Protocol for measuring membrane elasticity of mouse cardiomyocytes using atomic force microscopy

Summary

Atomic force microscopy (AFM) is a powerful technique that enables the determination of cellular mechanics at the single-cell level. Here, we present a protocol for using AFM to measure membrane mechanical properties of mouse ventricular cardiomyocytes. The key steps include isolation of mouse cardiomyocytes, cantilever preparation, modification and calibration, and measurement of force-deformation profiles, from which membrane Young’s moduli are quantified. The outcomes advance our knowledge of cardiomyocyte’s unique mechanical and dynamic properties.

Graphical abstract

Highlights

• •Steps for performing cardiomyocyte isolation and plating
• •Steps for setting up AFM for single-cell compression measurements
• •Guidance on measuring the force-deformation profiles of individual cardiomyocytes
• •Instructions for extracting the mechanical properties from force-deformation profiles

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Atomic force microscopy (AFM) is a powerful technique that enables the determination of cellular mechanics at the single-cell level. Here, we present a protocol for using AFM to measure membrane mechanical properties of mouse ventricular cardiomyocytes. The key steps include isolation of mouse cardiomyocytes, cantilever preparation, modification and calibration, and measurement of force-deformation profiles, from which membrane Young’s moduli are quantified. The outcomes advance our knowledge of cardiomyocyte’s unique mechanical and dynamic properties.

Before you begin

AFM was initially developed for high-resolution imaging and soon found significant applications in biological research.¹ AFM has been widely used for the measurement of the local mechanical properties of cells and tissues.^2,3,4,5 Cells interact and communicate with each other through mechanical, chemical, and electrical signaling pathways. Therefore, the mechanical interactions among cells are ubiquitous in biological systems, and the quantification of the mechanical properties of the cell is essential for understanding cell interactions in physiological and pathological conditions.

Cardiomyocytes (CMs) are known for their unique mechanical and dynamic properties, non-spherical rod shape, as well as their large size (100–150 μm long, 40–50 μm wide, and 15–25 μm tall).^6,7 Local stiffness of cells, as quantified by Young’s modulus, has been measured using AFM force-deformation profiles in diabetic mouse models,^8,9 and for testing lung cancer drug effects on rat CMs.¹⁰ Moreover, contractile force and strain have been extracted from the AFM measurement of height vs. time profiles in chick embryotic CMs,^11,12 rat CMs,^10,13 and human induced pluripotent stem cell-derived CMs.¹⁴ However, neither approach can effectively probe single-cell mechanics, as unmodified AFM probes only measure local mechanics at the nanometer scale. The time-dependent changes in cellular height measured with an unmodified probe provide data on the force and strain of the dynamic process but do not capture overall cellular mechanics, including those of quiescent CMs. Additional challenges include the quality control of the CMs, inconsistent cell-substrate adhesion, and accurate quantification of Young’s modulus, and cellular mobility under force, as CMs are intrinsically non-adherent and tend to contract spontaneously during measurements.

