Spatially resolved microrheology of heterogeneous biopolymer hydrogels using covalently bound microspheres
Long Hui Wong1, Nicholas A. Kurniawan1,2,@, Heng-Phon Too1,3,4,# and Raj Rajagopalan1,4,*
1Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, National University of Singapore, Singapore 117576;
2NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456;
3Department of Biochemistry, National University of Singapore, Singapore 117597 and
4Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117576.
#Communicating author; E-mail:
@Current address: FOM Institute AMOLF, Science Park 104, 1098XG Amsterdam,
The Netherlands.
*Current address: Dean of Faculty, Skolkovo Institute of Science and Technology, Moscow, The Russian Federation; E-mail:
Keywords: Particle-tracking; Microrheology; Biopolymer; Extracellular matrix; Microenvironment
Supplementary Figure 1
Schematic diagram illustrating the modification of COOH-microspheres to obtain NHS-microspheres, which in turn react with polymer containing amine groups to form covalent bonds.
Supplementary Figure 2
MSD against time lag of individualand ensemble-averaged surface-immobilized NHS- microspheres. Thetracking resolution of the confocal microscopy was determined at τ of 0.24 seconds, using , to be ~ 10 nm.
Supplementary Figure 3
(A) The concentration of Bovine Serum Albumin (BSA) protein found on NHS- and COOH- microspheres after multiple washing with urea. The Bicinchoninic acid (BCA) protein assay was used to quantify protein concentrations. *Student’s t-test (1-tailed, unpaired): p = 0.05 (B) Images of COOH- and NHS- microspheres left on collagen gel surface after dipping in 2 % SDS three times. Covalently-bound NHS- microspheres were not washed off the collagen surface while substantial washing of COOH- microspherescan be observed. The dotted red line indicated the dipping boundary with the upper regions showing number of microspheres left after dipping.
Supplementary Figure 4
MSD against time lag of 40 (each) NHS- and COOH-microspheres embedded in polyacrylamide gels with 2 different concentrations, 4.6 (w/v)%T and 10.5 (w/v)%T. 20 representative individual and the ensemble-averaged MSD of COOH- (red lines, full black line) and NHS- (blue lines, dashed black line) microspheres were presented on the left column.The ensemble-averaged and standard deviation of MSD at each time lag for COOH- (filled circle, dotted error bars) and NHS- (open circle, full line error bars) microspheres were tabulated on the right column. “Outlying” COOH- microspheres with higher recorded MSD compared to the bulk COOH- and NHS- microspheres contributed to the wider MSD distribution measured by COOH- microspheres.
Supplementary Figure 5
Elastic moduli of green NHS-microspheres modified with different concentration of EDC and NHS and the corresponding red normalizer microsphere (R10). The minimum concentration of EDC and NHS treatment should correspond to G1E-2 microspheres so as to generate microrheological information that is reliable. The 2-tailed, unpaired T-test of R10 normalizer microspheres, indicating batch-to-batch variability, gave a minimum p-value of 1.1 × 10-8. An α-value of 1.0 × 10-8 (*) was chosen to identify the surface modification required to give reliable microrheological reading that was interpretive of the solid material heterogeneity in the sample.
Supplementary Figure 6
MSD measurements of sample individual NHS- microspheres and ensemble-averaged for collagen at 1.5 mg/ml (black lines), 3.5 mg/ml (red lines), 5.5 mg/ml (green lines) and 7.5 mg/ml (blue lines). The ensemble-averaged MSD increased as collagen concentration decreased (1.5 mg/ml > 3.5 mg/ml > 5.5 mg/ml > 7.5 mg/ml) indicating the stiffness of gel increases with collagen concentrations.
Supplementary Figure 7
Elastic Moduli, G’(Pa) / Collagen Concentration (mg/ml)
1.5 / 3.5 / 5.5 / 7.5
NHS-microspheres / Ensemble-Averaged / 2.2 / 14.4 / 27.3 / 33.4
Standard Deviation / 0.9 / 5.7 / 7.0 / 7.6
COOH-microspheres / Ensemble-Averaged / 5.8 / 28.5 / 32.5 / 44.1
Standard Deviation / 2.6 / 16.0 / 9.7 / 13.1
Loss Moduli, G’’
(Pa) / Collagen Concentration (mg/ml)
1.5 / 3.5 / 5.5 / 7.5
NHS-microspheres / Ensemble-Averaged / 0.2 / 3.3 / 10.4 / 10.6
Standard Deviation / 0.08 / 2.2 / 3.3 / 4.0
COOH-microspheres / Ensemble-Averaged / 0.3 / 3.0 / 5.5 / 7.6
Standard Deviation / 0.2 / 2.7 / 4.4 / 6.0
Elastic and loss moduli of 40 individual NHS- and COOH- microspheres dispersed in various concentrations of collagen. The standard deviation in elastic moduli for COOH- was bigger compared to NHS- microspheres.
Supplementary Figure 8
(A) NHS-microspheres selected to monitor local mechanics at leading edge and around C6 glioma cell in 1.5 mg/ml collagen. The positions of the microspheres are highlighted in green circles. (B) Elastic and loss moduli obtained by tracking the trajectories of the selected NHS-microspheres.*Student’s t-test (2-tailed, unpaired): p = 1 × 10-7