We have applied time-of-flight secondary ion mass spectrometry (ToF-SIMS) and laser post-ionization secondary neutral mass spectrometry (laser-SNMS) to examine the immobilization process of PNA and its hybridization capability to unlabeled complementary DNA fragments, to characterize immobilized proteins, and to image intrinsic elements and molecules with subcellular spatial resolution in different types of frozen non-dehydrated biological samples. The possibilities and limitations of ToF-SIMS and laser-SNMS for imaging elements and molecules in biological samples are discussed. Furthermore possibilities for enhancing the detection sensitivity by using polyatomic and cluster primary ions and different laser post-ionization schemes, as well as ways of obtaining 3D molecular images from biological samples are described. The data shows that both ToF-SIMS and laser-SNMS are capable of imaging elements and molecules in complex biological samples and that they are very valuable tools in advancing applications in life sciences. It was found that cluster-ion bombardment is very useful for enhancing the molecular yield, while laser-SNMS resulted in much higher detection sensitivity for elements and specific molecules and is particularly well suited for imaging ultra-trace element concentrations in biological samples. (C) 2008 Elsevier B. V. All rights reserved.