Label-free Super-resolution Microscopy Research Articles

Label-free Super-resolution Microscopy for Long-term Monitoring the Dynamic Interactions of Cellular Organelles

Label-free Super-resolution Microscopy for Long-term Monitoring the Dynamic Interactions of Cellular Organelles

Polarization-insensitive high-numerical-aperture metalens for wide-field super-resolution imaging.

The development of super-oscillatory lens (SOL) offers opportunities to realize far-field label-free super-resolution microscopy. Most microscopes based on a high numerical aperture (NA) SOL operate in the point-by-point scanning mode, resulting in a slow imaging speed. Here, we propose a high-NA metalens operating in the single-shot wide-field mode to achieve real-time super-resolution imaging. An optimization model based on the exhaustion algorithm and angular spectrum (AS) theory is developed for metalens design. We numerically demonstrate that the optimized metalens with an NA of 0.8 realizes the imaging resolution (imaging pixel size) about 0.85 times the Rayleigh criterion. The metalens can achieve super-resolution imaging of an object with over 200 pixels, which is one order of magnitude higher than the unoptimized metalens. Our method provides an avenue toward single-shot far-field label-free super-resolution imaging for applications such as real-time imaging of living cells and temporally moving particles.

Label-free super-resolution microscopy based on non-diffraction superoscillation beam (NDSB) illumination

Due to the diffraction properties of light waves, optical microscopy suffers from a fundamentally limited resolution of ∼λ/2, where λ relates to the illumination wavelength. Though near-field methods capturing the evanescent waves can reach a resolution of several nanometers, the extremely short working distance has restrained their applications. Typical far-field super-resolution microscopies using fluorescence to monitor the labelled samples also have their limitations in applications because of their phototoxicity and complex preprocessing. We hereby proposed a label-free super-resolution microscopy based on non-diffraction superoscillation beam (NDSB) illumination. We developed a transmission-mode confocal optical microscopy and experimentally demonstrated an imaging resolution of 0.285 λ at the illumination wavelength of 632.8 nm. As NDSB owns the capabilities of self-healing and deep penetration in dense media, we could clearly visualize the fine complex structures while NDSB illuminated from the backside of a 175-µm-thick glass substrate. As a result of NDSB's excellent penetration and superoscillatory transverse size, the 3D super-resolution capability of the proposed microscopy was experimentally verified. Owing to the advantages of label-free imaging, far field, super resolution, self-healing behaviour and deep penetration, we believe the proposed NDSB microscopy bears the potential to become a promising tool for 3D super-resolution visualization in both biological and nonbiological samples.

Water-immersion supercritical metalens

Far-field super-resolution lenses are attractive due to their great potential in the application of label-free far-field optical super-resolution. In past decades, there has been a fast development in superoscillatory optical lenses, which allows engineering of optical point-spread functions in the far-field beyond the traditional Abbe diffraction limit. With the decrease of focal spot size, the sidebands will inevitably increase, especially when the spot size is close to 0.358λ/NA, where NA is the numerical aperture. However, it is of critical importance to reduce the spot size while maintaining comparatively small sidelobes for many practical applications. Here, we demonstrate a water-immersion supercritical metalens with a high-numerical aperture of 1.15. Experimental results show the generation of a super-resolution hot spot as small as 0.33λ, which is very close to the superoscillation criterion, at a distance of 60λ from the lens. More importantly, there is no significant sideband on the focal plane, and the strength of the sidelobes near the hot spot is lower than 13.3 % of the central lobe intensity. The deep compression of hot spot and the absence of large sidelobes offer a promising way to further improve the resolution of far-field label-free super-resolution microscopy.

Resolving plasmonic hotspots by label-free super-resolution microscopy.

The plasmonic hotspot of metal nanostructures has small dimension far beyond the optical diffraction limit. When trying to locate the hotspot using fluorescent probes, the localization is significantly distorted due to the coupling of emission and surface plasmon. A label-free technique can solve the problem, which uses hotspot emission as the native probe. We demonstrate a super-resolution microscopy investigation based on this idea. By modulating hotspot emission of crossed silver nanowires, which have a pair of plasmonic hotspots approximately 100 nm apart at the intersection, we precisely locate and separate them with nanometer precision. This label-free technique could be applied for analyzing hotspot distribution with high efficiency and precision.

Super-oscillatory metasurface doublet for sub-diffraction focusing with a large incident angle.

