Polarization-resolved super-resolution second-harmonic generation imaging based on multifocal structured illumination microscopy.

Polarization-resolved second-harmonic generation (PSHG) microscopy is widely used in investigating the structural and morphological alterations of collagen. However, the resolution of second-harmonic generation (SHG) imaging remains constrained by optical diffraction, resulting in the polarization extraction of collagen characteristics from the average properties of collagen fibers. In this study, multifocal structured illumination microscopy (MSIM) was combined with PSHG to achieve polarization-resolved super-resolution imaging of second-harmonic generation signals. For the first time to our knowledge, periodic structures with an average pitch of 277 nm were observed in mouse tail tendons using optical microscopy, and the orientation angle of fibrils within each period was found to exhibit an alternating arrangement along the axis in a regular pattern.

A reversible photoelectrochemical microsensor for dynamically monitoring sulfur dioxide in the epileptic brain.

Epilepsy is considered one of the most prevalent neurological disorders, yet the precise mechanisms underlying its pathogenesis remain inadequately elucidated. Emerging evidence implicates endogenous sulfur dioxide (SO2) in the brain as playing a significant role in epilepsy and associated neuronal apoptosis. Consequently, tracking the dynamic fluctuations in the levels of SO2 and its derivatives (SO32-/HSO3-) provides valuable insights into the molecular mechanisms underlying epilepsy, with potential implications for its diagnosis and therapeutic intervention. Nonetheless, the absence of reversible in vivo detection tools constitutes a formidable obstacle in the real-time monitoring of SO2 dynamics in the brain. In response to this challenge, we propose a novel approach involving a photoelectrochemical (PEC) microsensor capable of reversibly detecting SO2. This microsensor leverages a reversibly recognizing dye for SO2 and upconversion nanoparticles as the modulator of the excitation source for the photoactive material, enabling modulation of the photocurrent by the target. The reversible output of PEC signals allows for the monitoring of SO2 levels in real time in the brains of epileptic mice. This study reveals the patterns of SO2 level changes during epilepsy and provides insights into the neuroprotective mechanism of exogenous SO2.

Label-Free In Vivo Monitoring of Lipid Droplets for Real-Time Assessment of Adipose Activator-Induced Tumor Suppression.

To enhance our comprehension of the fundamental mechanisms driving tumor metabolism and metastasis, it is essential to dynamically monitor intratumoral lipid droplet (LD) and collagen processes in vivo. Traditional LD analysis in tumors predominantly relies on observations of in vitro cells or tissue slices, which unfortunately hinder real-time insights into the dynamic behavior of LDs during in vivo tumor progression. In this study, we developed a dual-modality imaging technique that combines coherent anti-Stokes Raman scattering (CARS) and second-harmonic generation (SHG) microscopy for in vivo monitoring of tumor LDs and collagen alterations, assisted by a murine breast cancer 4T1 cell-based dorsal skinfold window. Specifically, we accomplished real-time observations and quantitative analysis of the LD size, density, and collagen alignment within living tumors through CARS/SHG imaging. Additionally, our findings demonstrate that real-time LD monitoring provides a valuable means of assessing the efficacy of anticancer drugs in vivo. We evaluated the impact of adipose activators on lipid metabolism, oxidative stress, and tumor suppression by monitoring changes in LD size and density. Overall, this study highlights the potential of dual-modality CARS/SHG microscopy as a sensitive and flexible tool for antitumor therapeutic strategies.

In Vivo High-Contrast Biomedical Imaging in the Second Near-Infrared Window Using Ultrabright Rare-Earth Nanoparticles.

Intravital luminescence imaging in the second near-infrared window (NIR-II) enables noninvasive deep-tissue imaging with high spatiotemporal resolution of live mammals because of the properties of suppressed light scattering and diminished autofluorescence in the long-wavelength region. Herein, we present the synthesis of a downconversion luminescence rare-earth nanocrystal with a core-shell-shell structure (NaYF4@NaYbF4:Er,Ce@NaYF4:Ca). The structure efficiently maximized the doping concentration of the sensitizers and increased Er3+ luminescence while preventing cross relaxation. Furthermore, Ce3+ doping in the middle layer efficiently limited the upconversion pathway and increased downconversion by 24-fold to produce bright 1550 nm luminescence under 975 nm excitation. Finally, optimizing the inert shell coating of NaYF4:Ca and liposome encapsulation reduced the luminescence quenching impact by water and improved biological metabolism. Thus, our synthesized biocompatible, ultrabright NIR-II probes provide high contrast and resolution for through-scalp and through-skull luminescence imaging of mice cerebral vasculature without craniotomy as well as imaging of mouse hindlimb microvessels.

