Research of Pulmonary Fibrosis Lesions Based on FLIM and SHG Imaging Microscopy.

Two-photon fluorescence lifetime microscopy (TP-FLIM) is a powerful quantitative imaging technique that characterizes and analyzes the structure and function of biological samples through a combination of intensity and lifetime imaging. Because TP-FLIM is independent of the fluorescence signal intensity and the fluorophore concentration, it is widely used in high-throughput, high-content drug screening and clinical diagnostics. Second harmonic generation (SHG) imaging technology has the advantages of high spatial resolution and imaging depth inherent to nonlinear optical imaging. Second harmonics often appear in noncentrosymmetric structures. Collagen tissue in biological organisms is a good example of these structures, showing strong harmonic effects. Therefore, SHG has been widely used for imaging of specific tissue structure imaging. TP-FLIM technology is highly sensitive for quantitatively detecting changes in microenvironments. The objective of this study is to examine pathological pulmonary fibrosis slices using a combined approach of TP-FLIM and SHG technology. The fluorescence lifetime data of pulmonary collagen fibers are analyzed by using phasor plot analysis methods, and normal collagen fibers and fibrotic collagen fibers are distinguished by calculating the aspect ratio from the SHG images formed by the collagen fibers. Our study provides a new method for a deeper understanding of the pathological mechanisms and clinical diagnosis of pulmonary fibrosis and other collagen fiber-related disorders.

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.

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.

In vivo two-photon fluorescence lifetime imaging microendoscopy based on fiber-bundle.

Fluorescence lifetime imaging microendoscopy (FLIME) has been reported to investigate the physicochemical microenvironment in biological tissue. In this work, we designed a two-photon (TP) FLIME system based on a fiber-bundle glued with an achromatic mini-objective with 1.4-mm diameter, which was coupled to a standard TP microscope containing a dispersion precompensation module in the laser source. With 840 nm excitation, the field of view and lateral resolution of our system are 390 µm and 1.55 µm, respectively. To examine the capability of imaging in vivo, we obtained Z-stack (0-130 µm) TP-FLIME images from the intestine's surface of a mouse injected with squaraine dye. Further, we utilized the TP-FLIME system to image the kidney, liver, and xenografted tumor at 100-µm depth in vivo with cellular resolution, which features the distribution of cells and tissue structures with better contrast than intensity images. These results demonstrated that the proposed system is capable of measuring fluorescence lifetime in situ and provides a powerful tool to research the deep tissue microenvironment naturally.

Automated ExEm-spFRET Microscope.

Excitation­emission-spectral unmixing-based fluorescence resonance energy transfer (ExEm-spFRET) microscopy exhibits excellent robustness in living cells. We here develop an automatic ExEm-spFRET microscope with 3.04 s of time resolution for a quantitative FRET imaging. The user-friendly interface software has been designed to operate in two modes: administrator and user. Automatic background recognition, subtraction, and cell segmentation were integrated into the software, which enables FRET calibration or measurement in a one-click operation manner. In administrator mode, both correction factors and spectral fingerprints are only calibrated periodically for a stable system. In user mode, quantitative ExEm-spFRET imaging is directly implemented for FRET samples. We implemented quantitative ExEm-spFRET imaging for living cells expressing different tandem constructs (C80Y, C40Y, C10Y, and C4Y, respectively) and obtained consistent results for at least 3 months, demonstrating the stability of our microscope. Next, we investigated Bcl-xL-Bad interaction by using ExEm-spFRET imaging and FRET two-hybrid assay and found that the Bcl-xL-Bad complexes exist mainly in Bad-Bcl-xL trimers in healthy cells and Bad-Bcl-xL2 trimers in apoptotic cells. We also performed time-lapse FRET imaging on our system for living cells expressing Yellow Cameleon 3.6 (YC3.6) to monitor ionomycin-induced rapid extracellular Ca2+ influx with a time interval of 5 s for total 250 s.

Mcl-1 inhibits Mff-mediated mitochondrial fragmentation and apoptosis.

