Figure 1.
Laser tweezers active microrheology.
(A) Active microrheology instrumentation. A trapping laser (1064 nm) and particle position detection laser (785 nm) are combined and focused by the microscope objective into the device, which is mounted in a piezoelectric microscope stage. Forward scattered light is collected by the microscope objective and directed towards the QPD after passing through a filter designed to isolate the 785 nm beam. (B) Illustration of a laser trapped bead bound in a fibrous ECM and oscillated by laser tweezers. Both the trapping (red) and particle detection (orange) laser foci are incident onto the bead. As drawn the trapping beam is applying a leftward force to the bead. (C) Sample AMR loss modulus, G″, measured in water at room temperature reports a viscosity of 1±0.7 cP determined from a linear fit, R2 = 0.997.
Figure 2.
(A) Photograph of the assembled device containing a fibrin gel and media containing phenol red. CAD drawings showing: (B) trimetric view of the assembled device, (C) transverse section through a plane dissecting the lever arm, (D) vertical section through the center of the post. The device comprises a post (a) held by a cantilever arm (b), which houses a leadscrew (c) and spring-plunger assembly (d,e) held rigidly by a lead screw block (f). As the thumbscrew is rotated inwards, it pushes the lever arm (g) causing a counter clockwise rotation of the post as viewed from above. As the thumbscrew is rotate out, the spring-plunger assembly pushes the lever arm in the opposite direction causing clockwise rotation of the post. A stage insert (h) houses a Petri dish (i) containing the ECM.
Figure 3.
Microrheology reveals heterogeneity unresolved by parallel plate rheology.
Fibrin gels polymerized at 2.5, 5 and 10 mg/ml fibrin were measured by both AMR (A) and parallel plate rheology (B). Each curve in (A) represents AMR for a different bead measured at 5, 10, 20, 50, 100 and 200 Hz (black = G′, grey = G″). Errorbars represent the standard deviation of G for five sequential measurements. Errorbars in (B) represent the standard deviation in G measured for five gels. AMR measured for the three concentrations of fibrin demonstrate considerable overlap as compared to parallel plate rheology.
Table 1.
Comparison of fibrin parallel plate and microrheology.
Figure 4.
Microrheology reports local mechanical heterogeneity around a single cell.
An AoSMC cultured in a 3D fibrin gel polymerized at 2.5 mg/ml fibrin. Six beads proximal to the cell were chosen at random for AMR. Note the 10-fold difference in G′ between beads 4 and 6. Black = G′, grey = G″, scale bar = 20 µm.
Figure 5.
Microrheology within the strain gradient device.
AMR within a 2.5 mg/ml fibrin gel confirms location-dependent stiffening with rotation of the post. (A) FEA of shear strain, , in response to a 2.4° rotation of the post. (B) Stress-strain curves for parallel plate rheology strain sweeps of 2.5, 5, and 10 mg/ml fibrin gels report nonlinear stiffening. (C) AMR map of G′ in a region near the post. In the first 0.1 mm from the post, changes in G′ are largest in the circumferential direction. For each colored block, G′ is compared to that of the unrotated state (*p<0.05; †p<0.15). (D) AMR for a single bead in either region R1 or R2 as specified in (C). AMR was performed as the post was rotated by 0.8, 1.6 and 2.4°, and then again as the post was rotated back. In region R1, no stiffening was observed as compared to R2 where G′ increased with rotation and then decreased as the post was rotated back to its original position. (E) Stiffening in region R2 (triangles) exhibited hysteresis where the material stiffened and softened along different paths as the post rotated. Note that the material returned to its original stiffness as measured by AMR. No change in stiffness was observed in region R1 (circles). Each point is the mean G′ of all measured frequencies. Errorbars represent the standard deviation of 5 sequential measurements.
Figure 6.
Matrix deformation does not accompany stiffening as observed by confocal microcopy.
Maximum projection images computed from confocal stacks of fluorescently labeled fibrin gels polymerized within a Petri dish at 2.5 (A), 5 (B) , or 10 (C) mg/ml show decreasing pore size with increasing concentration. The 10 mg/ml gel exhibits fiber bundling. (D) A 2.5 mg/ml fibrin gel was polymerized within the shear gradient device. After rotation of the post by 2.4° there is a visible displacement in the gel in region R2 (see Fig. 5), with little deformation in matrix geometry. (E) Comparison of fibrin pore volume shows significant pore volume reduction with increasing fibrin concentration, but not with rotation of the post in region R2 of the device, where AMR reports 10-fold stiffening (*p<0.05).
Figure 7.
3D multiphoton orbital tracking shows nanostructural fiber changes.
(A) In 3D multiphoton orbital tracking the position of multiphoton fluorescence is calculated from intensity modulation of the point spread function as the laser is rapidly scanned in an orbit around the fiber. Fibers are tracked as the orbit is moved in the fiber direction by a linear ramp function. (B) Multiphoton images of a region in a fibrin gels before and after rotating the post by 2.4°. Fibrin was labeled with ANS (scale bar = 20 µm). (C) Tracked fiber before and after rotation of the post shows elongation and straightening. With no rotation the fiber has nanometer-scale structure. (D) MSD for the fiber midpoint in both the y and x directions with and without rotation of the post. Since the fiber runs nearly parallel to the x-axis of the orbital tracking coordinate system, the MSD is relatively flat along that axis, as compared to the y-axis where significant reduction in MSD is seen once the post is rotated.
Figure 8.
Aortic smooth muscle cells align with stiffness gradient.
AoSMCs were cultured in a fibrin gel polymerized at 2.5 mg/ml fibrin and subjected to rotation of the post by 2.4° on day 2. Cells were fixed and labeled with Alexa-488 Phalloidin on day 9 for confocal microscopy with a 10× microscope objective. (A) Far from the post cells are randomly oriented and relatively abundant. (B–H) Cells within several hundred microns from the post align in a direction more circumferential than radial. (I) a region in (C) imaged with a 60× water immersion objective. Scale bar = 300 µm (A–H), scale bar = 50 µm (I).