In this paper, we report the enhancement of resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy by a novel method of structured illumination of an excitation beam. Illumination by multiple excitation beams through the specific pupil apertures with high in-plane wave vectors leads to interference of diffracted light flux near the focal plane, resulting in the contraction of the point spread function (PSF) of the excitation. Light spot reduction by the suggested standing wave (SW) illumination method contributes to make up much lower depletion efficiency of the CW STED microscopy than that of the pulsed STED method. First, theoretical analysis showed that the full width at half maximum (FWHM) of the effective PSF on the detection plane is expected to be smaller than 25% of that of conventional CW STED. Second, through the simulation, it was elucidated that both the donut-shaped PSF of the depletion beam and the confocal optics suppress undesired contribution of sidelobes of the PSF by the SW illumination to the effective PSF of the STED system. Finally, through the imaging experiment on 40-nm fluorescent beads with the developed SW-CW STED microscopy system, we obtained the result which follows the overall tendency from the simulation in the aspects of resolution improvement and reduction of sidelobes. Based on the obtained result, we expect that the proposed method can become one of the strategies to enhance the resolution of the CW STED microscopy.
The feasibility of stimulated emission depletion (STED) microscopy using a solid immersion lens was investigated. First, the theoretical feasibility of the considered system is discussed based on a vectorial field algorithm that uses a stratified medium composed of a SIL air-gap and test sample. Using the simulation, we verified that evanescent waves with much higher spatial frequencies corresponding to the high numerical aperture in the air-gap can be utilized to achieve a higher resolution than a confocal fluorescent image without a depletion beam. The simulated expectation was supported by actual imaging on two types of samples: fluorescent beads with a 20 nm diameter and an actin sample with a filamentous structure. The lateral resolution of the system was determined to be 34 nm via the imaging results on the nano-beads. The system was quite promising for achieving nano-scale surface imaging of biological samples; an even higher resolution was achieved by adjusting the wavelength and the intensity of the depletion beam.
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