Effects of formalin fixation on tissue optical properties of in-vitro brain samples

Application of light spectroscopy based techniques for the detection of cancers have emerged as a promising approach for tumor diagnostics. In-vivo or freshly excised samples are normally used for point spectroscopic studies. However, ethical issues related to in-vivo studies, rapid decay of surgically excised tissues and sample availability puts a limitation on in-vivo and in-vitro studies. There has been a few studies reported on the application of formalin fixed samples with good discrimination capability. Usually formalin fixation is performed to prevent degradation of tissues after surgical resection. Fixing tissues in formalin prevents cell death by forming cross-linkages with proteins. Previous investigations have revealed that washing tissues fixed in formalin using phosphate buffered saline is known to reduce the effects of formalin during spectroscopic measurements. But this could not be the case with reflectance measurements. Hemoglobin is a principal absorbing medium in biological tissues in the visible range. Formalin fixation causes hemoglobin to seep out from red blood cells. Also, there could be alterations in the refractive index of tissues when fixed in formalin. In this study, we propose to investigate the changes in tissue optical properties between freshly excised and formalin fixed brain tissues. The results indicate a complete change in the spectral profile in the visible range where hemoglobin has its maximum absorption peaks. The characteristic bands of oxy-hemoglobin at 540, 580 nm and deoxy-hemoglobin at 555 nm disappear in the case of samples fixed in formalin. In addition, an increased spectral intensity was observed for the wavelengths greater than 650 nm where scattering phenomena are presumed to dominate.