Coronal sections of unfixed rat brain samples were prepared on a. at substrate in order to reveal hippocampal formation (CA1-4 pyramidal neurons) and adjacent neocortical white matter. We demonstrate the feasibility of using surface sensitive techniques such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) and scanning probe microscopy (SPM) to probe lipid distribution, as well as the subcellular features of neurons. In the same anatomical areas, the phase shift image in SPM is especially useful in revealing the cross-section of subcellular structures. We show that the phase shift images reveal distinctive subcellular features and ion images of CN and PO2 fragments from ToF-SIMS appear to de. ne some of the subcellular features. (C) 2008 Elsevier B. V. All rights reserved.