Cuifang Kuang

Effects of polarization on the de-excitation dark focal spot in STED microscopy

The size of the dark focal spot directly determines the resolution and stability of stimulated emission depletion (STED) microscopy. This paper investigates the relationship between the size of the dark focal spot and the polarization of the input light beam. The types of fundamental polarization are discussed, their effects on the dark focal spot are compared and the optimized mode for each kind of polarization is proposed. The results of the analysis provide the theoretical basis and reference for designing a STED system.

Phase encoding for sharper focus of the azimuthally polarized beam

We demonstrate that a sharper focal spot area can be generated (0.147λ(2)) by using an azimuthally polarized beam propagating through a vortex 0-2π phase plate than for radial polarization (0.17λ(2)) or for linear polarization (0.26λ(2)) under the same condition. Further research illustrates that such optimistic results can still be expected when condition limitations are liberalized. This will facilitate new approaches to get superresolution in confocal systems.

Microsphere based microscope with optical super-resolution capability

We experimentally demonstrated that the microsphere can discern the details of the object whose sizes are below the conventional diffractive limit and such super-resolution capability can be reinforced if semi-immersing the corresponding microspheres in liquid droplet, producing a sharper contrast and a comparatively smaller magnification factor. The microsphere is considered as a channel that connects the near-field evanescent wave and the transmission one in far field. A conjecture based on this is proposed to explain the mechanism of super-resolution and the corresponding phenomenon.

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.

Far-field super-resolution imaging using near-field illumination by micro-fiber

We propose an approach that uses a conventional optical microscope to achieve optical super-resolution. This approach will provide a direct, non-invasive, and far-field access in the observation of both metallic and non-metallic objects with a spatial resolution of tens-of-nanometres, achieved in a single snap shot. This method, combines near-field illumination by the micro-fiber and a passive spatial frequency shift by the sample itself, offering a promising approach to surface tomography imaging without localized field enhancement by surface plasmon polaritons. Our results present the potential of breaking the diffractive barrier using a conventional wide-field optical microscope within the visible spectrum.

Pushing phase and amplitude sensitivity limits in interferometric microscopy.

Sensitivity of the amplitude and phase measurements in interferometric microscopy is influenced by factors such as instrument design and environmental interferences. Through development of a theoretical framework followed by experimental validation, we show photon shot noise is often the limiting factor in interferometric microscopy measurements. Thereafter, we demonstrate how a state-of-the-art camera with million-level electrons full well capacity can significantly reduce shot noise contribution resulting in a stability of optical path length down to a few picometers even in a near-common-path interferometer.

Eliminating deformations in fluorescence emission difference microscopy.

We propose a method for eliminating the deformations in fluorescence emission difference microscopy (FED). Due to excessive subtraction, negative values are inevitable in the original FED method, giving rise to deformations. We propose modulating the beam to generate an extended solid focal spot and a hollow focal spot. Negative image values can be avoided by using these two types of excitation spots in FED imaging. Hence, deformations are eliminated, and the signal-to-noise ratio is improved. In deformation-free imaging, the resolution is higher than that of confocal imaging by 32%. Compared to standard FED imaging with the same level of deformations, our method provides superior resolution.

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.

Digital micromirror device-based laser-illumination Fourier ptychographic microscopy.

We report a novel approach to Fourier ptychographic microscopy (FPM) by using a digital micromirror device (DMD) and a coherent laser source (532 nm) for generating spatially modulated sample illumination. Previously demonstrated FPM systems are all based on partially-coherent illumination, which offers limited throughput due to insufficient brightness. Our FPM employs a high power coherent laser source to enable shot-noise limited high-speed imaging. For the first time, a digital micromirror device (DMD), imaged onto the back focal plane of the illumination objective, is used to generate spatially modulated sample illumination field for ptychography. By coding the on/off states of the micromirrors, the illumination plane wave angle can be varied at speeds more than 4 kHz. A set of intensity images, resulting from different oblique illuminations, are used to numerically reconstruct one high-resolution image without obvious laser speckle. Experiments were conducted using a USAF resolution target and a fiber sample, demonstrating high-resolution imaging capability of our system. We envision that our approach, if combined with a coded-aperture compressive-sensing algorithm, will further improve the imaging speed in DMD-based FPM systems.