Scanning acoustic microscopy of biological cryosections: the effect of local thickness on apparent acoustic wave speed
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Scanning acoustic microscopy of biological cryosections: the effect of local thickness on apparent acoustic wave speed Craig J. Williams1*, Helen. K. Graham2*, Xuegen Zhao1, Riaz Akhtar3, Christopher E.M. Griffiths2, Rachel E B Watson2, Michael J Sherratt2 and Brian Derby1 1 School of Materials, University of Manchester, Manchester, UK 2 Institute of Inflammation and Repair, Manchester Academic and Health Sciences Centre, University of Manchester, Manchester, UK 3 Centre for Materials and Structures, School of Engineering, University of Liverpool, UK. * Equal contributors ABSTRACT Scanning acoustic microscopy (SAM), when applied to biological samples has the potential to resolve the longitudinal acoustic wave speed and hence stiffness of discrete tissue components. The heterogeneity of biological materials combined with the action of cryosectioning and rehydrating can, however, create variations in section topography. Here, we set out to determine how variations in specimen thickness influence apparent acoustic wave speed measurements Cryosections (5μm nominal thickness) of human skin biopsies were adhered to glass slides before washing and rehydrating in water. Multiple regions (200x200 μm; n = 3) were imaged by SAM to generate acoustic wave speed maps. Subsequently co-localised 30x30 μm sub-regions were imaged by atomic force microscopy (AFM) in fluid. The images were then registered using Image J. Each pixel was allocated both a height and wave speed value before their relationship was then plotted on a scattergram. The mean section thickness measured by AFM was 3.48 ± 1.12 (SD) Pm. Regional height variations influenced apparent wave speed measurements. A 3.5 μm height difference was associated with a 400 ms-1 increase in wave speed. In the present study we show that local variations in specimen thickness influence apparent wave speed. We also show that a true measure of wave speed can be calculated if the thickness of the specimen is known at each sampling point. INTRODUCTION Alterations in the gross mechanical properties of soft tissues profoundly influence tissue function. For example, the physical properties of skin are known to change with exposure to environmental factors and age [1, 2], whilst the mechanical stiffening of arterial tissue, associated with ageing and diseases such as diabetes, can lead to hypertension, stroke and heart failure [3-5]. As a consequence, in part, of the complexity of tissue structure and composition the key compositional targets of age-related mechanical remodeling remain poorly defined. Hence there is a need to develop techniques that can investigate the mechanical properties of soft tissue at micron length scales [6]. Scanning acoustic microscopy (SAM) has previously been used to characterise soft cardiovascular tissues [7] such as blood vessels [9, 10] and heart valves [11]. Compared with
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nanoindentation, SAM enables relatively fast image and data acquisition at a high spatial resolution (around 1 μm at 1 GHz excitation), ease of sample preparation and the ability to
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