Research ArticleNANOROBOTS

Intracellular manipulation and measurement with multipole magnetic tweezers

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Science Robotics  13 Mar 2019:
Vol. 4, Issue 28, eaav6180
DOI: 10.1126/scirobotics.aav6180
  • Fig. 1 Multipole magnetic tweezers system.

    (A) Applications of magnetic micromanipulation ranging from organ level (13, 14), tissue level (16, 17), to embryo level (18) and single-cell level. (B) The multipole magnetic tweezers was integrated with a confocal microscope. Figures were adapted from (13, 14, 17, 18) with permissions. (C) Picture of the intracellular multipole magnetic tweezers integrated with the microscope stage. (D) Imaging light path and illumination light path. (E) Mechanical design of the multipole magnetic tweezers device. (F) Top stage includes yoke, three magnetic poles with three coils, and position control screws. (G) Bottom stage includes yoke, three magnetic poles with three coils, and structures for integration with confocal microscope.

  • Fig. 2 Magnetic force simulation and calibration.

    (A) Multiple 0.7-μm beads were introduced into the workspace for force quantification in different positions. All beads moved toward the +X direction when a magnetic force was applied toward +X. (B) Calculated magnetic force in +X fitted into the magnetic force model (Eq. 1). The dots show the experimentally measured force, and the line shows the force model calculated force. (C) 3D magnetic field constructed by experimental results on different focal planes. (D) Finite element simulated magnetic force exerted on the bead. (E) Force error map in the workspace of 40 μm by 40 μm by 20 μm.

  • Fig. 3 Spatial measurement of nuclear mechanics polarity.

    (A) Polarity of nuclear mechanics. (B to D) Magnetic bead introduced into the cell via endocytosis was moved from its initial position to target positions on cell nucleus. The bead’s trajectory inside the cell is shown as the red line in (C). (E) Experimentally measured nuclear deformation over time. (F) Apparent Young’s modulus measured from the minor and major axes of the cell nucleus. n = 10, error bars: std., P = 0.0017. (G) Experimentally measured polarity ratio, defined as the apparent Young’s modulus measured along the major axis over that measured along the minor axis, of T24 cells. The control group was treated with the drug solvent dimethyl sulfoxide (DMSO). n = 10, error bars: std. SD, *P = 0.008, P = 0.25 (N.S.), **P = 0.03. (H to J) Drug treatment effects in T24 cells. (H) Actin staining showed that the CD treatment disrupted the alignment of actin filaments (second and fourth images). (I) Staining of tubulin, the structural protein of microtubules, showed that microtubules were significantly knocked down after NC treatment (third and fourth images). (J) Costaining of nucleus, actin, and tubulin showed that the major axis of the cell nucleus aligned with actin filaments (first and third images), whereas the CD treatment disrupted the alignment of actin filaments (second and fourth images).

  • Fig. 4 Nuclear mechanics polarity in different Z planes.

    (A) Schematic of nuclear polarity measurement along the major and minor axes of the cell nucleus in two different Z planes. (B) Representative raw images of nuclear polarity measurement, corresponding to the schematic shown in (A). White circles indicate bead locations. (C) The measured apparent Young’s modulus results reveal that the major axis is significantly stiffer than the minor axis in both plane 1 and plane 2. n = 10 cells, error bar: SDs, *P < 0.001, **P < 0.001.

  • Fig. 5 Temporal measurement of nuclear stiffening.

    (A) The repeated application of 50-pN force on the nuclear envelope while nucleus deformation was recorded over time. (B) Magnetic bead displacement during force application and release. The force was applied on the nuclear envelope and released for the nucleus deformation to recover. Repeated loading and recovery was repeated five times in each measurement. Data were interpreted with the standard linear viscoelastic solid model shown in (C). (D and E) Effective elastic modulus and viscosity were quantified for each of the five measurements. (F) The fold change of effective elastic modulus and the viscosity after five cycles for force application and release. n = 10 cells, error bar: SD. The effective elastic modulus fold change is significantly larger than 1; one-sample t test, *P = 0.0003. The viscosity fold change showed no significant difference compared with 1; one-sample t test, P = 0.38. (G to I) Stiffening ratio is defined as the ratio of effective elastic modulus of the fifth measurement over that of the first measurement. (G) T24 cells after knocking down the nuclear envelope structural protein, lamin A/C via siRNA treatment. n = 10, error bar: std., *P = 0.0007. (H) T24 cells after anticytoskeleton drug treatments. n = 10, error bar: std., *P = 0.0002, P = 0.60 (N.S.). (I) Comparison of stiffening ratio, T24 versus RT4 cells. n = 10, error bar: std., *P = 0.04. (J) Western blot showing the lamin A/C expression after siRNA treatment, validating the knockdown effect. (K) Western blot showing lamin A/C and cytoskeleton differences between RT4 and T24 cells. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as control.

