Differential confocal Raman image subtraction microscopy for high spatial resolution imaging

Confocal Raman microscopy (CRM) is widely used in various fields because it provides topography and molecular fingerprint information on delicate structures in a non-destructive and label-free manner. However, CRM's limited spatial resolution and focusing stability have impeded its observation capabilities for finer structures. This paper proposes a differential confocal Raman image subtraction microscopy (DCRISM) method to address the issues above. This method improves spatial resolution by weighted subtraction of Raman images acquired from two pinholes of different sizes. Concurrently, differential confocal technology is employed to provide axial focus tracking, guaranteeing the stability of the focus during the imaging process. Compared to traditional CRM, the Raman spatial resolution is improved by about 19.5 % and 18.4 % in the lateral and axial directions, with an axial focusing capability of 2 nm. These results facilitate the application of CRM to the measurement of microstructures with dimensions approaching or even exceeding the diffraction limit.