The implementation of force volume AFM using micrometer-sized probes for the routine mapping of tissues elastic modulus needs methodical optimizations, in order to overcome the intrinsic slowness of the technique (in AFM, around ten minutes are necessary to collect a single image using a conventional microscope configuration) 3, 4, 16, adjust resolution and establish criteria to select regions of interest (ROIs) appropriately. Nevertheless, it has a great potential for screening large areas of biological samples with timescales compatible with many biological processes 21. The force volume AFM using micrometer-sized probes (beads, microspheres) does not provide such nanoscale resolution, due to the probe’s size. These probes have been implemented in dedicated setups for high-speed and high-resolution imaging to detect protein motion and study dynamic processes in biological membranes with spatiotemporal resolution (~ 1 nm lateral, ~ 0.1 vertical, 100 ms temporal resolution) 18, 19, 20. Nanometer-sized probes, with typical diameters below 10 nm, allow exploring sub-micrometer regions of the sample. In AFM it is possible, by selecting the probe spring constant and tip diameter, to define the relevant scales of the sample inspection. Mechanobiology, in fact, helps to understand how physical forces and mechanical properties affect the function of the molecules, organelles, cells and tissues 17. The detection of mechanical forces between the probe and the sample by AFM has enabled a deeper characterization of biological samples.
When used in force volume mode 3, 15, the atomic force microscope provides mechanical information as elastic modulus maps in a quantitative manner, with lateral resolution down to the nanometer (nm) and force sensitivity in piconewtons (pN) 16. Micro- and nano-mechanical information 3, 4, 5 has been used to address important biological questions from mechanosensing 6, 7, 8, 9, 10, to finding cancer fingerprints 4, 11, 12, targeting metastases 13, and unveiling the mechanism of mechanical failure in connective tissues 5, 14. Physiological and pathological modifications in the composition of the extracellular matrix critically influence the mechanical properties of living tissues, especially when components with support function are involved (e.g. The proposed preparation method ensures safe handling of the tissue sections guarantees the preservation of their micromechanical characteristics over time and makes it much easier to perform correlation experiments with different biomarkers independently. The choice of a separate reference sample stained for collagen allows correlating elastic modulus with collagen amount and position with high statistical significance. Collagen identification is obtained in a robust way and affordable timescales, through an optimal design of the sample preparation method and AFM parameters for faster scan with micrometer resolution.
In this work, force volume AFM is used to identify collagen-enriched domains, naturally present in human and mouse tissues, by their elastic modulus. The detection of micrometer-size, heterogeneous domains at different elastic moduli in tissue sections by AFM has remained elusive so far, due to the lack of correlations with histological, optical and biochemical assessments. The integration of AFM data with data of the molecular composition contributes to understanding the interplay between tissue biochemistry, organization and function. Force volume AFM can precisely capture the mechanical properties of biological samples with force sensitivity and spatial resolution. Changes in the elastic properties of living tissues during normal development and in pathological processes are often due to modifications of the collagen component of the extracellular matrix at various length scales.