Effects of tissue processing techniques in acoustical (1.2 GHz) and light microscopy

Abstract

In this study the influence of various tissue processing and staining techniques on the acoustical properties of liver tissue was investigated. A qualitative study was performed using ultrasound attenuation as the imaged parameter of a combined optical/acoustical microscope with a 1.2 GHz transducer. Images were made of three sets of adjacent liver sections (6 microns in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylin-eosin (HE), toluidine blue (TB) or non-stained; sectioned by a cryostat or by a paraffin microtome. It was concluded that the images obtained from cryostat sections were of much higher quality than those from paraffin sections. Images obtained from sections that were sectioned while embedded in paraffin displayed no detail at all. No consistent effect was noticed with respect to staining by HE or TB. Alcohol fixed sections gave more detailed images than formalin fixed sections. Formalin fixation in combination with cryostat sectioning yielded many cytoplasmic vacuoles.

Layer-Dependent Variation in the Anisotropy of Apparent Integrated Backscatter from Human Coronary Arteries

“…The samples were stored in saline at all times and were refrigerated at approximately 2°C between measurements. To minimize potential changes in physical and ultrasonic properties, the samples were not frozen or chemically fixed prior to scanning (van der Steen et al 1991; van der Steen et al 1992; Sasaki et al 1996; Hall et al 2000). All scans were performed as soon as feasible, typically beginning one day after death, and always completed no later than 8 days after death.…”

High-frequency photoacoustic microscopy of hematoxylin and eosin stained slides for veterinary oncology applications

“…These results are consistent with early studies showing that an increase in collagen content for different tissues causes a larger attenuation of pulse-echo diagnostic ultrasound (despite being tested at 1 MHz) 12 . It is possible to interpret the dark spot regions in the acoustic image as the paraffin artifacts previously recognized by 13 . Applying Costes' automatic threshold, we obtained the Pearson correlation coefficients r > 0.9 between white light and high-frequency acoustic images, confirming that tissue morphology details can be imaged acoustically at high (< 5 μm) resolution.…”

“…Applying Costes' automatic threshold, we obtained the Pearson correlation coefficients r > 0.9 between white light and high-frequency acoustic images, confirming that tissue morphology details can be imaged acoustically at high (< 5 μm) resolution. However, image quality can be influenced by tissue preparation technique, while embedding in paraffin for subsequent ultrasonic imaging has not been recommended in the past 13 . Although tissue H&E-stain produces no artefacts on high-frequency ultrasonic imaging detail 13 , our diffuse reflectance spectroscopy measurements show that the tissue mounted on a histology slide and exposed to high-frequency ultrasound releases H&E-stain towards the aqueous environment (Figure 3 B, bottom panel, black line).…”

“…However, image quality can be influenced by tissue preparation technique, while embedding in paraffin for subsequent ultrasonic imaging has not been recommended in the past 13 . Although tissue H&E-stain produces no artefacts on high-frequency ultrasonic imaging detail 13 , our diffuse reflectance spectroscopy measurements show that the tissue mounted on a histology slide and exposed to high-frequency ultrasound releases H&E-stain towards the aqueous environment (Figure 3 B, bottom panel, black line). This effect is presumed to be significant in photoacoustic imaging by causing an exogenous stain localized above the sample (in water) to absorb portion of excitation light pulse energy, thus acting as a strong background mask for PAM.…”

Effect of temperature and fixation on the optical properties of atherosclerotic tissue: a validation study of an ex-vivo whole heart cadaveric model

“…For ease of processing and analysis ex-vivo specimens of atherosclerotic tissue are commonly dissected from the myocardium, fixed with formalin and kept at low temperature for preservation. The effects of such processing on the acoustic properties of tissue have been studied extensively [18][19][20][21][22][23]. The knowledge of effects of such processing on tissue optics in the artery wall however is very scarce [24].…”