Combination of deep learning and 2D CARS figures for identification of amyloid-ß plaques.

In vivo imaging and accurate identification of amyloid-ß (Aß) plaque are crucial in Alzheimer's disease (AD) research. In this work, we propose to combine the coherent anti-Stokes Raman scattering (CARS) microscopy, a powerful detection technology for providing Raman spectra and label-free imaging, with deep learning to distinguish Aß from non-Aß regions in AD mice brains in vivo. The 1D CARS spectra is firstly converted to 2D CARS figures by using two different methods: spectral recurrence plot (SRP) and spectral Gramian angular field (SGAF). This can provide more learnable information to the network, improving the classification precision. We then devise a cross-stage attention network (CSAN) that automatically learns the features of Aß plaques and non-Aß regions by taking advantage of the computational advances in deep learning. Our algorithm yields higher accuracy, precision, sensitivity and specificity than the results of conventional multivariate statistical analysis method and 1D CARS spectra combined with deep learning, demonstrating its competence in identifying Aß plaques. Last but not least, the CSAN framework requires no prior information on the imaging modality and may be applicable to other spectroscopy analytical fields.

Super-Resolution Second-Harmonic Generation Imaging with Multifocal Structured Illumination Microscopy.

Second-harmonic generation (SHG) is a noninvasive imaging technique that enables the exploration of physiological structures without the use of an exogenous label. However, traditional SHG imaging is limited by optical diffraction, which restricts the spatial resolution. To break this limitation, we developed a novel approach called multifocal structured illumination microscopy-SHG (MSIM-SHG). By combination of SHG with MSIM, SHG-based super-resolution imaging of material molecules can be achieved, and this SHG super-resolution imaging has a wide range of applications for biological tissues and cells. MSIM-SHG achieved a lateral full width at half-maximum (fwhm) of 147 ± 13 nm and an axial fwhm of 493 ± 47 nm by imaging zinc oxide (ZnO) particles. Furthermore, MSIM-SHG was utilized to quantify collagen fiber alignment in various tissues such as the ovary, muscle, heart, kidney, and cartilage, demonstrating its feasibility for identifying collagen characteristics. MSIM-SHG has potential as a powerful tool for clinical diagnosis and biological research.

Deep-MSIM: Fast Image Reconstruction with Deep Learning in Multifocal Structured Illumination Microscopy.

Fast and precise reconstruction algorithm is desired for for multifocal structured illumination microscopy (MSIM) to obtain the super-resolution image. This work proposes a deep convolutional neural network (CNN) to learn a direct mapping from raw MSIM images to super-resolution image, which takes advantage of the computational advances of deep learning to accelerate the reconstruction. The method is validated on diverse biological structures and in vivo imaging of zebrafish at a depth of 100 µm. The results show that high-quality, super-resolution images can be reconstructed in one-third of the runtime consumed by conventional MSIM method, without compromising spatial resolution. Last but not least, a fourfold reduction in the number of raw images required for reconstruction is achieved by using the same network architecture, yet with different training data.

Fluorescence lifetime tracking and imaging of single moving particles assisted by a low-photon-count analysis algorithm.

Fluorescence lifetime imaging microscopy (FLIM) has been widely used in the field of biological research because of its high specificity, sensitivity, and quantitative ability in the sensing cellular microenvironment. The most commonly used FLIM technology is based on time-correlated single photon counting (TCSPC). Although the TCSPC method has the highest temporal resolution, the data acquisition time is usually long, and the imaging speed is slow. In this work, we proposed a fast FLIM technology for fluorescence lifetime tracking and imaging of single moving particles, named single particle tracking FLIM (SPT-FLIM). We used feedback-controlled addressing scanning and Mosaic FLIM mode imaging to reduce the number of scanned pixels and the data readout time, respectively. Moreover, we developed a compressed sensing analysis algorithm based on alternating descent conditional gradient (ADCG) for low-photon-count data. We applied the ADCG-FLIM algorithm on both simulated and experimental datasets to evaluate its performance. The results showed that ADCG-FLIM could achieve reliable lifetime estimation with high accuracy and precision in the case of a photon count less than 100. By reducing the photon count requirement for each pixel from, typically, 1000 to 100, the acquisition time for a single frame lifetime image could be significantly shortened, and the imaging speed could be improved to a great extent. On this basis, we obtained lifetime trajectories of moving fluorescent beads using the SPT-FLIM technique. Overall, our work offers a powerful tool for fluorescence lifetime tracking and imaging of single moving particles, which will promote the application of TCSPC-FLIM in biological research.