Myeloid cell leukemia-1 (Mcl-1) is involved in the regulation of mitochondrial fission and fusion. This report aims to explore whether Mcl-1 can interact with mitochondrial fission factor (Mff) and regulate Mff-mediated mitochondrial fragmentation and apoptosis. Fluorescence images of living cells coexpressing YFP-Mff and CFP-Mcl-1 showed that Mcl-1 markedly inhibited Mff-mediated mitochondrial fragmentation and apoptosis, suggesting that Mcl-1 played a key role in inhibiting mitochondrial fission. The cells coexpressing YFP-Mff and CFP-Mcl-1 exhibited consistent fluorescence resonance energy transfer (FRET) efficiency with that of the cells coexpressing CFP-Mcl-1 and YFP, demonstrating that Mcl-1 did not directly bind to Mff on mitochondria. Collectively, Mcl-1 inhibits Mff-mediated mitochondrial fission and apoptosis not via directly binding to Mff on mitochondria.

Enhanced multifocal structured illumination microscopy with desired optical sectioning capability and lateral resolution improvement.

Multifocal structured illumination microscopy (MSIM) can rapidly retrieve 3D structures of thick samples by using multi-spot excitation and detection. Although numerous super-resolution (SR) and optical sectioning (OS) methods have been introduced in this field, the existing OS-SR method in MSIM still has the difficulty in rejecting deep defocused light, which may lead to strong background signal in the retrieved results. To this end, an enhanced OS-SR method is proposed to simultaneously achieve the desired OS capability and significant resolution improvement in MSIM. The enhanced OS-SR image is obtained by combining the standard deviation image with the conventional OS-SR image in the frequency domain. The validity of the proposed method is demonstrated by simulation and experimental results.

Time-lapse FRET analysis reveals the ability of Bax dimer to trigger mitochondrial outer membrane permeabilization.

Bax oligomerization is essential for triggering mitochondrial outer membrane permeabilization (MOMP) in many apoptotic programs. However, it is controversial whether Bax dimer is sufficient to trigger MOMP. In this report, multiple Gaussian function-based FRET analysis (Multi-Gaussian FRET analysis) was used to dissect the dimerization and then tetramerization of Bax in relation to MOMP. Multi-Gaussian FRET analysis on the time-lapse FRET images of single living cells co-expressing CFP-Bax and YFP-Bax revealed that formation of mitochondrial Bax homodimers preceded MOMP within 3 min and Bax dimer transformed into tetramer within 6 min concomitantly with complete MOMP within 10 min, providing direct evidence in support of the sufficient ability of Bax dimers to trigger MOMP at least in natural cells.

Gaussian FRET two-hybrid assays for determining the stoichiometry of hetero-oligomeric complexes in single living cells.

Here we integrate multiple Gaussian-functions analysis into fluorescence resonance energy transfer (FRET) two-hybrid assays (Gaussian FRET two-hybrid assay) to determine the stoichiometric ratios of intracellular hetero-oligomers in single living cells. This method adopts in multiple Gaussian-functions to fit the E-count histograms of both donor- and acceptor-centric FRET efficiency (ED and EA) images of a single cell for obtaining the peak values (EDi and EAi), thus yielding the corresponding stoichiometric ratios (EDi/EAi) of intracellular hetero-oligomers. We performed Gaussian FRET two-hybrid assay for living Hela cells coexpressing different FRET tandem plasmids, and obtained consistent results with the expected values. Gaussian FRET two-hybrid assay for cells coexpressing Bad-CFP and Bcl-XL-YFP reveals that Bcl-XL binds with Bad to form a hetero-oligomeric complex with a stoichiometry of 2:1 on mitochondria.

ExEm-FRET two-hybrid assay: FRET two-hybrid assay based on linear unmixing of excitation-emission spectra.

Simultaneous linear unmixing of excitation-emission spectra (ExEm-unmixing)-based fluorescence resonance energy transfer (FRET) two-hybrid assay method, named as ExEm-FRET two-hybrid assay, was developed for evaluating the stoichiometric ratio of macromolecular complexes in living cells. Linear unmixing of the excitation-emission spectra (SDA) of cells obtains the weight factors of donor (WD), acceptor (WA) and acceptor sensitization (WS), yielding ED and EA (donor- and acceptor-centric FRET efficiency) images. ExEm-FRET two-hybrid assay employs pixel-to-pixel titration curves of ED/EA versus the free acceptor (Ca)/donor (Cd) concentration deduced from the three weight factors to obtain EA,max and ED,max (the maximal EA and ED), thus yielding the stoichiometric ratio (EA,max/ED,max) of donor-tagged protein to acceptor-tagged protein.

