Nonlinear Focal Modulation Microscopy

Wang

et al. 2019

Journal of Microscopy

Self Cite

Summary A superresolution fluorescence emission difference (FED) microscopy based on superoscillation excitation is investigated. The solid superoscillation excitation spot is produced by the radially polarised Laguerre–Gaussian beam (LG), and the donut superoscillation excitation spot is produced by the same LG beam with spiral phase modulation. We show that the superoscillation excitation can enhance the spatial resolution of FED microscopy. When the ratio of the pupil radius and the second moment radius of the LG3,1 beam is 0.85 and the subtractive factor is 0.3, the resolution can be enhanced about 2 times than the general confocal microscopy. Compared with the general FED microscopy, the resolution can be enhanced about 1.1 times. Our simulations also demonstrate that two‐view Richardson–Lucy (RL) deconvolution method can reduce the effect of side lobes, which is related to subtractive factor and pinhole. Lay Description The fluorescence emission difference (FED) microscopy is a high‐resolution light microscopy, which is based on the subtraction of images produced by two different confocal imaging modes. Here, the solid excitation spot is used in one imaging mode, and the donut excitation spot is in another imaging mode. There are various ways that the solid and donut excitation spots can be structured, but superoscillation excitation spots are still not used in the FED microscopy. Considering that the main lobe of superoscillation can be arbitrarily small in principle, we exploit the radially polarised Laguerre–Gaussian beam (LG) and spiral phase plate to produce superoscillation solid and donut spots. We present a detailed analysis about the performance of FED microscopy via superoscillation excitation. Our analysis is based on vector diffraction theory and supported by simulations of imaging. We find the proposed FED microscopy has better resolution than the confocal microscopy or the general FED microscopy excited by Gaussian beam. Especially, two‐view Richardson–Lucy (RL) deconvolution method can reduce the negative impact of strong side lobes, which is related to the subtractive factor and the size of pinhole located at the detector.

“…We used two‐view RL deconvolution in FED microscopy shown in Zhao et al . (). In this simulation, subtractive factor C is set to 0.9, and the diameter of pinhole D is set to 1.5 AU.…”

Section: Simulations and Discussionmentioning

confidence: 97%

Fluorescence emission difference microscopy by superoscillation excitation

Wang

et al. 2019

Journal of Microscopy

Self Cite

“…It maximizes the capabilities of Fourier domain OTF fusion of multiple emission PSFs in the spectral regime to improve the entire imaging quality, which can recover the otherwise hidden spatial information during the single beam scanning and confocal detection process. Compared with the temporal domain modulation of excitation modality that requires switching illumination pattern [ 43 ] or laser mode [ 44 ] with dual excitations procedure, the single scan method is simple, fast, and stable, and can avoid the use of the additional optical components and procedures in correcting the sample drifts between multiple sequential recordings. The single‐beam scanning mode using a simplified optics setup is compatible with the standard commercial or lab‐based laser scanning microscopes, and therefore may overcome the current bottleneck issue associated with the system complexity and stability.…”

Section: Discussionmentioning

confidence: 99%

“…Recent studies have indicated that UCNPs with unique nonlinear optical response and saturation properties can be developed as novel fluorescent probes, [ 32–37 ] especially for the sub‐diffraction imaging technologies. [ 38–41 ] Most recently, the dual NIR (980 nm excitation and 800 nm emission) working wavelength in UCNPs shows its utility in deep tissue super‐resolution imaging, [ 18,42 ] but the doughnut‐shaped PSF induces artifacts during the deconvolution process, as a certain band of frequency has been lost in the Fourier domain, [ 43,44 ] affecting the imaging quality. Ideally, simultaneously obtaining diverse PSFs using a simple setup, maximizing spatial information in the Fourier domain, would enhance the spatial resolution and overall imaging quality.…”

Section: Introductionmentioning

confidence: 99%

Heterochromatic Nonlinear Optical Responses in Upconversion Nanoparticles for Super‐Resolution Nanoscopy

Chen

et al. 2021

Advanced Materials

Point spread function (PSF) engineering by an emitter's response can code higher‐spatial‐frequency information of an image for microscopy to achieve super‐resolution. However, complexed excitation optics or repetitive scans are needed, which explains the issues of low speed, poor stability, and operational complexity associated with the current laser scanning microscopy approaches. Here, the diverse emission responses of upconversion nanoparticles (UCNPs) are reported for super‐resolution nanoscopy to improve the imaging quality and speed. The method only needs a doughnut‐shaped scanning excitation beam at an appropriate power density. By collecting the four‐photon emission of single UCNPs, the high‐frequency information of a super‐resolution image can be resolved through the doughnut‐emission PSF. Meanwhile, the two‐photon state of the same nanoparticle is oversaturated, so that the complementary lower‐frequency information of the super‐resolution image can be simultaneously collected by the Gaussian‐like emission PSF. This leads to a method of Fourier‐domain heterochromatic fusion, which allows the extended capability of the engineered PSFs to cover both low‐ and high‐frequency information to yield optimized image quality. This approach achieves a spatial resolution of 40 nm, 1/24th of the excitation wavelength. This work suggests a new scope for developing nonlinear multi‐color emitting probes in super‐resolution nanoscopy.

“…Sample handling approach, like expansion microscopy 6 , special inorganic nanoparticles labelling [7][8][9] , can be used to empower a conventional microscope with super-resolution imaging. Moreover, based on the optical geometry of confocal microscopy, the achieved developments such as image scanning microscopy 10,11 , focal modulation microscopy 12 , confocal rescan microscopy 13 has obtained remarkable resolution enhancement with the assistance of the special optical geometry and array detector. Nonetheless, the sample handling approach and the instrument modification approach still rely on complex sample processing process or special optical components and/or special inorganic nanoparticles with low labeling density.…”

Section: Mainmentioning

confidence: 99%

Non-linear scanning switch-off microscopy for super-resolution fluorescence imaging

Gao

Hou

Deng

et al. 2022

Preprint

Super-resolution (SR) microscopy provides a revolutionary approach to study cells and animals by breaking the diffraction limit of optical imaging. However, the popularity of the super-resolution microscope in biological sciences remains to be impeded by the high cost of hardware and/or the complexity of software. Here, we present a conceptually different non-linear scanning switch-off microscopy (nSSM) that exploits the omnipresent switch-off effect of fluorophores to enable super-resolution imaging beyond the diffraction limit. We develop a theoretical model of nSSM and experimentally implement the nSSM scheme with an unmodified confocal microscope. We also release a free code for the automatic reconstruction of super-resolution images. By measuring the PSF of the imaged DNA origami nanostructure and mammalian cytoskeleton structures, we demonstrate an SR resolution of ~ 100 nm that excels the optical resolution limit by over two folds. We further show the generality of nSSM using a range of commercially available fluorescent dyes and proteins to realize SR imaging in various settings. This nSSM methodology may in principle empower any confocal microscope to implement SR imaging to promote biological research.