Institutional permissions

All animal care and procedures were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of the University of California, Davis, and by National Institutes of Health guidelines. The experiments described in the protocol were conducted in a blinded fashion. The experimental mice are C57 BL/6 wild-type (WT) male and female mice (10–16 weeks old). Except specifically indicated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
NaCl	Sigma-Aldrich, USA	S5886
KCl	Sigma-Aldrich, USA	P5405
MgSO4	Sigma-Aldrich, USA	M2643
Na2HPO4	Sigma-Aldrich, USA	S9763
KH2PO4	Sigma-Aldrich, USA	P5379
NaHCO3	Sigma-Aldrich, USA	S6014
KHCO3	Sigma-Aldrich, USA	237205
MgCl2	Sigma-Aldrich, USA	M1028
CaCl2	Sigma-Aldrich, USA	C5670
NaOH	Sigma-Aldrich, USA	221465
HEPES	Sigma-Aldrich, USA	H4034
Taurine	Sigma-Aldrich, USA	T0625
D-glucose	Sigma-Aldrich, USA	G7021
Protease type XIV	Sigma-Aldrich, USA	P5147
Collagenase type II	Worthington, USA	LS004176
Fetal bovine serum	Fisher Scientific, USA	10-437-028
Bovine serum albumin	Sigma-Aldrich, USA	A9418-50G
Ketamine hydrochloride injection (100 mg/mL)	Vedco, Inc., USA	NDC 50989-161-06
Xylazine sterile solution (20 mg/mL)	Akorn Inc., USA	NDC59399-110-20
Heparin (1,000 USP units/mL)	Fresenius Kabi USA, LLC	NDC63323-540-57
Mouse Laminin	Corning Inc., USA	354232
Uncoated 50 mm dish no. 1.5 coverslip	MatTek Life Sciences, USA	P50G-1.5-14-F
Software and algorithms
GraphPad Prism	GraphPad Software, USA	https://www.graphpad.com
Origin Software	OriginLab Corp., USA	Version 6.1, https://originlab.com
BioRender	BioRender, Canada	https://BioRender.com
Python	Python Software Foundation	Python 3.8.10 (64-bit)
Image J	National Institute of Health, USA	ImageJ 1.54k, http://imagej.org
Igor Pro	WaveMetrics, USA	Version 6.20
MFP-3D AFM Systems	Oxford Instruments, United Kingdom	MFP-3D 090909 + 1312
Experimental models: Organisms/strains
C57BL/6 mice (Male and female, 10–16 weeks old)	The Jackson Laboratory	https://www.jax.org/
Other
Inverted microscope	Olympus, Tokyo, Japan	IX70
pH meter	Thermo Scientific, USA	Orion Star A111
Perfusion pump	Cole Parmer Instrument Co., USA	MasterFlex
Pressure monitor	World Precision Instruments, USA	BP-1
Water bath temperature controller	Fisher Scientific, USA	Model 9105
ELGA Veolia water purification system	Veolia Water Systems LTD, USA	Purelab Chorus PC1LSCXM2
0.22 μm filter	EMD Millipore Corporation, USA	SCGP00525
Langendorff apparatus	Harvard Apparatus, USA	EasyCell System for Cell Isolation, 73-4430
Nylon mesh cell strainer	Corning, USA	431752
Two-component epoxy glue	Devcon, USA	5 Min Epoxy in DevTube
Non-drying immersion oil	Cargille Labs, USA	Type DF formula: code 1261
Glass microsphere	Thermo Scientific, USA	Dry Soda Lime Glass Microsphere
AFM cantilever	Oxford Instruments, United Kingdom	AC240TSA-R3
CCD camera	Stocker & Yale	Mille Luce M1000
AFM	Oxford Instruments	MFP-3D
Optical microscope	Olympus America, USA	IX50
Isolation table	Hertzian	TS-140
Cell incubator	Eppendorf	New Brunswick Galaxy 170S
Scanning electron microscope	Hitachi High Technologies, USA	S-4100T FE-SEM

Materials and equipment

Solution preparations

Timing: 2–4 h

Note: Prepare all solutions using the ELGA Veolia water purification system (Purelab Chorus PC1LSCXM2) with a resistivity of 18.2 MΩ.cm.

Note: Prepare at 21°C–22°C.

Note: Filtration with a 0.22 μm filter.

Note: Store all solutions at 4°C (maximum time for storage is 7 days).

Note: Before use, warm the solution to 21°C–22°C.

• •Perfusion Buffer.

Reagent	Final concentration (mM)	Amount (g/L)
NaCl	113	6.604
KCl	4.7	0.350
MgSO4	1.2	0.296
Na2HPO4	0.6	0.161
KH2PO4	0.6	0.082
NaHCO3	12	1.008
KHCO3	10	1.001
Taurine	30	3.754
HEPES	10	2.383
pH 7.4 with NaOH

Store at 4°C, and the maximum time for storage is 7 days.

• •Perfusion Solution:

 Add 0.2 g glucose to 200 mL Perfusion Buffer.

• •Stop Solution:

 Add 2 mL fetal bovine serum and 3.75 μL 100 mM CaCl₂ stock to 20 mL Perfusion Solution, and adjust pH to 7.4 with NaOH.

• •Digestion Solution:

 Add 50 mg collagenase type II, 12.5 μL 100 mM CaCl₂ stock, and 2.5 mg protease type XIV to 50 mL Perfusion Solution, and adjust pH to 7.4 with NaOH.

• •Control Solution Buffer.

Reagent	Final concentration (mM)	Amount (g/L)
NaCl	113.5	7.802
KCl	4	0.298
MgSO4	1.2	0.296
Na2HPO4	0.62	0.166
Taurine	20	2.503
HEPES	10	2.383
pH 7.4 with NaOH

Store at 4°C, and the maximum time for storage is 7 days.

