Exploration of nanoscale tissue structures is crucial in understanding biological processes. Although novel optical microscopy methods have been developed to probe cellular features beyond the diffraction limit, nanometer-scale quantification remains still inaccessible for in situ tissue. Here we demonstrate that, without actually resolving specific geometrical feature, OCT can be sensitive to tissue structural properties at the nanometer length scale. The statistical mass-density distribution in tissue is quantified by its autocorrelation function modeled by the Whittle-Mateŕn functional family. By measuring the wavelengthdependent backscattering coefficient μ b (λ) and the scattering coefficient μ s , we introduce a technique called inverse spectroscopic OCT (ISOCT) to quantify the mass-density correlation function. We find that the length scale of sensitivity of ISOCT ranges from ~30 to ~450 nm. Although these subdiffractional length scales are below the spatial resolution of OCT and therefore not resolvable, they are nonetheless detectable. The subdiffractional sensitivity is validated by 1) numerical simulations; 2) tissue phantom studies; and 3) ex vivo colon tissue measurements cross-validated by scanning electron microscopy (SEM). Finally, the 3D imaging capability of ISOCT is demonstrated with ex vivo rat buccal and human colon samples.
Cell response to
matrix mechanics is well-known; however, the ability
to spatially pattern matrix stiffness to a high degree of control
has been difficult to attain. This study describes the use of maskless
photolithography as a flexible process for direct, noncontact gradient
patterning of photodegradable hydrogels with custom graphics. Any
input gray scale image can be used to directly chart hydrogel cross-link
density as a function of spatial position. Hydrogels can be patterned
with submicron resolution, with length scales within a single substrate
spanning several orders of magnitude. A quantitative relationship
between input grayscale image pixel intensity and output gel stiffness
is validated, allowing for direct gradient patterning. Such physical
gradient hydrogel constructs are rapidly produced in a highly controlled
fashion with measured stiffness ranges and length scales that are
physiologically relevant. Mesenchymal stem cells cultured on these
physical gradients matrices congregate and align orthogonal to the
gradient direction along iso-degraded lines. This approach results
in a robust and high-throughput platform to answer key questions about
cell response in heterogeneous physical environments.
Cross-linked
polyacrylamide hydrogels are commonly used in biotechnology
and cell culture applications due to advantageous properties, such
as the precise control of material stiffness and the attachment of
cell adhesive ligands. However, the chemical and physical properties
of polyacrylamide gels cannot be altered once fabricated. Here, we
develop a photodegradable polyacrylamide gel system that allows for
a dynamic control of polyacrylamide gel stiffness with exposure to
light. Photodegradable polyacrylamide hydrogel networks are produced
by copolymerizing acrylamide and a photocleavable ortho-nitrobenzyl (o-NB) bis-acrylate cross-linker. When
the hydrogels are exposed to light, the o-NB cross-links
cleave and the stiffness of the photodegradable polyacrylamide gels
decreases. Further examination of the effect of dynamic stiffness
changes on cell behavior reveals that in situ softening
of the culture substrate leads to changes in cell behavior that are
not observed when cells are cultured on presoftened gels, indicating
that both dynamic and static mechanical environments influence cell
fate. Notably, we observe significant changes in nuclear localization
of YAP and cytoskeletal organization after in situ softening; these changes further depend on the type and concentration
of cell adhesive proteins attached to the gel surface. By incorporating
the simplicity and well-established protocols of standard polyacrylamide
gel fabrication with the dynamic control of photodegradable systems,
we can enhance the capability of polyacrylamide gels, thereby enabling
cell biologists and engineers to study more complex cellular behaviors
that were previously inaccessible using regular polyacrylamide gels.
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