Nanomechanical and Morphological AFM Mapping of Normal Tissues and Tumors on Live Brain Slices Using Specially Designed Embedding Matrix and Laser-Shaped Cantilevers

Abstract

Cell and tissue nanomechanics has been intriguingly introduced into biomedical research, not only complementing traditional immunophenotyping and molecular analysis, but also bringing unexpected new insights for clinical diagnostics and bioengineering. However, despite the progress in the study of individual cells in culture by atomic force microscopy (AFM), its application for mapping live tissues has a number of technical limitations. Here, we elaborate a new technique to study live slices of normal brain tissue and tumors by combining morphological and nanomechanical AFM mapping in high throughput scanning mode, in contrast to the typically utilized force spectroscopy mode based on single-point probe application. This became possible due to the combined use of an appropriate embedding matrix for vibratomy and originally modified AFM probes. The embedding matrix composition was carefully developed by regulating the amounts of agar and collagen I to reach optimal viscoelastic properties for obtaining high-quality live slices that meet AFM requirements. AFM tips were rounded by irradiating them with focused nanosecond laser pulses, while the resulting tip morphology was verified by scanning electron microscopy. Live slices preparation and AFM investigation take only 55 min and could be combined with a vital cell tracer analysis or immunostaining, thus making it promising for biomedical research and clinical diagnostics.

Tissue mechanics modulate PCNP expression in oral squamous cell carcinomas with different differentiation

“…A number of pertinent studies have demonstrated that Young’s modulus can reflect the hardness of malignant cells or tissues, which are softer than normal tissue or cells. As a result, Young’s modulus is also thought to be an indication of changed cancerous tissue ( 40 ). The Young’s modulus of the tumor tissue in clear cell renal cell carcinomas was much lower than that of normal tissue, and the tumor tissue was significantly less fibrotic ( 41 ).…”

Atomic force microscopy in disease‐related studies: Exploring tissue and cell mechanics

“…The recently developed AFM embedding matrix and laser-shaped cantilever enable nanomechanical and morphological mapping of live brain sections, differentiating between normal and tumor tissue. Biopsy preparation and AFM detection require a mere 55 min and can be conveniently combined with critical cell tracer analysis or immunostaining, showcasing its potential for biomedical research and clinical diagnosis (Farniev et al, 2022).…”

Applications of scanning probe microscopy in neuroscience research

“…In a comparison of tissue sectioning techniques, vibratomed sections of living tissues have been shown to allow for the nanomechanical measurement of cells in tissues, while cryotomed sections inhibited the measurement of viable cells [83]. With an appropriate embedding matrix and modified AFM probes, measurement of the morphology and nanomechanics of live brain tissues has been demonstrated [84].…”