Injectable Gelled Multiple Emulsion for Glucose-Responsive Insulin Delivery.

A glucose-responsive insulin delivery system that sustains blood glucose equilibrium for an extended duration can address the low therapeutic window of insulin in diabetes treatment. Herein, insulin is loaded in a water-in-oil-in-water (W1/O/W2) gelled multiple emulsion using poly (4-vinylphenylboronic acid) (PVPBA) homopolymer as an effective emulsifier. The gelled multiple emulsion exhibits a high encapsulation efficiency (99%), enhanced stability and remarkable shear-thinning behavior, making it easy to inject. Under hyperglycemic conditions, the gelled emulsion system instantly binds to glucose molecules and reduces the hydrogen bonds of the PVPBA homopolymer, resulting in insulin release. In a streptozotocin-induced type 1 diabetic mouse model, a single subcutaneous injection of the gelled emulsion rapidly responds to high blood glucose concentration (BGC) and release insulin in a glucose dependent manner, thus prolonging the antihyperglycemic effect compared with free insulin.

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.

Enzyme-Silenced Nanosponges Prolong Intratumoral Lifetime to Facilitate Intercellular Relay Drug Delivery and Treatment Efficacy.

The clinical application of nanomedicines faces the dilemma of improved safety but restricted efficacy due to the poor intratumoral bioavailability of chemotherapeutics. We here design an enzyme-silenced nanosponge that shares a long-term lifespan to reversibly exhale/inhale doxorubicin (DOX) for continuous intercellular relay delivery and improved intratumoral retention. The nanosponge is composed of a cationic lipid overlaying a hyaluronic acid derivative polyampholyte core for enveloping of DOX and hyaluronidase-1-targeted siRNA (siHyal1), and a lipoprotein shell decorated with fusion peptide 4F-tLyP-1 that was fused with apolipoprotein A-I (apoA-I) mimetic peptide 4F and tLyP-1 for tumor homing and extravasation into the tumor interstitium. Triggered by the intra/intercellular pH variation, the nanosponge core could reversibly swell in endo/lysosome (pH 5.0) for DOX release. Owing to the deprotonation, the nanosponge core shrinks back in cytoplasm (pH 7.4) for DOX reloading and continues the behavior after being secreted to the extracellular matrix (pH 6.8) via Golgi apparatus, which dramatically improves intratumoral DOX retention and availability. Concurrently, the intratumoral lifespan of the nanosponge is prolonged by siHyal1-specific silencing, ensuring spatiotemporal consistency of carrier and drug when shuttling multilayer tumor cells. As a result, the nanosponge achieves efficient tumor inhibition in 99.1% of tumor spheroids and 80.1% of orthotopic tumor models. Collectively, this study provides an intelligent nanosponge design for active intercellular relay drug delivery, achieving improved intratumoral bioavailability of drugs and amplified chemotherapy on solid tumors.

Nanoparticle-Mediated RNA Therapy Attenuates Nonalcoholic Steatohepatitis and Related Fibrosis by Targeting Activated Hepatic Stellate Cells.

