Collagen is a main constituent of the extracellular matrix, and its content, organization, and
crosslinking status affect significantly tissue mechanical properties. Cells continuously sense
their local mechanical environment via interactions that impact significantly cell fate and the
development and progression of numerous diseases. Non-linear imaging, especially through the
combined use of second harmonic generation (SHG) and two-photon excited fluorescence
(TPEF), has the potential to provide detailed information regarding collagen organization,
content, and crosslinking with sub-micron level resolution in a non-invasive and label-free
manner. However, the crosslink fluorescence properties that are correlated with tissue stiffness
aren’t well understood. Thus, we characterized the TPEF emission over 680 to 920 nm
excitation and 400 to 780 nm emission of three types of collagen gels: a) plain collagen gels, b)
collagen gels with a 250 mM ribose solution for five days prior to gelation, and c) following
gelation. Uniaxial tensile testing of gels created using the same protocols was performed by a
custom-built testing system. We identified two components, which peak at 440 nm and 510
nm, that were significantly correlated with the exponential stiffening detected in collagen gels
treated with ribose following gelation. Thus, TPEF information acquired at these emission
ranges can be used in future studies and in combination with SHG measurements to develop
detailed mathematical models that aim to predict tissue micromechanical properties. The latter
will be useful in helping us to understand mechanosensitive cell behaviors that ultimately
dictate the progress of diseases such as fibrosis and cancer.
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