Abstract

The advent of 4pi microscopy broke the conventional optical resolution limit in the axial direction of the microscope. In combination with fluorescence microscopy it broadened the knowledge of cell biology at the expense of perturbing the samples with extrinsic fluorescent labels. In contrast, Raman microscopy acquires the molecular fingerprint of the sample without the need of extrinsic labels, and therefore improving its resolution can make an even greater impact. Here, we take advantage of the improved axial resolution of a 4pi configuration to form a 4pi Raman microscope. With this microscope we independently and simultaneously analyzed different nanolayers in a multilayer stack. We identified their chemical composition and retrieved their relative sub-wavelength optical separation with a precision of 6 nm.