We theoretically show that the shot-noise-limited sensitivity of stimulated Raman scattering (SRS) microscopy, which enables high-contrast vibrational imaging, is similar to that of coherent anti-Stokes Raman scattering microscopy. We experimentally confirm that the sensitivity of our SRS microscope is lower than the shot-noise limit only by <15 dB, which indicates that the high-sensitivity of SRS microscopy is readily available.
The performance of nonlinear structured illumination microscopy (NSIM), which has theoretically infinite optical resolution, largely depends on nonlinear phenomena. Unfortunately, nonlinear effects of excitation saturation and photoswitchable fluorophores suffer from photobleaching and slow switching time, respectively. In contrast, stimulated emission depletion (STED) is also a nonlinear phenomenon, having fast switching time and being potentially harmless. We propose NSIM based on STED, which has structured excitation light and structured STED light, and both structured illuminations have the same pitch and orientation in the sample plane. Theoretical analysis shows that two-structured illumination having the same grating vector can efficiently increase nonlinearity caused by the STED effect and improve optical resolution. We also found that nonlinearity depends on the phase difference between two-structured illumination made by excitation and STED light and that opposite phase is the most efficient to increase harmonic strength. The feasibility study shows that our method can theoretically achieve optical resolution of 1/7 of wavelength with commercially available laser over large field of view.
We propose a time-domain approach for fluorescence lifetime measurements using nonlinear fluorescence microscopy constructed of a pump-probe setup with two-wavelength laser pulses. Nonlinear fluorescence signals generated by fluorescence reduction due to stimulated emission were detectable through a lock-in technique. Changing the time delay between the two-wavelength pulses enables acquisition of a time-resolved nonlinear fluorescence signal, which directly reflects the fluorescence lifetime of the sample and is thus applicable to fluorescence lifetime imaging. We also quantitatively demonstrate that nonlinear fluorescence microscopy possesses better optical resolution than conventional laser-scanning fluorescence microscopy. Experimental trials indicate that straightforward fluorescence lifetime imaging with high optical resolution is readily available.
Super-resolving nonlinear fluorescence microscopy with a pump-probe setup that utilizes repetitive stimulated absorption and stimulated emission caused by two-color beams was developed. The resulting nonlinear fluorescence that undergoes such a repetitive stimulated transition is detectable as a signal via the lock-in technique. The signal is produced by a multiplicative combination of incident beams, which leads to an improvement of the optical resolution. A phenomenological interpretation of the nonlinear fluorescence process that offers estimation of signal properties is provided with rate equations. The proposed method is demonstrated to provide the scalability of the optical resolution.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.