Guangyuan Zhao

Resolution enhancement of saturated fluorescence emission difference microscopy

Fluorescence emission difference microscopy (FED) obtains resolution-enhanced images by subtracting acquired solid and doughnut confocal images. Because of the mismatch of the outer contours of the two subtraction components, negative values are inevitable in the conventional FED method, giving rise to deformations. In this study, by using a saturation effect, we obtain imaging results with a profile-extended solid and center-shrunken doughnut point spread function. Owing to the nonlinear effect, two better-matched saturated images not only eliminate the deformations, but also enhance the resolving ability and signal to noise ratio compared to conventional FED. Simulations based on the saturated model of rhodamine 6G, as well as experiments on biological samples, are presented to verify the capability of the proposed concept, while experimental results show the unprecedented resolving ability of the saturated FED method.

3D fluorescence emission difference microscopy based on spatial light modulator

We report three-dimensional fluorescence emission difference (3D-FED) microscopy using a spatial light modulator (SLM). Zero phase, 0–2π vortex phase and binary 0-pi phase are loaded on the SLM to generate the corresponding solid, doughnut and z-axis hollow excitation spot, respectively. Our technique achieves super-resolved image by subtracting three differently acquired images with proper subtractive factors. Detailed theoretical analysis and simulation tests are proceeded to testify the performance of 3D-FED. Also, the improvement of lateral and axial resolution is demonstrated by imaging 100nm fluorescent beads. The experiment yields lateral resolution of 140nm and axial resolution of approximate 380nm.

Saturated absorption competition microscopy

Owing to the advantage of being non-invasive in observing living samples, far-field optical microscopy is widely used in the life sciences, but the existence of the diffraction barrier leads to the poor imaging of samples with spatial features smaller than approximately half the wavelength of the probes. This limit has been overcome by a number of pointwise scanning optical imaging techniques, such as stimulated emission depletion microscopy (STED) and saturated excitation microscopy (SAX). Here, we introduce the concept of saturated absorption competition (SAC) microscopy as a simple means of providing sub-diffraction spatial resolution in fluorescence imaging. Our approach can be physically implemented in a confocal microscope by dividing the input laser source into a time-modulated primary excitation beam and a doughnut-shaped saturation beam and subsequently employing a homodyne detection scheme to select the modulated fluorescence signal. Herein, we provide both a physico-chemical model of SAC and experimentally demonstrate a transverse spatial resolution of 1.5- to 2-fold that of confocal.

Resolution-enhanced SOFI via structured illumination

By analyzing the statistics of the temporal fluctuations from the blinking emitters, super-resolution fluctuation imaging (SOFI) achieves super-resolution while imposing fewer constraints on the blinking behavior of the probes and is more suitable for low signal-to-noise ratio acquisition than localization methods. However, determined by the square root of cumulation orders, the resolution improvement of SOFI highly restricts its promotion into high-resolution observations. In this Letter, abandoning the default flat illumination in stochastic imaging methods, we introduce structured illumination (SI) (e.g., Gaussian or sinusoidal pattern) into SOFI (SI-SOFI) to render greatly enhanced resolution. Through simulation with parameters of both real acquisition procedures and microscope properties, we examine the feasibility of SI-SOFI and obtain a resolution improvement of four-six folds at just second-order cumulation compared to wide-field imaging. In addition, a practical pathway for the SI-SOFI reconstruction is offered.

3D point scanning super-resolution microscopy via polarization modulation.

We report a new approach to achieving super-resolution in point-scanning microscopy through polarization modulation for the first time, to the best of our knowledge. By modulating linearly polarized incident light, the emission extent of fluorescent dyes changes periodically, adding sparsity in each recording, which contributes to the super-resolution reconstruction. To recover the super-resolution result, a sparse penalty-based deconvolution method is implemented onto the polarization-modulated dataset subsequently. By simply inserting a vortex half-wave retarder into a typical confocal microscope, we obtain the super-resolution experimental results of both nuclear pore complex proteins and tubulins in vero cells, which evidence a sub-diffraction resolution of λ/5. In addition, three-dimensional (3D) super-resolution on spatial distributed single molecules is simulated, where the significant resolution improvement in both lateral and axial directions further confirms its capacity in 3D imaging applications. Considering no constraint on fluorescence dyes and easy implementation in a point-scanning microscope, we envision that the polarization-modulated confocal microscope would be a helpful alternative in biological imaging.

A comprehensive description of diffraction phase microscopy

Diffraction Phase Microscopy (DPM) is a widely used quantitative phase imaging method, whose common-path nature endows it with low noise and high sensitivity. Current applications of DPM include biological topography as well as biological dynamics for its nondestructive feature. Many different forms of DPM based on the original idea have appeared according to the different demands. In this paper, both the principle and the DPM classification will be given a comprehensive description. Furthermore, the future trend of DPM development is also discussed.