Analysis of optical properties of the mouse cranium--implications for in vivo multi photon laser scanning microscopy

Trans-cranial imaging is the least invasive method for optical in vivo studies of structures in the mouse brain and has found wide application over the last few years. An important issue is how and to what extent the cranium and the tissue between the cranium and the focal point detract from the quality of the recorded images. Here we address this issue by recording transmission images in wild type mice at five wavelengths in the visible and near-infrared spectrum. The recorded laser scanning microscopic images were analyzed pixel by pixel in order to quantify the light attenuation and shading as function of the location of the focal point relative to the cranium. Additional images demonstrate the effects of the mouse crania on the images of fluorescent microspheres in the low micrometer range. The results of this study demonstrate that light attenuation by the cranium, though with typical losses of less than 20% of the incident light, induces shading effects during the imaging process. Geometrical shapes and sizes in the images of the recorded objects may differ substantially depending on whether they have been recorded trans-cranially or not. This is true even for comparatively large structures such as cell somata. Our results call for a more realistic appraisal of the potential of the trans-cranial imaging approach, particularly when it comes to absolute measurements of sizes and shapes of small objects. As trans-cranial imaging has found wide use in contemporary research it is important that the results be interpreted with due caution.