Compatibility of staining protocols for bone tissue with Raman imaging.

We report the use of Raman microscopy to image mouse calvaria stained with hematoxylin, eosin and toluidine blue. Raman imaging of stained specimens allows for direct correlation of histological and spectral information. A line-focus 785 nm laser imaging system with specialized near-infrared (NIR) microscope objectives and CCD detector were used to collect approximately 100 x 450 micro m Raman images. Principal components analysis, a multivariate analysis technique, was used to determine whether the histological stains cause spectral interference (band shifts or intensity changes) or result in thermal damage to the examined tissue. Image analysis revealed factors for tissue components and the embedding medium, glycol methacrylate, only. Thus, Raman imaging proved to be compatible with histological stains such as hematoxylin, eosin and toluidine blue.