Supplementary Materials

  • robotics.sciencemag.org/cgi/content/full/4/28/eaav6180/DC1

    Text S1. Calculation of the force required for intracellular measurements

    Text S2. Magnetic bead position control

    Text S3. Nuclear deformation under different image capturing rates

    Text S4. Supplementary methods and materials

    Fig. S1. Cell viability test and success rate of cell endocytosis.

    Fig. S2. Generalized predictive control.

    Fig. S3. Trajectory tracking performance using PID and GPC.

    Fig. S4. Intracellular navigation performance.

    Fig. S5. Viscoelastic parameters of major and minor axes of T24 cells.

    Fig. S6. Staining of actin and tubulin for single T24 cells.

    Fig. S7. Staining of actin and tubulin for validation of the drug treatment effects in RT4 cells.

    Fig. S8. Nuclear deformation under different image capturing frame rates.

    Fig. S9. Top down and side view of the device and scanning electron microscope images of the microbead and pole tip.

    Fig. S10. Mean square displacement over lag time for bead position tracking.

    Table S1. Key parameters in system design.

    Table S2. Key parameters in the generalized predictive controller.

    Table S3. Representative 3D magnetic micromanipulation systems.

    Movie S1. System design.

    Movie S2. Position control of magnetic bead.

    Movie S3. Intracellular navigation and nuclear polarity measurement.

    Movie S4. Intracellular force application on cell nucleus for stiffening measurement.

    Movie S5. Simultaneous bright-field and fluorescence imaging.

    Movie S6. Raw video of nuclear deformation and recovery after 50-pN force application.

    Movie S7. Raw video of multibead navigating inside the silicone oil in calibration experiments.

    References (6774)

  • Supplementary Materials

    The PDF file includes:

    • Text S1. Calculation of the force required for intracellular measurements
    • Text S2. Magnetic bead position control
    • Text S3. Nuclear deformation under different image capturing rates
    • Text S4. Supplementary methods and materials
    • Fig. S1. Cell viability test and success rate of cell endocytosis.
    • Fig. S2. Generalized predictive control.
    • Fig. S3. Trajectory tracking performance using PID and GPC.
    • Fig. S4. Intracellular navigation performance.
    • Fig. S5. Viscoelastic parameters of major and minor axes of T24 cells.
    • Fig. S6. Staining of actin and tubulin for single T24 cells.
    • Fig. S7. Staining of actin and tubulin for validation of the drug treatment effects in RT4 cells.
    • Fig. S8. Nuclear deformation under different image capturing frame rates.
    • Fig. S9. Top down and side view of the device and scanning electron microscope images of the microbead and pole tip.
    • Fig. S10. Mean square displacement over lag time for bead position tracking.
    • Table S1. Key parameters in system design.
    • Table S2. Key parameters in the generalized predictive controller.
    • Table S3. Representative 3D magnetic micromanipulation systems.
    • References (6774)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). System design.
    • Movie S2 (.mp4 format). Position control of magnetic bead.
    • Movie S3 (.mp4 format). Intracellular navigation and nuclear polarity measurement.
    • Movie S4 (.mp4 format). Intracellular force application on cell nucleus for stiffening measurement.
    • Movie S5 (.mp4 format). Simultaneous bright-field and fluorescence imaging.
    • Movie S6 (.mp4 format). Raw video of nuclear deformation and recovery after 50-pN force application.
    • Movie S7 (.mp4 format). Raw video of multibead navigating inside the silicone oil in calibration experiments.

    Files in this Data Supplement:

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