Super-Resolution Microscopy with Nonlinear Fluorescence Responses for Signal Localization

Abstract

Nonlinear optical responses of specimens enable super-resolution microscopy by confining the excitation and detection volumes of fluorescence to spatial regions smaller than the diffraction limit. These responses arise under specific illumination conditions, such as increased light intensity or specific excitation wavelengths, which induce non-linear emission behaviors in fluorescent molecules. This review highlights laser-scanning and light-sheet microscopy as key platforms where nonlinear optical effects have been applied to significantly enhance spatial resolution. In addition to these platforms, structured illumination microscopy (SIM), an established super-resolution technique, can further broaden its capabilities by incorporating nonlinear optical responses—a direction also highlighted in this review. Selective-plane activation SIM (SPA-SIM), for instance, integrates light-sheet illumination and structured illumination using photoswitchable fluorescent proteins, enabling the selective observation of specific planes with high spatial resolution. This approach effectively suppresses background signals and enables three-dimensional super-resolution imaging even in thick or dense specimens, such as cell spheroids.