Characterization of MS-FRAP. (A) Experimental setup. Arrows designate directionality (colored lines) along optical paths. (B) In vivo schematic of multiscale measurement regions for each MS-FRAP component (MP-FRAP and SFA-FRAP) overlaid on an MPM image (white, interstitial matrix; black, cells). Scale bar = 30 μm. (C and D) Bleach volumes in 10% agarose containing FITC-dextran2M after a single bleach from each FRAP modality. Axial (left) and radial (right) profiles are shown with overlaid Gaussian fits. Loss of fluorescence appears where bleaching occurred. The bleach volumes at both scales are axially confocal and radially concentric. Scale bars = 1 μm in C, 10 μm in D.

Resolving two diffusing components with MS-FRAP. (A) For MP-FRAP in vivo, a single-component recovery model shows poor fits at early time points. (B) A two-component model is able to fit recovery curves well, with lower Gaussian-distributed residuals (not shown). Accurate fitting to early time points is critical because the greatest signal/noise ratio exists during initial recovery of fluorescence. The abscissa is plotted on a logarithmic scale to highlight the temporal dynamics of the initial recovery. (C) Multiphoton-FRAP measured the diffusion coefficients for FITC-IgG at the interface between two physical phases (agarose gel and aqueous solution). (D) Diffusion coefficients at the interface determined by a two-component fluorescence recovery model were statistically indistinguishable from diffusion measured solely within either phase (gel, p = 0.83; solution, p = 0.37). (E) For comparison, diffusion coefficients were determined by MP-FCS, known to resolve two-component diffusion. Diffusion coefficients measured by MP-FCS at the interface are not statistically different from those shown in D (gel, p = 0.28, solution, p = 0.15). For each bar, n ∼ 9.

Diffusion coefficients measured by MS-FRAP in gels and in vivo. (A) Increasing collagen lowers the effective diffusion rate quantified by SFA-FRAP (p < 0.01). (B) SFA-FRAP shows a difference in vivo between normal and neoplastic tissue (p < 0.01), but not between tumor types (p = 0.61). (C and E) Two-component diffusion of FITC-IgG in gels at short path lengths. Generally, decreasing collagen or increasing hyaluronan content reduced the slow phase fraction, whereas the diffusion coefficients in each phase were largely unaffected. Collagen gels lacking hyaluronan demonstrated a greater diffusion coefficient in the fast phase and larger slow phase fraction. (D and F) In vivo two-component diffusion of FITC-IgG. In D, no significant difference is observed for either phase across tissue types. In F, the slow diffusing fraction shows differences between tissue types. A similar trend of phase fractions is seen as in the gels (matched shading), possibly due to differences in estimated collagen/hyaluronan content.

Measured tortuosity (λ) by MS-FRAP in gels and in vivo. (A, B, D and E) Path-length dependent tortuosities in interstitial matrix-mimetic gels and in vivo tissue. MP-FRAP measures diffusion in the interstitial matrix between cells while confocally concentric SFA-FRAP probes both the interstitial spaces and cellular geometries. Tortuosity >1 indicates increased hindrance to diffusion. Higher collagen content in gels increases the interstitial tortuosity (λ_int), or interstitial hindrance, reflected in both λ_SFA (A) and λ_MP (D) (p < 0.01). A similar effect is observed with λ_MP in E between tumor types previously shown to differ in collagen content (p < 0.01). There is a pronounced difference in λ_SFA between the normal tissue and the tumors shown in B (p < 0.01). (G) Cellular hindrance (λ_cell), defined as the geometric tortuosity in the literature, across gels is nonexistent (λ_cell ∼ 1) due to acellularity (p = 0.85). (H) Diffusion in normal subcutaneous muscle demonstrates a cellular hindrance near 1 given the anisotropic arrangement of muscle fibers, whereas those in LS174T (p < 0.01) and HSTS26T (p < 0.05) are significantly greater suggesting increased cellularity. (C, F and I) Selective cytotoxic treatment of mice bearing LS174T tumors with diphtheria toxin showed a significant decrease in cellular hindrance (p = 0.01), which arises from a decrease in λ_SFA (p < 0.01) but not in λ_MP (p = 0.39) because the matrix is not affected. Tortuosity >1 indicates increased hindrance to diffusion. For each group, n = 3 in gels and n = 4 in vivo.