Chronic liver injury and inflammation triggered by metabolic abnormalities initiate the activation of hepatic stellate cells (HSCs), driving fibrosis and parenchymal dysfunction, culminating in disorders such as nonalcoholic steatohepatitis (NASH). Unfortunately, there are currently no approved drugs capable of effectively treating NASH due to the challenges in addressing fibrosis and restoring extracellular matrix (ECM) homeostasis. We discovered a significant up-regulation of interleukin-11 (IL-11) in fibrotic livers using two well-established murine models of NASH. To leverage this signaling pathway, we developed a nanoparticle (NP)-assisted RNA interfering approach that specifically targets activated HSCs (aHSCs), blocking IL-11/ERK signaling to regulate HSC transdifferentiation along with fibrotic remodeling. The most potent NP, designated NP-AEAA, showed enhanced accumulation in fibrotic livers with NASH and was primarily enriched in aHSCs. We further investigated the therapeutic efficacy of aHSC-targeting NP-AEAA encapsulating small interfering RNA (siRNA) against IL11 or its cognate receptor IL11ra1 (termed siIL11@NP-AEAA or siIL11ra1@NP-AEAA, respectively) for resolving fibrosis and NASH. Our results demonstrate that both siIL11@NP-AEAA and siIL11ra1@NP-AEAA effectively inhibit HSC activation and resolve fibrosis and inflammation in two well-established murine models of NASH. Notably, siIL11ra1@NP-AEAA exhibits a superior therapeutic effect over siIL11@NP-AEAA, in terms of reducing liver steatosis and fibrosis as well as recovering liver function. These results constitute a targeted nanoparticulate siRNA therapeutic approach against the IL-11 signaling pathway of aHSCs in the fibrotic liver, offering a promising therapeutic intervention for NASH and other diseases.

Recent Advances in Strategies for Addressing Hypoxia in Tumor Photodynamic Therapy.

Photodynamic therapy (PDT) is a treatment modality that uses light to target tumors and minimize damage to normal tissues. It offers advantages including high spatiotemporal selectivity, low side effects, and maximal preservation of tissue functions. However, the PDT efficiency is severely impeded by the hypoxic feature of tumors. Moreover, hypoxia may promote tumor metastasis and tumor resistance to multiple therapies. Therefore, addressing tumor hypoxia to improve PDT efficacy has been the focus of antitumor treatment, and research on this theme is continuously emerging. In this review, we summarize state-of-the-art advances in strategies for overcoming hypoxia in tumor PDTs, categorizing them into oxygen-independent phototherapy, oxygen-economizing PDT, and oxygen-supplementing PDT. Moreover, we highlight strategies possessing intriguing advantages such as exceedingly high PDT efficiency and high novelty, analyze the strengths and shortcomings of different methods, and envision the opportunities and challenges for future research.

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.

Human serum albumin gradient in serous ovarian cancer cryosections measured by fluorescence lifetime.

Human serum albumin (HSA) is a depot and carrier for many endogenous and exogenous molecules in blood. Many studies have demonstrated that the transport of HSA in tumor microenvironments contributes to tumor development and progression. In this report, we set up a multimodal nonlinear optical microscope system, combining two-photon excitation fluorescence, second harmonic generation, and two-photon fluorescence lifetime imaging microscopy. The fluorescence lifetime of a small squaraine dye (SD) is used to evaluate HSA concentrations in tumor tissue based on specific binding between SD and HSA. We used SD to stain the cryosections from serous ovarian cancer patients in high-grade (HGSOC) and low-grade (LGSOC), respectively, and found a gradient descent of HSA concentration from normal connective tissue to extracellular matrix to tumor masses from 13 to 2 µM for LGSOC patients and from 36 to 12 µM for HGSOC patients. We demonstrated that multimodal nonlinear optical microscopy can obtain similar results as those from traditional histologic staining, thus it is expected to move to clinical applications.

Sponge particulates for biomedical applications: Biofunctionalization, multi-drug shielding, and theranostic applications.

Sponge particulates have attracted enormous attention in biomedical applications for superior properties, including large porosity, elastic deformation, capillary action, and three-dimensional (3D) reaction environment. Especially, the tiny porous structures make sponge particulates a promising platform for drug delivery, tissue engineering, anti-infection, and wound healing by providing abundant reservoirs of broad surface and internal network for cargo shielding and shuttling. To control the sponge-like morphology and improve the diversity of drug loading, some optimized preparation techniques of sponge particulates have been developed, contributing to the simplified preparation process and improved production reproducibility. Bio-functionalized strategies, including target modification, cell membrane camouflage, and hydrogel of sponge particulates have been applied to modulate the properties, improve the performance, and extend the applications. In this review, we highlight the unique physical properties and functions, current manufacturing techniques, and an overview of spongy particulates in biomedical applications, especially in inhibition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity. Moreover, the current challenges and prospects of sponge particulates are discussed rationally, providing an insight into developing vibrant fields of sponge particulates-based biomedicine.