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Magnetic twisting cytometry PowerPoint Presentation
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Magnetic twisting cytometry

Magnetic twisting cytometry

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Magnetic twisting cytometry

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  1. Session 10 Magnetic twisting cytometry

  2. Approach • Torque applied at surface of cells with magbeads • Used to determine cell mechanics • Cellular viscoelasticity (Fabry, Fredberg) • Mechanotransduction (Wang, Ingber)

  3. Technique • Coercive field applied briefly in one direction • Sinusoidal field applied in perpendicular direction • Torque applied: t = m x H M is magnetic moment H is sinusoidal external field

  4. Apparatus A) Microscope stage with electromagnetic coils B) Twist causes magbead rotation and translation C) Human airway smooth muscle cells with magbeads

  5. Magnetic Measurement • Magnetometer detects remanent magnetic field, B • Twisting field rotates magbeads • Magbead’s m turned away from magnetometer • Decrease in B field from horizontal direction indicates degree of twist

  6. Torque Response • For magbeads: T(t) = c H(t) cos (t) c = 0.41 Pa/gauss H(t) = H sin (t) (t) = B(t)/B0 Histamine added First 10 cycles Pre-histamine Last 20 cycles Post-histamine

  7. Particle Tracking Measurement • Fast, real-time image analysis • Individual particle tracking better than aggregated magnetometry • Pixel-to-displacement, intensity weighted algorithm of centroid position per frame

  8. Firm Integrin and CSK connection • Ligand-bound integrins actively resist the twist

  9. Full CSK Needed for Twist Resist • CSK Inhibitors • Acrylamide (Acr) inhibits IF • Nocodozal (Noc) inhibits MT • Cytochalasin D (Cyt) inhibits actin • Full inhibition of CSK needed for “free” twisting

  10. Rheological Measurements • Complex Modulus: (s, Fourier transform) • Magnetometry: G*(s) = T(s) / (s) • Optical Tracking: G*(s) = T(s) / x(s) • Storage and Loss Moduli: G*(s) = G’(S) + iG’’(s) • Hysteresivity : h (s) = G’’(S) /G’(s)

  11. Histamine Stiffens hASMCs • Histamine activates myosin, leads to SM contraction • Steady baseline before adding histamine • Storage modulus increased 2.2X • Loss modulus increased 3X • Transient hysteresistivity response

  12. DibutryrlcAMP softens hASMCs • DBcAMP inhibits myosin, leads to SM relaxation • G’ falls to 30% • G’’ decreased to 45% • H increased 1.5X

  13. Wide Variability in Cells • Not donor-to-donor, day-to-day, or culture-to-culture, but cell-to-cell variability • Wide distribution in data necessitates large sample populations DBcAMP-softening Histamine-stiffening

  14. Baseline Affects Stiffening/Softening • “Soft” baseline cells: More histamine-stiffening, less DBcAMP-softening • “Hard” baseline cells: More DBcAMP-softening, less histamine-stiffening