We propose a novel physical mechanism for breaking the diffraction barrier in the far field. Termed fluorescence emission difference microscopy (FED), our approach is based on the intensity difference between two differently acquired images. When fluorescence saturation is applied, the resolving ability of FED can be further enhanced. A detailed theoretical analysis and a series of simulation tests are performed. The validity of FED in practical use is demonstrated by experiments on fluorescent nanoparticles and biological cells in which a spatial resolution of <λ/4 is achieved. Featuring the potential to realize a high imaging speed, this approach may be widely applied in nanoscale investigations.
The resolution of conventional optical equipment is always restricted by the diffraction limit, and improving on this was previously considered improbable. Optical super-resolution imaging, which has recently experienced rapid growth and attracted increasing global interest, will result in applications in many domains, benefiting fields such as biology, medicine and material research. This review discusses the contributions of different researchers who identified the diffractive barrier and attempted to realize optical super-resolution. This is followed by a personal viewpoint of the development of optical nanoscopy in recent decades and the road towards the next generation of optical nanoscopy.
A novel method is proposed for generating sharper fluorescent super-resolution spot by azimuthally polarized beam in stimulated emission depletion (STED) microscopy. The incoherent superposition of azimuthally polarized beam with five-zone binary phase plate and the same beam with quadrant 0/πphase plate can yield a tightly focused doughnut spot surrounded completely and uniformly. And azimuthally polarized beam modulated by a vortex 0-2π phase plate works as pump beam. Compared with known effective excitation spot yielded by circular polarized STED beam, the azimuthally polarized beam result is shaper, as well as energy-saving, costing only ~50% of the energy cost by circular polarized beam. A STED beam of less intensity has the potential to reduce fluorescence photobleaching and photodamage in living cell imaging. In addition, the influence of Ez absence as well as FWHM of pump beam in the focal field is discussed.
We have designed and built a time-gated continuous wave stimulated emission depletion (CW-STED) nanoscopy to visualize microstructures beyond the diffraction limit. An off-line time-gating detection was performed with the help of time-correlated single-photon counting technique. Experimental results showed that before time-gating, the resolution of our system was about 75 nm with a depletion beam (592 nm) power of 200 mW. By using the off-line time-gating detection, the resolution was further improved to 38 nm. Biological samples were also used to test the performance of our time-gated CW-STED, and a resolution of 70 nm was achieved with a depletion beam (592 nm) power of 85 mW. Detailed principles of time-gated CW-STED were discussed in the text. The time-gated STED provides a better resolution with finite laser power.
A novel method is proposed for generating a three-dimensional dark spot surrounded uniformly and completely by light in all three dimensions. The superposition of a radially polarized beam with a circular π phase plate and the same radially polarized beam with a quadrant 0/π phase plate can yield a better three-dimensional dark spot. The coherent and incoherent superpositions of these two beams are demonstrated and discussed in detail and the new effective full width at half maximum (FWHM) of a super-resolution focal spot is discussed. Compared with some former results, the dark spot yielded by this proposed method proved to be more uniformly surrounded by light. Only one type of polarized beam is applied. It facilitates the process of finding a suitable polarization for the pump beam in a super-resolution fluorescent system. Axial resolution has a significant improvement.
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