• •Control Solution:

 Add 0.4 g glucose and 0.2 g bovine serum albumin to 200 mL Control Solution Buffer, pH to 7.4 with NaOH.

• •Calcium Tolerance Solution I:

 Add 50 μL 100 mM CaCl₂ stock to 25 mL Control Solution to a final concentration of 0.2 mM/L Ca²⁺, adjust pH to 7.4 with NaOH.

• •Calcium Tolerance Solution II:

 Add 125 μL 100 mM CaCl₂ stock to 25 mL Control Solution to a final concentration of 0.5 mM/L Ca²⁺, adjust pH to 7.4 with NaOH.

• •Calcium Tolerance Solution III:

 Add 250 μL 100 mM CaCl₂ stock to 25 mL Control Solution to a final concentration of 1.0 mM/L Ca²⁺, adjust pH to 7.4 with NaOH.

• •Tyrode’s Solution for AFM Measurements.

Reagent	Final concentration (mM)	Amount (g/L)
NaCl	138	8.065
MgCl2	1	0.095
CaCl2	2	0.222
KCl	4	0.298
NaH2PO4	0.33	0.040
D-Glucose	10	1.802
HEPES buffer	10	2.383
pH 7.4 with NaOH
Sterile filtering with 0.22 μm filter

Store at 4°C, and the maximum time for storage is 7 days.

Refer to the key resources table “Reagent or Resource” for the list of materials and equipment.

Step-by-step method details

Mouse ventricular cardiomyocyte isolation and preparation

Timing: 1–2 h

We describe the detailed operational steps to perform isolation of mouse ventricular myocytes (VCMs) in this section.

Note: Preheat a water bath (Model 9105, Fisher Scientific, USA) of the Langendorff apparatus to 37°C.

• 1.Measure and record the body weight of the mouse to the nearest gram.
• 2.Inject 80 mg/kg of ketamine and 5 mg/kg of xylazine intraperitoneally to adult mice (both male and female, 12–16 weeks).
• 3.Assess level of anesthesia by firm toe pinch.
• 4.Inject 300 USP unit of sodium heparin intraperitoneally.
• 5.Perform a midline thoracotomy and heart excision, which takes about 30–60 s.
• 6.Transfer the heart to a petri dish containing Perfusion Solution and wash the heart to remove the blood, which takes about 10–30 s.
• 7.Trim the connective tissues and atria using fine scissors in about 30–60 s.
• 8.Cannulate the heart through aorta onto a Langendorff apparatus prefilled with Perfusion Solution, which takes about 30–60 s.
• 9.Retrogradely perfuse the heart with the Perfusion Solution at 37°C, circulated by an electrical perfusion pump (MasterFlex, Cole Parmer Instrument Co., USA). A schematic illustration of the procedure is shown in Figure 1.
• 10.
  Washout the blood by perfusion for 3 min.

  • a.
    Adjust the perfusion speed at a flow rate about 4–6 mL/min.
  • b.
    Control the initial pressure at ≈60 mmHg using pressure monitor (BP-1, World Precision Instruments, USA) connected to the Langendorff apparatus.
  • c.
    Decrease flow rate if pressure increases.
• 11.
  Load Digestion Solution into the Langendorff pump apparatus.

  • a.
    Continue monitoring the declining perfusion pressure for 15–20 min during tissue digestion by collagenase type II, until the pressure is below 30 mmHg.
  • b.
    Check the color of the heart surface, and the softness of heart tissue to determine the digestion level.
• 12.
  Remove the heart from the perfusion apparatus and transfer to a petri dish containing stop solution.

  • a.
    Mince the left ventricle and mechanically dissociate the tissue with forceps.
  • b.
    Triturate the dissociated tissue with gentle pipetting.
• 13.Harvest isolated VCMs by filtering through 100 μm nylon mesh cell strainer.
• 14.Pellet VCMs by allowing cells to settle with gravity and removing supernatant.
• 15.Replace with Calcium Tolerance Solution I and incubate VCMs for 15 min at 21°C–22°C.
• 16.Repeat this twice, each time changing the solution to Calcium Tolerance Solution II and III. Keep the final volume of Calcium Tolerance Solution III to be 1-2 ml for cell plating.

Figure 1.

Schematic illustration of mouse thoracotomy surgery, heart excision, cannulation and perfusion

Plating mouse ventricular CMs for AFM measurements

Timing: 1–2 h

We describe the operational steps to plate the isolated mouse VCMs onto the glass coverslip dishes for AFM experiments in this section.

