Focal Modulation Microscopy: Principle and Techniques

Focal modulation microscopy (FMM) is an emerging single-photon fluorescence microscopy technique that can provide superior image contrast with sub-micron spatial resolutions at large penetration depths in highly scattering media such as biological tissues, mainly by preserving the signal-to-background ratio (SBR). To achieve this, FMM utilizes the coherence property of the light source, through a spatio-temporal modulation scheme to differentially phase modulate segments of the excitation beam. These segments of the beam, when being focused by the objective lens, generate an intensity modulation exclusively at the focal region. Demodulation of the collected fluorescence signal at the designated modulation frequency could allow us to discriminate the in-focus fluorescence from the multiple-scattered background, hence greatly enhance the SBR compared to confocal microscopy. More importantly, the penetration depth of FMM can be significantly improved as the degradation of the image contrast is considerably much slower, and thus could potentially revolutionize the clinical and biomedical applications of FMM for in vivo highresolution visualization of biological specimens. Up to date, a penetration depth up to はどど 航兼 has been demonstrated with biological specimens.