Quantitative intrinsic auto-cathodoluminescence can resolve spectral signatures of tissue-isolated collagen extracellular matrix

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.

A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

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For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated

Clearly defined error bars
State explicitly what error bars represent (e.g. SD, SE, CI) Our web collection on statistics for biologists may be useful.

Software and code
Policy information about availability of computer code Data collection CL and SE data was obtained using Attolight Rosa 4634 CL-SEM microscope. For LC MS/MS analysis, peptides were resuspended and separated by reversed-phase chromatography on a Dionex Ultimate 3000 RSLC nanoUPLC system in-line connected with an Orbitrap Q Exactive mass spectrometer (Thermo Fischer Scientific). Fluorescence data was collected using Zeiss LSM 700 laser scanning confocal microscope. The Masson's trichrome data was obtained using Olympus AX70 microscope. Data analysis CL/SEM sata analysis was performed using Digital Surf MountainsMap software with Attolight CL spectroscopy package (v7.4). Spectral histograms were plotted and analyzed using OriginPro 8.0 (from OriginLab). The mass spec data was processed and inspected with the Scaffold 4 software (Proteome Software) to obtain emPAI values for each sample, and finally plotted using Prism 4 software. The open source DAVID online software (https://david.ncifcrf.gov/) was used to select proteins identified with the GO-terms for extracellular space and extracellular region.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information. Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The data that support the plots within this manuscript and other findings of this study are available from corresponding author upon reasonable request.

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Life sciences study design
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Sample size
No sample-size calculation was performed for the CL-SEM study.
Data exclusions In the hyperspectral CL data scans, all the measurement points with an intensity below 10 counts (recorded over 20 ms integration times) were excluded to avoid false peak fitting and deconvolution results. These points resulted from the CCD detector dark noise, and were marked as 'NM' (non-measurable points). Presented spectral histograms do not contain these 'NM' data points.

Replication
Each presented sample was CL-probed 40'000 times to obtain a data set over a whole cross-section area. Each sample type (CFC, bovine, fibrin, humanized scaffolds) was scanned at three different areas showing very similar spectral responses for particular samples. However, the heterogeneity of these samples prevents direct comparison side-by-side. The study has been designed that way, to demonstrate a capability of the CL technique in characterization of biomaterials, and not to provide an absolute information about the studied materialsthis is the scope of another manuscript in preparation.
Randomization Each sample type was selected with an increase in molecular heterogeneity. To demonstrate the CL-technique capacity in molecular identification, we have firstly scanned a reference fibrin and bovine/rat collagen samples, and further performed characterization of CFC and human-cell remodeled CFC samples after 2 and 4 weeks of remodeling. The three different areas per sample in CL characterization were selected randomly on corresponding cross-sections.

Blinding
Sample bio-engineering process and preparation for CL imaging were performed by HML and MSZ was blinded during the CL imaging.
Reporting for specific materials, systems and methods

Mycoplasma contamination
The cells were not tested for mycoplasma.
Commonly misidentified lines (See ICLAC register) Cells used in this manuscript were isolated primary human bladder smooth muscle cells, no misidentified line is applicable