Serial section Raman tomography with 10 times higher depth resolution than confocal Raman microscopy

Confocal Raman microscopy enables 3D imaging of various samples solely based on chemical contrast. However, optical artifacts impair resolution and image quality in subsurface imaging. With serial section Raman tomography, we show that serial ultrathin and semithin sectioning by ultramicrotomy can successfully be combined with subsequent confocal Raman imaging. This new 3D Raman imaging technique reaches a depth resolution of up to 100 nm, which is about 10‐fold better than in confocal Raman microscopy. Structurally complex and optically inhomogeneous samples can not only be imaged, but also be used to quantify structural parameters. Serial section Raman tomography is a promising method for materials science and possibly also for life sciences. Serial section Raman tomography successfully combines serial sectioning by ultramicrotomy with subsequent confocal Raman imaging. This new 3D Raman imaging technique reaches a depth resolution of up to 100 nm, which is about 10‐fold better than in confocal Raman microscopy. Structurally complex and optically inhomogeneous samples can not only be imaged with serial section Raman tomography, but also be used to quantify structural parameters.