Snapshot temporal compressive light-sheet fluorescence microscopy via deep denoising and total variation priors.

We present a snapshot temporal compressive light-sheet fluorescence microscopy system to capture high-speed microscopic scenes with a low-speed camera. A deep denoising network and total variation denoiser are incorporated into a plug-and-play framework to quickly reconstruct 20 high-speed video frames from a short-time measurement. Specifically, we can observe 1,000-frames-per-second (fps) microscopic scenes when the camera works at 50 fps to capture the measurement. The proposed method can potentially be applied to observe cell and tissue motions in thick living biological specimens.

Revealing the structure and organization of intercellular tunneling nanotubes (TNTs) by STORM imaging.

Tunneling nanotubes (TNTs) are nanoscale, actin-rich, transient intercellular tubes for cell-to-cell communication, which transport various cargoes between distant cells. The structural complexity and spatial organization of the involved components of TNTs remain unknown. In this work, the STORM super-resolution imaging technique was applied to elucidate the structural organization of microfilaments and microtubules in intercellular TNTs at the nanometer scale. Our results reveal different distributions of microfilaments and intertwined structures of microtubules in TNTs, which promote the knowledge of TNT communications.

Multifunctional Hollow MnO2 @Porphyrin@Bromelain Nanoplatform for Enhanced Photodynamic Therapy.

Photodynamic therapy (PDT) has been showing great potential in cancer treatment. However, the efficacy of PDT is always limited by the intrinsic hypoxic tumor microenvironment (TME) and the low accumulation efficiency of photosensitizers in tumors. To address the issue, a multifunctional hollow multilayer nanoplatform (H-MnO2 @TPyP@Bro) comprising manganese dioxide, porphyrin (TPyP) and bromelain (Bro), is developed for enhanced photodynamic therapy. MnO2 catalyzes the intracellular hydrogen peroxide (H2 O2 ) to produce oxygen (O2 ), reversing the hypoxic TME in vivo. The generated O2 is converted into singlet oxygen (1 O2 ) by the TPyP shell under near-infrared light, which can inhibit tumor proliferation. Meanwhile, the Bro can digest collagen in the extracellular matrix around the tumor, and can promote the accumulation of H-MnO2 @TPyP@Bro in the deeper tumor tissue, further improving the therapeutic effect of PDT. In addition, MnO2 can react with the overexpressed glutathione in TME to release Mn2+ . Consequently, Mn2+ not only induces chemo-dynamic therapy based on Fenton reaction by converting H2 O2 into hydroxyl radicals, but also activates the Mn2+ -based magnetic resonance imaging. Therefore, the developed H-MnO2 @TPyP@Bro nanoplatform can effectively modulate the unfavorable TME and overcome the limitations of conventional PDT for cancer diagnostic and therapeutic.

Lipophilic Red-Emitting Carbon Dots for Detecting and Tracking Lipid Droplets in Live Cells.

Lipid droplets (LDs), a dynamic organelle, are of vital importance in regulating the storage of neutral lipids and energy homeostasis. The aberrant expression of LDs is found to be highly associated with diverse metabolic diseases. Thus, detecting and monitoring LDs are essential to study the pathological and physiological processes of LDs in living bodies. However, it remains challenging to obtain suitable imaging probes to track LDs in vivo. Fortunately, the emergence of carbon dots (CDs), which are fluorescent nanomaterials with good biocompatibility and high stability, has provided us an unprecedented choice. In this work, CDs were synthesized via a solvothermal treatment of commercial reagents, 3-dimethylaminophenol. Interestingly, the prepared CDs show an intense red emission in non-hydrogen-bonding solution and have strong LD-targeting ability without any postmodification of ligands. Moreover, due to their low phototoxicity and excellent photostability, CDs were successfully applied to track the dynamics of LDs in live cells and image LDs in different cell lines and lipid-rich tissues. Overall, this work here proposed an LD-specific red-emitting CD probe, which will be of great value for learning more about LD-associated behaviors and diseases.

