Abstract
By analyzing isolated collagen gel samples, we demonstrated in situ detection of spectrally deconvoluted auto-cathodoluminescence signatures of specific molecular content with precise spatial localization over a maximum field of view of 300 µm. Correlation of the secondary electron and the hyperspectral images proved ~40 nm resolution in the optical channel, obtained due to a short carrier diffusion length, suppressed by fibril dimensions and poor electrical conductivity specific to their organic composition. By correlating spectrally analyzed auto-cathodoluminescence with mass spectroscopy data, we differentiated spectral signatures of two extracellular matrices, namely human fibrin complex and rat tail collagen isolate, and uncovered differences in protein distributions of isolated extracellular matrix networks of heterogeneous populations. Furthermore, we demonstrated that cathodoluminescence can monitor the progress of a human cell-mediated remodeling process, where human collagenous matrix was deposited within a rat collagenous matrix. The revealed change of the heterogeneous biological composition was confirmed by mass spectroscopy.
Highlights
By analyzing isolated collagen gel samples, we demonstrated in situ detection of spectrally deconvoluted auto-cathodoluminescence signatures of specific molecular content with precise spatial localization over a maximum field of view of 300 μm
No image drift correction was applied to the presented result, yet correlation of individual fibers on the secondary electron and cathodoluminescence images was possible despite a slight sample drift
We have presented the capability of spectrally resolved cathodoluminescence microscopy in the identification and localization of extracellular matrix (ECM) protein complexes in bioengineered soft biological samples by detecting only the intrinsic emission
Summary
By analyzing isolated collagen gel samples, we demonstrated in situ detection of spectrally deconvoluted auto-cathodoluminescence signatures of specific molecular content with precise spatial localization over a maximum field of view of 300 μm. Resolving a complex fibrillar tissue structure in situ, becomes extremely challenging since different types of crosslinked collagens and interacting proteins might be involved In this case, typical characterization techniques, such as histology, immunostaining[10], scanning electron microscopy (SEM) or diffraction-limited optical methods based on fluorescence or second-harmonic generation (SHG)[11,12,13] cannot provide unambiguous compositional information. We show that spectrally resolved cathodoluminescence microscopy can unveil material composition and its spatial distribution in extracellular matrices of collagen isolated from bovine and rat sources, without using antibodies, nanolabels, or fluorophores to enhance the optical signal This capability appears to provide a useful characterization method, especially in the case when no specific antibodies are available, and when characterization of structural properties of complex biological systems is challenging.
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