Raman spectroscopic investigation of frozen and deparaffinized tissue sections of pediatric tumors: neuroblastoma and ganglioneuroma

Raman spectroscopy is a nondestructive technique that can provide information at the molecular level about the biochemicals in tissues. We have investigated the cellular regions in neuroblastoma and ganglioneuroma using Raman spectroscopy and compared their spectral characteristics with those of the corresponding normal adrenal gland. Thin sections from both the frozen and the corresponding formalin‐fixed paraffin‐processed (FFPP) tissues were studied in conjunction with the pathological examination of the tissues. Investigation of the spectral data shows that the normal adrenal gland tissues have higher levels of carotenoids, lipids, and cholesterol compared with the neuroblastoma and ganglioneuroma frozen tissues. However, in comparison with the frozen tissues, the FFPP tissues show a significant alteration of several biochemicals, including the complete removal of carotenoids, lipids, and cholesterol in the adrenal tissues. A quantitative analysis using chemometric methods of principal component analysis and discriminant function analysis of the Raman spectral data obtained from the frozen tissues show a clear‐cut classification among pathological groups with high sensitivity and specificity. We have validated the classification results of the FFPP tissues against a training set data obtained from the archived FFPP tissues of nine other patients. The validation process correctly identified and grouped the data with the training set of normal adrenal gland (>97% of the time) and neuroblastoma (100% of the time) tissues, whereas the validation was not so strong for ganglioneuroma. This study shows that Raman spectroscopy combined with chemometric methods can be successfully used to distinguish neuroblastoma and ganglioneuroma at cellular level in frozen tissue sections. This study also shows that formalin fixation and paraffinization/deparaffinization of tissues can alter their biochemical composition. Copyright © 2012 John Wiley & Sons, Ltd.     

Triggered Functional Dynamics of AsLOV2 by Time-Resolved Electron Paramagnetic Resonance at High Magnetic Fields

We present time-resolved Gd−Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter-residue distances during a protein's mechanical cycle in the solution state. TiGGER makes use of Gd-sTPATCN spin labels, whose favorable qualities include a spin-7/2 EPR-active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C-terminus and N-terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light-activated long-range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.