Corrigendum: Super-Resolution Microscopy: Shedding New Light on In Vivo Imaging.

[This corrects the article DOI: 10.3389/fchem.2021.746900.].

Super-Resolution Microscopy: Shedding New Light on In Vivo Imaging.

Over the past two decades, super-resolution microscopy (SRM), which offered a significant improvement in resolution over conventional light microscopy, has become a powerful tool to visualize biological activities in both fixed and living cells. However, completely understanding biological processes requires studying cells in a physiological context at high spatiotemporal resolution. Recently, SRM has showcased its ability to observe the detailed structures and dynamics in living species. Here we summarized recent technical advancements in SRM that have been successfully applied to in vivo imaging. Then, improvements in the labeling strategies are discussed together with the spectroscopic and chemical demands of the fluorophores. Finally, we broadly reviewed the current applications for super-resolution techniques in living species and highlighted some inherent challenges faced in this emerging field. We hope that this review could serve as an ideal reference for researchers as well as beginners in the relevant field of in vivo super resolution imaging.

Multistage Cooperative Nanodrug Combined with PD-L1 for Enhancing Antitumor Chemoimmunotherapy.

Combinatorial CpG oligonucleotide (CPG) and chemotherapy drug represent a promising approach to reactivate immune system. However, these two agents possess different physicochemical properties, hindering the application of direct self-assembly of these two cargos into a single nanostructure. Here, a multistage cooperative nanodrug is developed by the direct self-assembly of cis-platinum (CDDP, Pt), l-arginine (l-Arg, R), and CPG (defined as PtR/CPG) for antitumor chemoimmunotherapy. First, the CDDP can induce cell apoptosis. Meanwhile, CDDP also promotes the production of H2 O2 , catalyzing the conversion of l-Arg into nitric oxide (NO). The generated NO decreases the multidrug resistance of cells toward CDDP. Thus, the synergistic effects of CDDP and NO can trigger immunogenic cell death to produce tumor-associated antigens (TAAs). The TAAs and CPG will induce the maturation of dendritic cells (DCs) and enhance antigen presentation ability of DCs. In this way, the PtR/CPG can reverse the immunosuppressive microenvironment, sensitizing tumors to immune checkpoint inhibitors mediated by the programmed death-ligand 1 (PD-L1) antibody. Furthermore, the PtR/CPG combined with the PD-L1 antibody decreases the exhaustion and dysfunction of cytotoxic T lymphocytes to elicit durable systemic immune response. As a result, the prepared PtR/CPG nanodrug in combination with PD-L1 may be highly significant for cancer immunotherapy.

Rational design of a prodrug to inhibit self-inflammation for cancer treatment.

Photothermal therapy (PTT) has been extensively used as an effective therapeutic approach against cancer. However, PTT can trigger the proinflammatory response of dendritic cells (DCs) and macrophages to release proinflammatory cytokines, which can simulate tumor regeneration and further hinder subsequent therapy. Hence, an effective therapeutic system, comprising gold nanoparticle modified Cu2ZnSnS4 nanocrystals and aspirin (Au-CZTS/Asp), was developed to co-deliver PTT agents and inflammatory inhibitors for the synergistic treatment of cancer. Au-CZTS with high near infrared (NIR) photothermal conversion abilities can effectively induce apoptosis and tumor ablation under NIR light. Furthermore, Asp can inhibit the activation of the cGAS-STING pathway in DCs and the polarization of macrophages to intercept the PTT mediated inflammatory responses. Therefore, the as-prepared Au-CZTS/Asp can effectively realize the integration of tumor treatment and recovery. Simultaneously, the Au-CZTS/Asp with ultrasmall size can be rapidly cleared to reduce biotoxicity and side effects. In addition, the Au-CZTS/Asp showed excellent photoacoustic (PA) imaging properties around the tumor in vivo. Thus, our study provides a potential platform for a nano-prodrug that is viable for cancer diagnostic-treatment-recovery integration.

Virtual single-pixel imaging-based deconvolution method for spatial resolution improvement in wide-field fluorescence microscopy.