• 17.
  During the VCM isolation, prepare the laminin-coated MatTek dishes.

  • a.
    Pipette 500 μL of 0.04 mg/mL mouse laminin (Corning Inc., USA) onto the glass coverslip region of the MatTek 50 mm dish (MatTek Life Sciences, USA).
  • b.
    Spread evenly.
  • c.
    Let the dishes sit at 21°C–22°C for at least 30 min.
  • d.
    Aspirate out the laminin and let the surface dry completely.
• 18.
  Plate 1 mL of the isolated VCMs onto the prepared dish.

  • a.
    Let the cells attach for 15 min at 37°C with 5% CO₂ in a cell incubator.
  • b.
    Use an inverted microscope (Olympus, Tokyo, Japan) to verify cell attachment after incubation.
• 19.
  Change the plating solution to Tyrode’s Solution.

  • a.
    Gently aspirate the plating solution, removing any unattached VCMs from the dish.
  • b.
    Add 5 mL of Tyrode’s Solution to the dish.
  • c.
    Use an inverted microscope (Olympus, Tokyo, Japan) to observe the cell density, as shown in Figure 2.

CRITICAL: To achieve single-cell AFM compression measurements, VCMs must be sufficiently spread out. If the density is too high, neighboring cells may interact and interfere with the measurements. Perform a gentle wash by replacing the Tyrode's solution to wash away loosely attached cells. Repeat as needed until reaching the density shown in Figure 2.

Figure 2.

Ideal cardiomyocyte plating density

A bright-field optical microscopy image (10X objective) of surface-supported live VCMs in Tyrode’s solution. Note that a low cell coverage or sufficient separation among cells is observed to assure single-cell compression. Scale bar = 40 μm.

Cantilever modification for AFM-based single-cell mechanics

Timing: 24 h

We describe the operational steps to set up the AFM system and the cantilever to prepare for AFM single-cell mechanics measurement. Figure 3A shows a schematic diagram for the AFM set-up for single-cell compression.

• 20.
  Follow our previously reported protocols for single-cell-based measurements.^15,16,17 Use an MFP-3D AFM system (Oxford Instruments, United Kingdom) coupled with an inverted microscope (Olympus America, USA).

  • a.
    Mount a gold-coated AC240 (k = 2 N/m, Oxford Instruments, United Kingdom) cantilever onto the AFM cantilever holder; then, insert the holder into the AFM head.

    CRITICAL: The selection of cantilever is important to assure researchers to attain desired compression under practical loading force. The gold-coating enhances the reflectivity of the cantilever, thus benefits the initial laser alignment and subsequent AFM photodiode detection. The stiffness of the cantilever k = 2N/m, assures a near 100% deformation of cardiomyocytes under 3–4 μN load.

  • b.
    Open the data acquisition software Igor Pro (WaveMetrics, USA).
  • c.
    Focus the laser on the reflective area of the cantilever. Then, maximize the SUM signal of the photodiode and zero the deflection signal.
  • d.
    Measure the intensity-frequency profile of the cantilever to determine the thermal frequency (f₀), which serves to calculate the spring constant (k₀) via the thermal tuning method,¹⁸ and as a reference value for subsequent steps.
• 21.Use a spatula to deposit approximately 5–10 mg of 40 μm glass microspheres (Thermo Scientific, USA) onto the center of a clean MatTek dish.
• 22.Disperse the microspheres by gently tapping the side of the MatTek dish.
• 23.
  Mount MatTek dish on the AFM stage.

  • a.
    Use a dropper or pipette to carefully drop one droplet (15–20 μL) of non-drying immersion oil (Cargille Labs, USA) onto the center of the 60x objective lens of the inverted microscope (Olympus America, USA).
  • b.
    Secure the MatTek dish on the AFM stage.
  • c.
    Bring the 60x objective into contact with the glass base of the dish.
  • d.
    Use the microscope’s fine focus knobs to bring the microspheres into clear view.
  • e.
    Remove the AFM head to create space for working with the glue in the following steps.
• 24.
  Attachment of microsphere to cantilever.

  • a.
    On a clean glass surface, use a clean plastic pipette to mix the two-component 5-min epoxy glue (Devcon, USA) in a 1:1 ratio.

    • i.
      Immediately after mixing, use a clean pipette tip to carefully pick up approximately 1 μL of the glue mixture.
    • ii.
      Draw a thin glue line across the glass bottom of the MatTek dish, perpendicular to the cantilever’s long axis, slightly off-center, as shown in Figures 3B and 3C.
  • b.
    Place the AFM head back on the stage and align the AFM cantilever directly above the glue line.

