z: Laser light scatters off the bead Quadrant Photodetector reads Slide 10 CCD camera Optical Layout: Koehler Illumination 50/50 beam splitter filter tube lens, f=100mm Pigtail fiber connector Collimating optical Lens fiber Upper 100x objective lens Diode Laser sample chamber Lower 100x objective lens fiber light polarizing beam splitter cube BFP Imaging lens, f=50mm Advanced quadrant Visualization q1q2and Control photo 3DFM University of Hamburg, Russ q3q4 diode Taylor, Summer mirror 04 Slide 11 CCD camera Optical Layout: Laser tracking 50/50 beam splitter filter tube lens, f=100mm Pigtail fiber connector Collimating optical Lens fiber Upper 100x objective lens Diode Laser sample chamber Lower 100x objective lens fiber light polarizing beam splitter cube BFP Imaging lens, f=50mm Advanced quadrant Visualization q1q2and Control photo 3DFM University of Hamburg, Russ q3q4 diode Taylor, Summer mirror 04 Slide 12

5 Optical Tracking Incident beam (maximum in center) Bead Scattering Resulting pattern (maximum in center) Slide 13 Z Tracking, QPD Image Theoretical Image Measured Image Slide 14 X/Y Tracking, QPD Response Right Left = X, Top Bottom = Y 1 normalized intensity x - displacement (microns) Slide 15

6 3D Tracking: Bead Capture Bead 2.8 microns in diameter attached to cilium Two beats uncaptured Several captured Note background (XY) Note focus (Z) Slide 16 Initial Magnetic Design Increasing >4.071e e-001 : 4.071e e-001 : 3.701e e-001 : 3.331e e-001 : 2.961e e-001 Field : 2.591e e-001 : 2.221e e-001 : 1.851e e-001 : 1.481e e-002 Density : 1.111e e-002 : 7.412e- <3.712e- Density Plot: B, Slide 17 Initial Magnetic Implementation Pairs of coils driven in common drive poles Front View Top View Slide 18

7 Bead Pulled in Orbit Each pole energized in turn Slide 19 Initial Direct Manipulation Interface Slide 20 Force Magnitudes and Directions Current Status 1pN on 1 micron bead Force Dependencies Increases as 3 rd power of bead radius Decreases as 5 th power of pole separation Getting Higher Forces 2 micron bead 2^3 = 8 pn ¼ pole separation 4^5 = 1 nn (next design) Slide 21

8 3DFM: Useful Force Metrics Thermal forces: ~0.1pn Biological motor (kinesin, myocin): <8pn Cell motility lower limit: 100pn ---- Limit of Laser Tweezer forces ---- Antibody/antigen: 200pn Molecular motor systems (cilia):??? Kinetochores (chromosome sep): >500pn Cell motion by actin polymerization: 2nn Slide 22 3DFM: Version 2 Magnetics Slide 23 3D Position Control XY: Queensgate closed-loop positioner Z: Piezo stack Slide 24

9 3DFM: Whole System Slide 25 3DFM: Close-up of Cores Slide 26 3DFM: 2 nd -Generation Slide 27

10 3dFM: 2 nd -Generation Cores Slide 28 User Interface and System Diagram from CS point of view Nested Feedback Control Systems Distributed, Real-Time System 2D User Interface for parameter controls 3D User Interface to view data and control Challenges Slide 29 D DFM Control System XYZ Xlate Model Slide 30

11 D DFM Control System Bead Tracking XYZ Xlate Two processes: Track at >= 5kHz Log and transmit Similar to AFM feedback Slide 31 D DFM Control System Several more processes: 2D GUI (Java/Swing) 3D Visual GUI (VTK) 3D Haptic GUI (VRPN) Similar to nanomanipulator 3D User Interface XYZ Xlate Slide 32 D DFM Control System An additional process: Capture/send video Similar to nm + Optical Optical Overlay XYZ Xlate Model Slide 33

12 D DFM Control System Magnet Control An additional process: Control Magnets Beyond what we ve done before XYZ Xlate Model Slide 34 D DFM Control System N more processes: Fit model to image data Simulated microscopes Overlaid 3D visuals Core 3 Research needed XYZ Xlate Model Analysis & Model Overlay 3DFM University of Hamburg, Russ Taylor, Summer 04 Slide 35 D DFM Control System Make it work this way too: Re-analysis Use new analysis tools on stored data Training new users Replay Burst-Mode Storage Make it work this way too: Pairs of scientists on same experiment Remote use of facilities Calling in the expert Shared Use Slide 36

