Quantitative acoustic microscopy at 250 MHz for unstained ex vivo assessment of retinal layers

Abstract

Few quantitative acoustic microscopy (QAM) investigations have been conducted on the vertebrate retina. However, quantitative assessment of acoustically-related material properties would provide valuable information for investigating several diseases. We imaged 12-μm sections of deparaffinized eyes of rdh4 knockout mice (N = 3) using a custom-built acoustic microscope with an F-1.16, 250-MHz transducer (Fraunhofer IBMT) with a 160-MHz bandwidth and 7-μm lateral beamwidth. 2D QAM maps of ultrasound attenuation (UA) and speed of sound (SOS) were generated from reflected signals. Scanned samples then were stained using hematoxylin and eosin and imaged by light microscopy for comparison with QAM maps. Spatial resolution and contrast of QAM maps of SOS and UA were sufficient to resolve anatomic layers within the 214 μm thick retina; anatomic features in QAM maps corresponded to those seen by light microscopy. UA was significantly higher in the outer plexiform layer (420±70 dB/mm) compared to the inner nuclear layer (343±22 dB/mm). SOS values ranged between 1696±56 m/s for the inner nuclear layer and 1583±42 m/s for the inner plexiform layer. To the authors’ knowledge, this study is the first to assess the UA, and SOS of retina layers of vertebrate animals at high frequencies. [NIH Grant R21EB016117 and Core Grant P30EY019007.]