    • i.
      Lower the cantilever until the tip contacts the glue, allowing 2–3 s for glue transfer.
    • ii.
      Lift the cantilever away from the glue and measure the free cantilever’s new resonance frequency (f₁).
  • c.
    Position the cantilever above the center of a selected glass microsphere on the dish.

    • i.
      Lower the cantilever until the glue-covered tip contacts the microsphere, making sure the microsphere is centered at the cantilever tip’s end.
    • ii.
      Allow the cantilever tip to remain in contact with the microsphere for at least 30 min to ensure attachment.
    • iii.
      Lift the microsphere-modified cantilever off the surface and measure its resonance frequency (f₂). Figure 4 shows the scanning electron microscope images of centrally positioned microsphere.
• 25.
  Spring constant determination of microsphere-modified cantilever:

  • a.
    Apply the added-mass method, as described in Equation 1,¹⁸ to calculate the spring constant (K₁).

    $${\mathrm{k}}_1=\frac{{\left(2\pi \right)}^2{\mathrm{M}}_1}{\left(\frac{1}{{\mathrm{f}}_2^2}-\frac{1}{{\mathrm{f}}_1^2}\right)}$$ (Equation 1)

    Note: The mass of the microsphere (M₁) is calculated using its diameter and density. The microsphere has 40 μm diameter and it is made of soda-lime glass whose density is 2500 kg/m.^3,19 F₁ and f₂ are measured in steps 19 and 20, respectively.

  • b.
    Calibrate the spring constant (K₁) for the position of the microsphere on the cantilever.

    $$k_2=k_1{\left(\frac{l_0}{l_1}\right)}^3$$ (Equation 2)

    • i.
      Capture an image of the modified cantilever using the 10X objective.
    • ii.
      Use ImageJ software (NIH, USA) to measure the distance from the cantilever’s base to its apex (L₀), and the distance from the cantilever’s base to the center of the attached microsphere (L₁), as shown in Figure 3D.
    • iii.
      Use these measurements (l₁, l₀, and K₁) to calculate the final spring constant (K₂), following Equation 2.²⁰ From this point forward, the cantilever modified with the microsphere is referred to as “compression probe”.

      CRITICAL: Step 24 must be completed within 5 min after dispensing the glue on the MatTek Dish to avoid premature curing. Alternatively, glues with longer curing times (up to 30 min) are available, providing more time for probe preparation. This alternative runs the risk of sphere rotation during curing, leading to glue-coated spheres, instead of clean ones. We recommend using fast-curing glue. Complete the attachment of the glass microsphere to the cantilever at least 24 h before single-cell mechanics measurements to ensure full curing of the glue. The complete curing was checked my measuring the resonance frequency with time until ready.

Figure 3.

Schematic diagram for single-cell mechanics measurements by AFM

(A) Schematic diagram illustrating the use of AFM for single-cell mechanics measurements.

(B) Glue line at the bottom of the MatTek dish was moved till its center is under the cantilever probe.

(C) Cantilever was lowered till the tip apex was poking into the glue.

(D) A glass sphere-modified cantilever with lengths well defined.

Figure 4.

Scanning electron microscope (SEM) images of a modified AFM cantilever with a 40 μm microsphere

Images were acquired using an S-4100T FE-SEM (Hitachi High Technologies America, California) at an accelerating voltage of 3 kV.

(A) Top view of the cantilever (scale bar: 100 μm).

(B) Bottom view showing the centrally positioned microsphere (scale bar: 23.1 μm).

Single-cell mechanics measurements

Timing: 2–4 h

We describe the operational steps to perform mechanics measurement on single cardiomyocytes using AFM.

• 26.Mount the compression probe and MatTek dish containing VCMs in Tyrode’s solution to the instrument, refer to step 23. Refer to Figure 3A for a schematic diagram for the AFM set-up for single-cell compression.
• 27.
  Place the AFM back on the stage and then lower the compression probe until it is fully submerged in the Tyrode’s solution.

  • a.
    Adjust the SUM and deflection signals accordingly.
  • b.
    Carefully lower the compression probe until contacts the glass base of the MatTek dish.
• 28.
  Prepare for single-cell compression measurements:

  • a.
    Compression probe sensitivity. Obtain a deflection vs. z-piezo movement curve on the glass bottom of the MatTek dish. The slope yields the Deflection Inverse Optical Lever Sensitivity (InvOLs), typically ∼80 nm/V. Note that the acceptable range for Delf InvOLs is < 100 nm/V and for the noise level is < 100 pN.
  • b.
    Spring constant Input. Enter the spring constant of the compression probe (K₂), into the data acquisition software and click on “save”.

