Effect of substrate choice and tissue type on tissue preparation for spectral histopathology by Raman microspectroscopy

Abstract

Raman spectroscopy is a non-destructive, non-invasive, rapid and economical technique which has the potential to be an excellent method for the diagnosis of cancer and understanding disease progression through retrospective studies of archived tissue samples.Historically, biobanks are generally comprised of formalin fixed paraffin preserved tissue and as a result these specimens are often used in spectroscopic research. Tissue in this state has to be dewaxed prior to Raman analysis to reduce paraffin contributions in the spectra. However, although the procedures are derived from histopathological clinical practice, the efficacy of the dewaxing procedures that are currently employed is questionable. Ineffective removal of paraffin results in corruption of the spectra and previous experiments have shown that the efficacy can depend on the dewaxing medium and processing time. The aim of this study was to investigate the influence of commonly used spectroscopic substrates (CaF 2 , Spectrosil quartz and low-E slides) and the influence of different histological tissue types (normal, cancerous and metastatic) on tissue preparation and to assess their use for spectral histopathology. Results show that CaF 2 followed by Spectrosil contribute the least to the spectral background. However, both substrates retain paraffin after dewaxing. Low-E substrates, which exhibit the most intense spectral background, do not retain wax and resulting spectra are not affected by paraffin peaks. We also show a disparity in paraffin retention depending upon the histological identity of the tissue with abnormal tissue retaining more paraffin than normal.

Raman Micro-Spectroscopy for Rapid Screening of Oral Squamous Cell Carcinoma

“…A 100x water immersion objective (LUMPlanF1,Olympus,N.A. : 1.0) was used to focus the laser on the sample, immersed in distilled water, providing a spatial resolution of ~1μm (Fullwood et al, 2014;.…”

Spectral pre and post processing for infrared and Raman spectroscopy of biological tissues and cells

“…However, it may be argued that greater consistency of spectral information is achieved when sections are deparaffinised. Deparaffinising also allows post-staining of the sections, although it has been demonstrated that the efficiency of the deparaffinisation process can depend on the tissue pathology [32]. Nevertheless, it can also be argued that, even for research purposes, protocols for such tissue processing should be maintained as close as possible to those currently employed in the clinical environment.…”

Raman spectroscopy for screening and diagnosis of cervical cancer

“…However, it may be argued that greater consistency of spectral information is achieved when sections are deparaffinised. Deparaffinising also allows post-staining of the sections, although it has been demonstrated that the efficiency of the deparaffinisation process can depend on the tissue pathology [23].…”

“…The x, y co-ordinates of each recorded cell were saved and, after Raman recording at 532 nm, the slides were Pap stained and each cell was revisited to verify whether it was a superficial, intermediate, parabasal or white blood cell. Spectroscopic substrates, such as calcium fluoride, are commonly used for research purposes and although they reduce the presence of confounding contributions of the substrate [22][23][24], they are considerably more expensive and thus not really a viable choice for population screening applications such as cervical cancer screening. Although 785 nm is commonly used for biological applications of Raman spectroscopy, glass shows a strong background at this wavelength so to use glass as a substrate for Raman spectroscopy, spectra must be recorded using shorter wavelengths such as 532 nm.…”