13 Distributed, Real-time System RTS3 RTS1 RTS2 Slide 37 3DFM: 2D User Interface Measurement and computation output Viscosity estimates over time Histogram of bead position System Parameters Force magnitude for manipulation Video capture resolution and rate Visualization mode (volume render, isosurface) Sharing controls to enable live collaboration Connect to instrument and collaborator(s) Private vs. Shared state view Slide 38 3DFM: 3D User Interface 3D graphics display experiment results 2D live video stream of context and bead 1D trace of bead annotated with viscosity 2D extracted surface of bead s course 3D confocal or COSM fluorescence data 3D force-feedback control Enable pulling bead with certain force Enable trapping bead at certain location Slide 39

14 VTK UI Prototype Parameter menus Bead Histogram Yellow trace Green estimate Translucent volume swept by bead Complicated path from Brownian motion simulator Slide 40 Newer 3DFM-UI Includes Video Slide 41 3DFM: CS Challenges Data Visualization Overlaying volume, surface, line-trace data: both visually and haptically Displaying surfaces with uncertain borders Computation and Rendering Real-time volume convolution and display (COSM) Incremental updates of a subset of the volume Measurement and Control Theory Tracking the bead, estimation of forces, viscosity and other system state parameters Slide 42

15 Experiments Cystic Fibrosis Study of mucus clearance Study of cilia motion and forces Study of viscosity Bacterial Motility Study of proteins causing bacteria to move Study of forces needed to stall bacteria Slide 43 Initial Experiment Target: CF CF gene controls Cl - and Na + transport through cells Affects airway secretions (mucin) Mucociliary clearance is the first line of defense against inhaled particulates, aerosols, etc. Particulate-laden mucus transported by cilia beating in a mucus-free periciliary liquid (PCL) to the glottis where it is expelled and swallowed Slide 44 Nanomachines and Biology: Lungs and Microfluidics Pathogens (bacteria, viruses, etc.) enter lungs Cleared by flow of mucus Flow generated by beating cilia Slide 45

16 MucoCilliary Clearance and Cystic Fibrosis Rheology of mucus/gene therapy barriers Forces of Cilia Feedback System: mucus volume/viscosity regulation (ASL) (PCL) Cilia Normal mucus flow = Good Silent CF mucus = Bad 3DFM University of Hamburg, Russ Taylor, Summer 04 Slide 46 Cystic Fibrosis: Cilia Dynamics, Exquisite Engineering Arranged Filaments and coordinated motor dynamics Slide 47 Cystic Fibrosis: Cilia Dynamics, Exquisite Engineering Arranged Filaments and coordinated motor dynamics Slide 48

17 Cilia Dynamics, Exquisite Engineering: How do they move? Kinesin motors move processively: chemical motors! Vale, Science 288, 88 (2000) 3DFM University of Hamburg, Russ Taylor, Summer 04 Slide 49 Cilia Dynamics and coupled motion From single cilia to coordination: metachronal beating from hydrodynamic coupling Levit-Gurevich, PNAS 96, (1999) Slide 50 Cilia Dynamics and MC Clearance Open Questions: Levit-Gurevich, PNAS 96, (1999) (ASL) (PCL) Cilia Single Cilia: Beat Pattern Force Generation Slide 51

18 Cilia Dynamics and MC Clearance Open Questions: Levit-Gurevich, PNAS 96, (1999) (ASL) (PCL) Cilia System Questions: Coupling to mucus Regulation and Feedback Slide 52 Cilia Dynamics: Complex 3d trajectories Proposed model of cilia beat pattern from literature P = Power Stroke Slide 53 Tracked cilium beating at 15 Hz Slide 54

19 Cilia Dynamics: Measuring Forces Apply force as square wave to bead See change in Bead Center, Amplitude Y-displacement (um) attached bead displacement Applied Voltage time (sec.) Slide 55 Cilia Dynamics: Measuring Forces Apply force as square wave to bead See change in Bead Center, Amplitude V applied 0.5V applied Y-displacement (um) attached bead displacement Applied Voltage time (sec.) Power (db) frequency (Hz) Slide 56 Extracting Viscosity Bead moved by Brownian motion (not magnets) -130 Depends on power spectrum Size of bead Power spectral density, 10log(S(f)) frequency (Hz) Slide 57

20 Bacterial motility Bacteria motility Actin polymerization Within cell Cell is also moving Sped up 60X Julie Theriot lab Stanford University Slide 58 Bead Motility Bead covered with ActA protein Polymerizes actin trail behind it Moves just like bacteria Questions: Force to stall it? Will it re-start? Can we guide it? Lisa Cameron Slide 59 Slide 60

21 Credits for non-unc Inclusions Cilia internal structure diagram: King s College London Bacterial Motility: Julie Theriot s laboratory at Stanford Bead Motility: Lisa Cameron, work done at Julie Theriot s lab Slide 61