    Note: Igor uses the entered spring constant (K₂), along with the probe’s deflection InvOLS and the raw deflection vs. z-piezo curve, to generate the force vs. z-piezo movement curve, also known as force-distance curve.

  • c.
    Baseline correction. Acquire a force-distance curve on the glass-base, and select two points within its baseline and click the function of “polynomial virtual baseline correction” in Igor to obtain a corrected, linear baseline.
• 29.
  Perform single-cell compression measurements and record time-lapse video:

  • a.
    Set compression speed and z-piezo range.

    • i.
      In Igor Pro, set the compression speed to 2 μm/s. An acceptable range is between 0.5 and 5 μm/s.

      CRITICAL: Speeds higher than 5 μm/s could introduce hydrodynamic forces, potentially affecting the accuracy of force measurements.^15,16,17,21 Conversely, lower than 0.5 μm/s leads to longer experiment and could subject the AFM to more thermal drifts.

    • ii.
      Set the z-piezo range to ≥27 μm to ensure a complete compression of VCMs (typical VCM height of 17–25 μm).

      Note: The initial probe is positioned at ∼2 μm above the center of the cell, and the movement range was then set to 2 μm plus cellular height.

  • b.
    Acquire reference and target VCM force-distance curves. Our team developed a custom-made macro to extract relevant values from the force-distance curves in-situ. The code is available at GitHub (https://github.com/admoralesm/AFM-tools.git).

    • i.
      Reference curve (glass base). Select debris-free region near (∼10 μm) the targeted VCM and perform a force-distance measurement. Identify and select the contact region of the force-distance curve and click “Set surface” in the macro.

      Note: This assumes the glass is infinitely stiff and stablishes a zero-force baseline by recording the reference values for deflection (Delf₀) and z-piezo (Z₀) at contact point. With these settings, the cantilever bending (Defl) is proportional to the z-piezo displacement (Δz).

    • ii.
      VCM compression curve. (Start recording time-lapse video before compression, see step 29b(i)). Position the compression probe above the central region of the VCM and perform a force-distance curve measurement on the VCM to maximum cellular relative deformation (ε ∼0.75 for VCMs). Then, use the cursor in Igor to select two points of the resulting force-distance curve: the probe-cell contact point, Z₁ (Defl₁) and the maximum deflection (maximum force) reached, Z₂ (Delf₂₎. Macro records these two points and uses it for future plotting.
  • c.
    Record time-lapse video.

    • i.
      Start recording using a high-resolution video camera (Olympus America, USA) connected to the bright-field microscope just before compressing the VCM.
    • ii.
      Figure 5 reveals 4 snapshots obtained from Methods video S1 (166 s) at relevant points. Figure 5A shows the cell next to the compression probe before compression. Figure 5B shows the probe above the center of a VCM. Figure 5C shows the maximum deformation, where the probe reaches near the glass surface and the VCM bulges outward. Figure 5D shows the cardiomyocyte immediately after compression and probe retreat, i.e., nearly a full recovery.

      CRITICAL: Perform and complete all measurements within two hours to ensure cell viability and minimize the time cells were outside the incubator.

• 30.
  Plot Generation. The macro is specifically programmed to generate the following plots after finishing the compression cycle for each target cell.

  • a.
    VCM height at point of compression. Use Equation 3 to extract the cellular height.

    $$Z_H=Z_0-Z_1$$ (Equation 3)
  • b.
    Force vs. z-piezo movement.

    • i.
      The macro generates a full force-distance profile (approach and retreat) for each compressed VCM.
    • ii.
      Figure 6 shows the full force vs. z-profile, which aids on the determination of mechanical properties and cell adhesion.
  • c.
    Force vs. relative deformation(ε).

    • i.
      Use the force vs. z-piezo profile and Z_H values, and Equation 4 to plot force vs. ε profile of the VCM.

      $$\epsilon =\frac{\left(\Delta z\right)}{Z_H}$$ (Equation 4)
    • ii.
      Figure 7A shows the full force vs. ε profile of a compressed VCM.

Figure 5.

Snapshots revealing critical moments during single-cell compression

(A) t = −32 s, i.e., before the AFM was aligned and brought in contact with the cell.

