Estimation-free spatial-domain image reconstruction of structured illumination microscopy

Structured illumination microscopy(SIM)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditional Wiener-filtering-based reconstruction algorithm operates in the Fourier domain,it requires prior knowledge of the sinusoidal illumination patterns which makes the time-consuming procedure of parameter estimation to raw datasets necessary,besides,the parameter estimation is sensitive to noise or aberration-induced pattern distortion which leads to reconstruction artifacts.Here,we propose a spatial-domain image reconstruction method that does not require parameter estimation but calculates patterns from raw datasets,and a reconstructed image can be obtained just by calculating the spatial covariance of differential calculated patterns and differential filtered datasets(the notch filtering operation is performed to the raw datasets for attenuating and compensating the optical transfer function(OTF)).Experiments on reconstructing raw datasets including nonbiological,biological,and simulated samples demonstrate that our method has SR capability,high reconstruction speed,and high robustness to aberration and noise.

Multiplexed stimulated emission depletion nanoscopy(mSTED)for 5-color live-cell long-term imaging of organelle interactome

Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation.Here,we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity.By separating live-cell fluorescent probes with similar spectral properties using phasor analysis,our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures.The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.

Signal recovery method based on co-prime array

A signal waveform recovery method based on the co-prime array is investigated to extract the waveform of the desired signal from spatial interferences in narrowband scenarios. The direction of arrivals (DOAs) of the desired signal and interference signals are estimated with the compressive sensing approach based on angle grids, and the signal power together with the noise power are estimated. Thereafter, a modified steepest descent (SD) method is derived to recover the waveform of the desired signal and interferences utilizing the estimated power and directions. The recovered waveform of the desired signal is the output of the proposed method. The situation in which the signals are not on the predefined angle grids is also considered. The DOAs estimated via the predefined angle grids are corrected based on the maximum likelihood (ML) angle estimation. Compared to the existing beamforming methods on co-prime array, the proposed method can obtain the waveform of the desired signal. Simulation results demonstrate that the proposed method can achieve good performance in signal waveform recovery and output signal to noise ratio. © 2017 Beijing Institute of Aerospace Information.

Super-resolution microscopy based on parallel detection

Image scanning microscopy based on pixel reassignment can improve the confocal resolution limit without losing the image signal-to-noise ratio(SNR)greatly[C.J.R.Sheppard,"Super resolution in confocal imaging,"Optik 80(2)53-54(1988).C.B.Miller,E.Jorg,"Image scanning microscopy,"Phys.Reu.Lett.104(19)198101(2010).C.J.R.Sheppard,s.B.Mehta,R Heintzmann,"Superresolution by image scanning microscopy using pixel reassignment,"Opt.Lett.38(15)28892892(2013)].Here,we use a tailor-made optical fiber and 19 avalanche pho-todiodes(APDs)as parallel detectors to upgrade our existing confocal microscopy,termed as parallel-detection super resolution(PDSR)microscopy.In order to obtain the correct shift value,we use the normalized 2D cross correlation to calculate the shifting value of each image.We characterized our system performance by imaging fuorescence beads and applied this system to observing the 3D structure of biological specimen.

Phase-only pattern synthesis based on gradient-descent optimization

By applying phase-only technique in array antenna pattern synthesis, antenna arrays can form desired patterns with the use of phase shifters only. A novel phase-only pattern synthesis algorithm is proposed for the passive phased array seeker. This algorithm synthesizes the main beam of the antenna pattern through least-squares approximation, thus minimizing the errors between the actual and the desired main beams. The synthesis problem can be solved by applying gradient-descent optimization. The item for suppressing side lobes is added to the above synthesis problem. To obtain a side lobe level as low as possible, the algorithm assigns different weights to different directions in the side lobe region. The algorithm is run repeatedly and the weights are adjusted adaptively according to the normalized power in the side lobe directions. Detailed examples are presented to demonstrate the accuracy and effectiveness of the proposed approach.