Based on the delicate interference behavior of light in the far field, the optical super-oscillatory phenomenon has been successfully applied in non-invasive sub-diffraction focusing and super-resolution imaging in recent years. However, the optical super-oscillatory field is particularly sensitive to the change of incident angle, leading to a limited field of view for super-resolution imaging. In this paper, a super-oscillatory metasurface doublet is proposed to achieve far-field sub-diffraction focusing with an incident angle of up to 25°. The constructed doublet, consisting of high-aspect-ratio rectangular nanopillars with high efficiency, is further demonstrated through a full-wave simulation, and the numerical results indicate that the sub-diffraction foci with about 0.75 times of the diffraction limit is achieved for different incident angles. The proposed super-oscillatory metasurface doublet may find intriguing applications in label-free super-resolution microscopy and optical precise fabrication.

Structural Changes and Dynamics of Disassembling Microtubules Revealed by Label-Free Super-Resolution Microscopy

Microtubules are a key part of the eukaryotic cytoskeleton typically composed of linear chains of head-to-tail connected tubulin dimers denoted as protofilaments that are connected laterally to form a hollow cylindrical filament. Due to highly dynamic ends, microtubules undergo alternating periods of assembly and disassembly, also known as dynamic instability, which is essential for a number of microtubule functions. Electron micrographs provide still images of the disassembling microtubule suggesting a structural transition at the microtubule tip characterized by disintegration of the lateral bonds of the tubulin lattice before the longitudinal depolymerization.

Label-Free Super-Resolution Microscopy By Means of Transient Absorption Saturation

In recent decades, super-resolution optical microscopy has become an attractive research topic that typically has been developed based on fluorescent labelling that becomes the essential tool for breaking diffraction-limited in biological imaging. However, deep tissue and label-free imaging remain challenging, especially for tick and highly scattering biological specimens [1]. With the development of ultrashort pulse laser sources, non-linear optical (NLO) light-matter interaction has acquired a central role in optical microscopy for label-free imaging due to efficient signal generation in the non-linear process [2]. The advantages of NLO approaches include the reduction of scattering due to the use of near-infrared radiation, which enables high penetration depth and reduces the aberrations introduced by the specimen [1]. Non-linear interaction comprises the generation of sum frequencies, high-harmonic generation (SHG/ THG), Raman scattering, two-photon fluorescence and others [3]. Transient absorption, also known as pump-probe microscopy is a non-linear optical imaging technique that can probe the dynamics of the excited states. [1]. In this work, we perform super-resolution label-free imaging using our custom-built near-infrared pump-probe microscope. Two femtosecond pulsed laser beams, generated by an OPO pumped by a Ti:sapphire laser (Chameleon Ultra II and compact OPO Coherent), are coupled into a commercial laser scanning confocal microscope (Nikon A1 MP). We explore the saturation of transient absorption using a doughnut-shaped beam in the STED-like approach to saturate the absorption [3] and forward SHG and SFG processes to achieve label-free imaging at an improved spatial resolution. [1] Astratov V. (2019). “Label-Free Super-Resolution Microscopy”. Springer Nature.[2] Sheppard. C.J.R. (2020). Multiphoton Microscopy: a personal historical review with some future prediction. Biomed. Opt.[3]Zanini, G. et al. 2019. Label-Free Optical Nanoscopy of Single-Layer Graphene. ACS Nano.

Superoscillation: from physics to optical applications

The resolution of conventional optical elements and systems has long been perceived to satisfy the classic Rayleigh criterion. Paramount efforts have been made to develop different types of superresolution techniques to achieve optical resolution down to several nanometres, such as by using evanescent waves, fluorescence labelling, and postprocessing. Superresolution imaging techniques, which are noncontact, far field and label free, are highly desirable but challenging to implement. The concept of superoscillation offers an alternative route to optical superresolution and enables the engineering of focal spots and point-spread functions of arbitrarily small size without theoretical limitations. This paper reviews recent developments in optical superoscillation technologies, design approaches, methods of characterizing superoscillatory optical fields, and applications in noncontact, far-field and label-free superresolution microscopy. This work may promote the wider adoption and application of optical superresolution across different wave types and application domains.

Label-free super-resolution with coherent nonlinear structured-illumination microscopy

Structured-illumination microscopy enables up to a two-fold lateral resolution improvement by spatially modulating the intensity profile of the illumination beam. We propose a novel way to generalize the concept of structured illumination to nonlinear widefield modalities by spatially modulating, instead of field intensities, the phase of the incident field while interferometrically measuring the complex-valued scattered field. We numerically demonstrate that for second-order and third-order processes an almost four- and six-fold increase in lateral resolution is achievable, respectively. This procedure overcomes the conventional Abbe diffraction limit and provides new possibilities for label-free super-resolution microscopy.