(B) Probe was in contact with the top of the cell, with center-to-center alignment. We define this moment as t = 0 s.

(C) At t = 9 s, the cardiomyocyte reached its maximum deformation.

(D) At t = 180 s, AFM probe retreated away from the cell. Scale bar = 20 μm.

Snapshots are from Methods video S1.

Figure 6.

Raw data, i.e., Force vs. z-movement, acquired from a healthy live VCM, following the protocol discussed

Red trace represents approach, and blue trace represents retreat. Data are taken from Methods video S1.

Figure 7.

A typical force-relative deformation profile, from which Em is extracted

(A) A force versus relative deformation profile of the VCM shown in Methods video S1, with experimental data (solid blue) and non-linear least square fitting using Equation 5 (broken blackline).

(B) Scheme of a cross-sectional view of a VCM revealing key subcellular components contributing to cellular mechanics.

(C) A schematic diagram illustrates possible cellular deformation upon compression.

Methods video S1. Compression of cardiomyocytes during AFM measurement, related to step 25

The compression probe approached the cardiomyocyte, compressed the cell and retreated from the cell.

Analyses of force-deformation profiles to quantify cellular mechanical properties

Timing: 1–5 weeks

We describe the steps to perform AFM data analyses to quantify cardiomyocyte mechanical properties.

• 31.
  Bin cells by their morphology and responses to external forces. Review the videos of each cell compression and categorize the measurements into two groups (bins).

  • a.
    VCMs that remain quiescent during the duration of the recorded video.
  • b.
    VCMs that display contractile activity during and or after compression. This work focuses on (a) as contraction sometimes causes misalignment.
• 32.Cell-probe adhesion. The adhesion force corresponds to the negative peak in the force-distance curve during retraction, representing the adhesive force between the cell and the AFM glass microsphere. In Figure 6, the raw data, i.e., force vs. piezo movement, was recorded, from which the adhesive force was measured to be 0.36 μN.
• 33.
  Quantification of the membrane Young’s modulus of the sarcolemma (E_m) using the method reported by Lulevich et al.¹⁵

  • a.
    Assumptions.

    • i.
      The cross-section of the VCM in the central region is circular, as illustrated in Figure 7B.
    • ii.
      The cell is filled with incompressible fluid.
    • iii.
      The membrane of living VCMs, also referred to as the sarcolemma, is impermeable during compression.
  • b.
    Nonlinear least-square fitting. Use Python to perform a nonlinear fitting on the contact region of the force vs. ε profile of the VCM using Equation 5,¹⁵ and extract the Em corresponding to the stretching of sarcolemma.

    $$\mathrm{F}=2\pi \frac{{\mathrm{E}}_{\mathrm{m}}}{1-{\mathrm{v}}_{\mathrm{m}}}\mathrm{h}{\mathrm{R}}_0{\epsilon }^3$$ (Equation 5)

    Note: The Poisson ratio of the membrane (v_m) is 0.5; the lipid bilayer thickness (h) used is 4 nm, and the radius from the cross section (R₀) is half of Z_H. The code for the fitting using Python can be found at GitHub (https://github.com/admoralesm/AFM-tools.git).

  • c.
    Extract Young’s modulus of the sarcolemma (E_m) as 22.0 ± 4.3 MPa (mean ± standard error), from a dataset (n = 6), fitting range of ε = 0–0.55.

    Note:Figure 7A shows the quality of fit for a typical curve, yielding E_m 20.0 Mpa, with a reduced Χ² = 1.27E⁻¹⁵, and R² = 0.97. Data is further contextualized with the Figure 5 (Snapshots from Methods video S1), force vs. z-piezo (Figure 6), and force vs. ε (Figure 7), with a measured the cellular height of 22.3 μm. The results agree with prior work by our team: the Em measured was of 10–30 MPa for T-cells,¹⁵ 1.5–3.1 MPa for human breast cancer,¹⁶ and 0.9 ± 0.4 MPa in neuronal cells.²¹ As anticipated, VCMs exhibit strong elastic compliance, thus on the high side of E_m.

Expected outcomes

The outcomes from the protocols above include direct measurements of cellular height at the point of compression (Z_H); a time-lapse bright-field microscopy video, as shown in Figure 5 and Methods video S1; force-distance profiles (F vs. z), as shown in Figure 6; and force vs. relative deformation profiles (F vs. ε), as shown in Figure 7.