Fluorescence interference structured illumination microscopy for 3D morphology imaging with high axial resolution

Imaging three-dimensional,subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy.However,trade-offs exist between axial resolution and other important technical indicators,such as temporal resolution,optical power density,and imaging process complexity.We report a new imaging modality,fluorescence interference structured illumination microscopy(FI-SIM),which is based on three-dimensional structured illumination microscopy for wide-field lateral imaging and fluorescence interference for axial reconstruction.FI-SIM can acquire images quickly within the order of hundreds of milliseconds and exhibit even 30 nm axial resolution in half the wavelength depth range without z-axis scanning.Moreover,the relatively low laser power density relaxes the requirements for dyes and enables a wide range of applications for observing fixed and live subcellular structures.

Far-field super-resolution chemical microscopy

Far-feld chemical microscopy providing molecular electronic or vibrational fingerprint information opens a new window for the study of three-dimensional biological,material,and chemical systems.Chemical microscopy provides a nondestructive way of chemical identification without exterior labels.However,the diffraction limit of optics hindered it from discovering more details under the resolution limit.Recent development of super-resolution techniques gives enlightenment to open this door behind far-field chemical microscopy.Here,we review recent advances that have pushed the boundary of far-field chemical microscopy in terms of spatial resolution.We further highlight applications in biomedical research,material characterization,environmental study,cultural heritage conservation,and integrated chip inspection.

Dual-modulation difference stimulated emission depletion microscopy to suppress the background signal

Stimulated emission depletion(STED)nanoscopy is one of the most well-developed nanoscopy techniques that can provide subdiffraction spatial resolution imaging.Here,we introduce dual-modulation difference STED microscopy(dmdSTED)to suppress the background noise in traditional STED imaging.By applying respective time-domain modulations to the two continuous-wave lasers,signals are distributed discretely in the frequency spectrum and thus are obtained through lock-in demodulation of the corresponding frequencies.The background signals can be selectively eliminated from the effective signal without compromise of temporal resolution.We used nanoparticle,fixed cell,and perovskite coating experiments,as well as theoretical demonstration,to confirm the effectiveness of this method.We highlight dmdSTED as an idea and approach with simple implementation for improving the imaging quality,which substantially enlarges the versatility of STED nanoscopy.

Simple and efficient delivery of cellimpermeable organic fluorescent probes into live cells for live-cell superresolution imaging

Numerous commercial organic fluorophores with excellent optical properties are precluded from live-cell superresolution imaging due to poor cell permeability.Here,we develop a simple but effective strategy that renders cells permeable to cell-impermeable,organic fluorescent probes by using a novel peptide vehicle,PV-1.By simple coincubation with PV-1,22 different cell-impermeable,organic fluorescent probes were efficiently delivered into live cells and specifically labeled a variety of organelles.Moreover,PV-1 can simultaneously transfer up to three different probes into live cells.By using PV-1 and these cell-impermeable fluorescent probes,we obtained multicolor,longterm,live-cell superresolution images of various organelles,which allowed us to study the dynamic interactions between them.PV-1,together with these organic fluorescent probes,will greatly broaden the applications of superresolution imaging technology in diverse live-cell studies and opens up a new avenue in the design and application of peptide vehicles.

“Xanthene” is a premium bridging group for xanthenoid dyes

Novel xanthenoid dyes by replacing the central oxygen atom of the xanthene dyes with less electronrich bridging groups have been intensively sought after primarily for their long spectral wavelengths.However, the new scaffolds are likely prone to nucleophilic attack at their central methane carbon, as the result of the reduced electron density of the fluorochromic scaffolds. We envisage that the bridging group may be harnessed to sterically shield the central methane carbon from incoming nucleophiles and render high stability and synthesized xantheno-xanthene dyes. Additionally, the xantheno-bridging group can be modified via electrophilic aromatic substitution to introduce functionalities, e.g., sulfonate groups.

Author Correction:Simple and efficient delivery of cell-impermeable organic fluorescent probes into live cells for live-cell superresolution imaging

Because dfTAT(Nat Methods,2014,11(8):861−867)and 3TAT(Traffic,2018,19(6):421−435)have been reported to be able to deliver SNAP-surface 488 into live cells,our claim that“PV-1 is the first vehicle that can transfer cellimpermeable organic fluorescent probes into live cells with satisfactory efficiency for imaging,requiring only the coincubation of the probes with PV-1”is inaccurate.To address this discrepancy,we have made the following corrections to the article on the recommendations of the editor.