Super-resolution optical microscopy, sometimes called optical nanoscopy, refers to a new kind of far-field optical microscopy which allows optical imaging with a resolution higher than the well-known resolution limit (or the Abbe diffraction limit) which stood for more than a century. The reported approaches that break the resolution limit involve either effectively reducing the focus region of the excitation light or stochastic separation and precise localization of single fluorophores. Super-resolution optical microscopy provides unprecedented opportunities for tackling outstanding fundamental questions in life science, medicine, materials, and many others that require non-invasive imaging with molecular level spatial resolution.

The image reconstruction process in super-resolution structured illumination microscopy (SIM) is investigated. The structured pattern is generated by the interference of two Gaussian beams to encode undetectable spectra into detectable region of microscope. After parameters estimation of the structured pattern, the encoded spectra are computationally decoded and recombined in Fourier domain to equivalently increase the cut-off frequency of microscope, resulting in the extension of detectable spectra and a reconstructed image with about two-fold enhanced resolution. Three different methods to estimate the initial phase of structured pattern are compared, verifying the auto-correlation algorithm affords the fast, most precise and robust measurement. The artifacts sources and detailed reconstruction flowchart for both linear and nonlinear SIM are also presented.

Carbohydrates on cell surfaces play a crucial role in a wide variety of biological processes, including cell adhesion, recognition and signaling, viral and bacterial infection, inflammation and metastasis. However, owing to the large diversity and complexity of carbohydrate structure and nongenetically synthesis, glycoscience is the least understood field compared with genomics and proteomics. Although the structures and functions of carbohydrates have been investigated by various conventional analysis methods, the distribution and role of carbohydrates in cell membranes remain elusive. This review focuses on the developments and challenges of super-resolution imaging in glycoscience through introduction of imaging principle and the available fluorescent probes for super-resolution imaging, the labeling strategies of carbohydrates, and the recent applications of super-resolution imaging in glycoscience, which will promote the super-resolution imaging technology as a promising tool to provide new insights into the study of glycoscience.

Low-light camera is an indispensable component in various fluorescence microscopy techniques. However, choosing an appropriate low-light camera for a specific technique (for example, single molecule imaging) is always time-consuming and sometimes confusing, especially after the commercialization of a new type of camera called sCMOS camera, which is now receiving heavy demands and high praise from both academic and industrial users. In this tutorial, we try to provide a guide on how to fully access the performance of low-light cameras using a well-developed method called photon transfer curve (PTC). We first present a brief explanation on the key parameters for characterizing low-light cameras, then explain the experimental procedures on how to measure PTC. We also show the application of the PTC method in experimentally quantifying the performance of two representative low-light cameras. Finally, we extend the PTC method to provide offset map, read noise map, and gain map of individual pixels inside a camera.

In the past two decades, various super-resolution (SR) microscopy techniques have been developed to break the diffraction limit using subdiffraction excitation to spatially modulate the fluorescence emission. Photomodulatable fluorescent proteins (FPs) can be activated by light of specific wavelengths to produce either stochastic or patterned subdiffraction excitation, resulting in improved optical resolution. In this review, we focus on the recently developed photomodulatable FPs or commonly used SR microscopies and discuss the concepts and strategies for optimizing and selecting the biochemical and photophysical properties of PMFPs to improve the spatiotemporal resolution of SR techniques, especially time-lapse live-cell SR techniques.

Optical microscopy allows us to observe the biological structures and processes within living cells. However, the spatial resolution of the optical microscopy is limited to about half of the wavelength by the light diffraction. Structured illumination microscopy (SIM), a type of new emerging super-resolution microscopy, doubles the spatial resolution by illuminating the specimen with a patterned light, and the sample and light source requirements of SIM are not as strict as the other super-resolution microscopy. In addition, SIM is easier to combine with the other imaging techniques to improve their imaging resolution, leading to the developments of diverse types of SIM. SIM has great potential to meet the various requirements of living cells imaging. Here, we review the recent developments of SIM and its combination with other imaging techniques.

Far-field fluorescence microscopy has made great progress in the spatial resolution, limited by light diffraction, since the super-resolution imaging technology appeared. And stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM) can be grouped into one class of the super-resolution imaging technology, which use pattern illumination strategy to circumvent the diffraction limit. We simulated the images of the beads of SIM imaging, the intensity distribution of STED excitation light and depletion light in order to observe effects of the polarized light on imaging quality. Compared to fixed linear polarization, circularly polarized light is more suitable for SIM on reconstructed image. And right-handed circular polarization (CP) light is more appropriate for both the excitation and depletion light in STED system. Therefore the right-handed CP light would be the best candidate when the SIM and STED are combined into one microscope. Good understanding of the polarization will provide a reference for the patterned illumination experiment to achieve better resolution and better image quality.

We generated a super-resolution optical tube by tightly focusing a binary phase modulated azimuthally polarized laser beam. The binary phase modulation is achieved by a glass substrate with multi-belt concentric ring grooves. We also characterized the 3D beam profile by using a crossshaped knife-edge fabricated on a silicon photo-detector. The size of the super-resolution dark spot in the tube is 0.32λ, which remains unchanged for ~ 4λ within the tube. This optical tube may find applications in super-resolution microscopy, optical trapping and particle acceleration.

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 100 nm fluorescent beads. The experiment yields lateral resolution of 140 nm and axial resolution of approximate 380 nm.