Supercritical Angle Fluorescence and Raman Techniques in Biophotonics

ABSTRACT Fluorescence and Raman microscopy have gained popularity in bioimaging due to their unique ability to provide three‐dimensional images at excellent contrast in both label and label‐free manners. Currently, conventional optical microscopy techniques make it relatively easy to measure these two mechanisms of a sample's contrast but can access a limited range of resolutions. The recent developments in optical microscopy for the interface through detection manipulation can easily access resolution down to the diffraction limit that utilizes evanescent waves (EW). For example, EW‐based supercritical angle (SA) microscopy can offer much greater axial confinement information than the existing total internal reflection (TIR) techniques and allow for structural and functional imaging with and without the need for fluorescent dyes and labels. This review begins by exploring the fundamental principles of SA fluorescence (SAF) and Raman (SAR) microscopy, shedding light on the underlying physical mechanisms and the advantages they offer over conventional imaging techniques. We delve into the optical principles and instrumentation, highlighting the role of EWs and the critical angle in the excitation and detection of fluorescence signals and Raman scattering from samples. In conclusion, this review article provides a comprehensive overview of the current state of SAF and SAR microscopy in biophotonics.