Zhaotai Gu

Breaking the Diffraction Barrier Using Fluorescence Emission Difference Microscopy

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.

Sharper fluorescent super-resolution spot generated by azimuthally polarized beam in STED microscopy

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.

Time-gated stimulated emission depletion nanoscopy

Abstract. 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.

Optical super-resolution by subtraction of time-gated images

Applying image subtraction strategy together with time-gated detection, we can reveal finer details in a fixed cell sample using moderate light intensities. The subtractive imaging is obtained from the subtraction of a suitably weighted short lifetime intensity-image from a long lifetime one, while the weight coefficient can be selected using the analysis of an optical transfer function. Compared with other subtractive methods, ours is regarded as an optimized variation of gated stimulated emission depletion microscopy and can be implemented in a simpler manner, providing lateral super-resolution, and it is theoretically possible to further expand its application scope.

Generation of a 3D isotropic hollow focal spot for single-objective stimulated emission depletion microscopy

Single-objective stimulated emission depletion (STED) microscopy has long suffered from resolution anisotropy due to the elongated shape of the hollow STED spot along the optical axis. In this paper, we place a dielectric mirror behind the sample and use spatial light modulators to shape the illumination beam so that both the reflected beam and the incident beam converge to the same point and interfere with each other. By specially setting the reflection property of the dielectric mirror, we show that a 3D isotropic hollow focal spot can be generated while using only one objective lens. When this approach is introduced into STED microscopy, simulated results demonstrate that an axial resolution enhancement of 3.65-fold together with a lateral resolution improvement of 1.14-fold compared with conventional single-objective STED microscopy can be reached.

A method for generating a three-dimensional dark spot using a radially polarized beam

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 ? phase plate and the same radially polarized beam with a quadrant 0/? 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.