Nonlinear optical microscopy improvement by focal-point axial modulation

Among the most important challenges of microscopy—even more important than the resolution enhancement, especially in biological and neuroscience applications—is noninvasive and label-free imaging deeper into live scattering samples. However, the fundamental limitation on imaging depth is the signal-to-background ratio in scattering biological tissues. Here, using a vibrating microscope objective in conjunction with a lock-in amplifier, we demonstrate the background cancellation in imaging the samples surrounded by turbid and scattering media, which leads to more clear images deeper into the samples. Furthermore, this technique offers the localization and resolution enhancement as well as resolves ambiguities in signal interpretation, using a single-color laser. This technique is applicable to most nonlinear as well as some linear point-scanning optical microscopies.