Limitations

The model focuses on the extraction of membrane stiffness. The stiffness of other internal cellular structures, such as myofibrils, sarcomeres, nuclei, and cytoskeleton, requires further data analysis and modeling.

Troubleshooting

Problem 1

VCM quality control is critical to AFM recordings. The isolation procedures of mouse VCMs need to be followed strictly during the experiments (Steps 1–16).

Potential solution

Heart excision, trimming and cannulation need to be rapid and precise. Heart excision should be properly performed to ensure the remaining ascending aorta section is long enough for cannulation. The aorta should be securely cannulated, ensuring that there is no leak, to maintain proper retrograde perfusion. The concentrations of collagenase type II may need to be adjusted and optimized with each new batch of collagenase. Successful VCM isolations are determined by high percentage of healthy VCMs, characterized by the rod-shape with sharp and distinct edges, clear striations, and clean surfaces under bright-field illumination.

Problem 2

VCMs must be firmly adhered to the surface to assure accurate measurement of single cell mechanics. Under weak attachment, cells may move during compression, resulting in inaccuracy in data acquisition and quantification of cellular mechanics (Step 17–19).

Potential solution

Assuring cell adhesion by coating the glass cover slip of the dish with laminin, fibronectin, or poly-l-lysine, followed by seeding cells, and allowing for 15 min incubation. Test the cell adhesion by gently moving the dish to ensure VCMs are firmly adhered to the glass cover slip.

Problem 3

VCMs are known for their cylindrical morphology, but the two ends of the cells may deviate from this geometry. Choosing the appropriate compression site on VCMs is important to keep the measurement results consistent (Step 29).

Potential solution

To ensure accurate mechanical measurements, the compression should take place in the middle of the VCM, which are firmly adhered to the cover slip. Avoid compression at the edge or two ends of the cells.

Problem 4

For cantilever modification, the center of the sphere needs to be aligned with the center of the cantilever long axis. Misalignments may occur while gluing the microsphere to the apex of the cantilever, leading to twisting of the tip apex, and inaccuracy in measurements (Step 20–25).

Potential solution

Operate with the view of optical microscopy to assure the initial center-to-center alignment. View and correct any shift in position during and after gluing to assure the maintaining of the alignment. Finally, the modified probes are inspected via SEM for high resolution verification.

Problem 5

Materials could attach to the glass microspheres after compression, which alters the spring constant, and bead’s functionality. The former impacts the measurement accuracy, while the latter impacts the adhesive force values (Step 27–29).

Potential solution

Constantly inspect the probe for any attachment of debris. Perform gentle rinses with ELGA Veolia water and ethanol as needed to remove any contaminants.

Resource availability

Lead contact

Further information and/or requests for reagents/materials should be directed to the lead contact, Xiao-Dong Zhang (xdzhang@ucdavis.edu).

Technical contact

Further information and technical details should be directed to the technical contact, Xiao-Dong Zhang (xdzhang@ucdavis.edu).

Materials availability

This study did not generate new unique reagents or animal models.

Data and code availability

The data supporting the current study have not been deposited in a public repository because they are only used for the publication of this manuscript but are available from the corresponding author on request. The code is available in https://github.com/admoralesm/AFM-tools.git.

Acknowledgments

We thank our laboratory members, Yunbo Zheng, Minyuan Wang, Zack Xu, Diana Ramirez, Saswati Panda, Subach Chaudhry, Terell Keel, Noah Haugh, and Sara Ismet, for their constructive comments.

This study was supported by National Institutes of Health (NIH) R56 HL138392, NIH R01 HL158961, and American Heart Association (AHA) 23SFRNPCS1061606 (X.-D.Z.); NIH R01 HL085727, HL085844, HL170520, and HL152055 and AHA 23SFRNCCS1052478 and 23SFRNPCS1060482 (N.C.); and NIH F31 Predoctoral Fellowship HL168956 (D.A.D.). This publication is based upon work supported by the National Science Foundation of the United State of America while G.-Y.L. served at the foundation.

Author contributions

Conceptualization and design, N.C., G.-Y.L., and X.-D.Z.; experiments and data analyses, A.D.M.M., D.A.D., X.-D.Z., V.T., A.K., E.O., Y.H., and N.Z.; manuscript writing and figure creating, X.-D.Z., A.D.M.M., D.A.D., and G.-Y.L.; resources, N.C., G.-Y.L., and X.-D.Z.

Declaration of interests

The authors declare no competing interests.