Deconvolution technique has been widely used in fluorescence microscopy to restore fine structures of biological samples. However, conventional deconvolution methods usually achieve little contrast enhancement in dense structures that have the intervals close to the Rayleigh criterion. Herein, we developed a novel deconvolution method, termed virtual single-pixel imaging (v-SPI). Differing from existing deconvolution methods, v-SPI aims to retrieve the less blurred image directly, not the sample distribution which cannot be actually obtained. And the result can be retrieved simply by solving a linear matrix in spatial domain. In addition, the proposed method has no requirement of calibrating parameters of microscope system. Simulation and experimental results demonstrated that the proposed v-SPI method can enhance the contrast of dense structures significantly and acquire a 24% increase in resolution.

Laser-Induced Periodic Ag Surface Structure with Au Nanorods Plasmonic Nanocavity Metasurface for Strong Enhancement of Adenosine Nucleotide Label-Free Photoluminescence Imaging.

The label-free detection of biomolecules by means of fluorescence spectroscopy and imaging is topical. The developed surface-enhanced fluorescence technique has been applied to achieve progress in the label-free detection of biomolecules including deoxyribonucleic acid (DNA) bases. In this study, the effect of a strong enhancement of photoluminescence of 5'-deoxyadenosine-monophosphate (dAMP) by the plasmonic nanocavity metasurface composed of the silver femtosecond laser-induced periodic surface structure (LIPSS) and gold nanorods or nanospheres has been realized at room temperature. The highest value of 1220 for dAMP on the Ag-LIPSS/Au nanorod metasurface has been explained to be a result of the synergetic effect of the generation of hot spots near the sharp edges of LIPSS and Au nanorod tips together with the excitation of collective gap mode of the cavity due to strong near-field plasmonic coupling. A stronger plasmonic enhancement of the phosphorescence compared to the fluorescence is achieved due to a greater overlap of the phosphorescence spectrum with the surface plasmon spectral region. The photoluminescence imaging of dAMP on the metasurfaces shows a high intensity in the blue range. The comparison of Ag-LIPSS/Au nanorod and Ag-LIPSS/Au-nanosphere metasurfaces shows a considerably higher enhancement for the metasurface containing Au nanorods. Thus, the hybrid cavity metasurfaces containing metal LIPSS and nonspherical metal nanoparticles with sharp edges are promising for high-sensitive label-free detection and imaging of biomolecules at room temperature.

Optimized approach for optical sectioning enhancement in multifocal structured illumination microscopy.

Multifocal structured illumination microscopy (MSIM) is the parallelized version of image scanning microscopy (ISM) that is created by using many excitation spots, which provides a two-fold resolution enhancement beyond the diffraction limit with a frequency of 1 Hz per 3D picture, but scattered and out-of-focus light in thick samples degrades MSIM optical sectioning performance. Herein, we introduce a new optical sectioning method in MSIM via illumination fluctuation. The proposed method suppresses the out-of-focus light by taking full advantage of the statistic property of MSIM raw data and has no requirement of changing the system setup or projecting more illumination patterns. Experimental results demonstrate that the method can reduce the out-of-focus light by 7.25 times in optical sectioning image.

Rapid 3D image scanning microscopy with multi-spot excitation and double-helix point spread function detection.

Refocusing after Scanning using Helical phase engineering (RESCH) microscopy has previously been demonstrated to provide volumetric information from a single 2D scan. However, the practical application of this method is challenging due to its limited image acquisition speed and spatial resolution. Here, we report on a combination of RESCH and multifocal structured illumination microscopy (MSIM) to improve the image acquisition speed and spatial resolution. A phase mask is introduced to modulate the conventional point spread function (PSF) to the double-helix PSF (DH-PSF), which provides volumetric information, and meanwhile, sparse multifocal illumination patterns are generated by a digital micromirror device (DMD) for parallel 3D subdiffractive imaging information acquisition. We also present a strategy for processing the collected raw data with a Richardson-Lucy deconvolution and pixel reassignment algorithm to improve the spatial resolution of the depth estimation and imaging performance. The proposed 3D image scanning microscopy can record 3D specimen information and the corresponding depth information from a single multi-spot 2D planar scan, which ensures faster data acquisition, larger field of view, and higher spatial resolution than RESCH. Finally, we demonstrate the capability of our system with actual experiments.