Investigating cartilage-related diseases by polarization-resolved second harmonic generation (P-SHG) imaging.

Establishing quantitative parameters for differentiating between healthy and diseased cartilage tissues by examining collagen fibril degradation patterns facilitates the understanding of tissue characteristics during disease progression. These findings could also complement existing clinical methods used to diagnose cartilage-related diseases. In this study, cartilage samples from normal, osteoarthritis (OA), and rheumatoid arthritis (RA) tissues were prepared and analyzed using polarization-resolved second harmonic generation (P-SHG) imaging and quantitative image texture analysis. The enhanced molecular contrast obtained from this approach is expected to aid in distinguishing between healthy and diseased cartilage tissues. P-SHG image analysis revealed distinct parameters in the cartilage samples, reflecting variations in collagen fibril arrangement and organization across different pathological states. Normal tissues exhibited distinct χ33/χ31 values compared with those of OA and RA, indicating collagen type transition and cartilage erosion with chondrocyte swelling, respectively. Compared with those of normal tissues, OA samples demonstrated a higher degree of linear polarization, suggesting increased tissue birefringence due to the deposition of type-I collagen in the extracellular matrix. The distribution of the planar orientation of collagen fibrils revealed a more directional orientation in the OA samples, associated with increased type-I collagen, while the RA samples exhibited a heterogeneous molecular orientation. This study revealed that the imaging technique, the quantitative analysis of the images, and the derived parameters presented in this study could be used as a reference for disease diagnostics, providing a clear understanding of collagen fibril degradation in cartilage.

RepE: unsupervised representation learning for image enhancement in nonlinear optical microscopy.

We present an unsupervised learning denoising method, RepE (representation and enhancement), designed for nonlinear optical microscopy images, such as second harmonic generation (SHG) and two-photon fluorescence (TPEF). Addressing the challenge of effectively denoising images with various noise types, RepE employs an encoder network to learn noise-free representations and a reconstruction network to generate denoised images. It offers several key advantages, including its ability to (i) operate without restrictive statistic assumptions, (ii) eliminate the need for clean-noisy pairs, and (iii) requires only a few training images. Comparative evaluations on real-world SHG and TPEF images from esophageal cancer tissue slides (ESCC) demonstrate that our method outperforms existing techniques in image quality metrics. The proposed method provides a practical, robust solution for denoising nonlinear optical microscopy images, and it has the potential to be extended to other nonlinear optical microscopy modalities.

Elafin as a Prognostic Marker in Esophageal Squamous Cell Carcinoma: A Pilot Study Using Three-Dimensional Imaging and Genomic Profiling.

Esophageal cancers are globally the sixth deadliest malignancy, with limited curative options. The association of high serum elafin levels, a molecule produced by epithelial cells, with esophageal squamous cell carcinoma (ESCC) risk is established, but its link to poor ESCC prognosis remains unclear. To explore this question, we first used three-dimensional confocal imaging to create a model of the spatial distribution of elafin inside locoregional ESCC tissues. Then, after analyzing data obtained from whole-genome microarrays for ESCC cell lines and their more invasive sublines, we performed in vitro experiments using RNA sequencing to identify possible elafin-related pathways. Three-dimensional tissue imaging showed elafin distributed as an interweaved-like fibrous structure in the stroma of tissue obtained from patients with high serum levels of elafin and poorer prognoses. By contrast, the signal was confined inside or around the tumor nest in patients who had lower serum levels and better survival. The analysis of a TCGA dataset revealed that higher levels of elafin mRNA in stage I-IIIA ESCC patients were associated with shorter survival. The in vitro studies revealed that elafin promoted ESCC cell proliferation, migration, and invasion via the epithelial-mesenchymal transition pathway. Thus, elafin inhibition could potentially be used therapeutically to improve survival in patients with locoregional ESCC.

Administration of mRNA-Nanomedicine-Augmented Calvarial Defect Healing via Endochondral Ossification.

Large-area craniofacial defects remain a challenge for orthopaedists, hastening the need to develop a facile and safe tissue engineering strategy; osteoconductive material and a combination of optimal growth factors and microenvironment should be considered. Faced with the unmet need, we propose that abundant cytokines and chemokines can be secreted from the bone defect, provoking the infiltration of endogenous stem cells to assist bone regeneration. We can provide a potent mRNA medicine cocktail to promptly initiate the formation of bone templates, osteogenesis, and subsequent bone matrix deposition via endochondral ossification, which may retard rapid fibroblast infiltration and prevent the formation of atrophic non-union. We explored the mutual interaction of BMP2 and TGFß3 mRNA, both potent chondrogenic factors, on inducing endochondral ossification; examined the influence of in vitro the transcribed polyA tail length on mRNA stability; prepared mRNA nanomedicine using a PEGylated polyaspartamide block copolymer loaded in a gelatin sponge and grafted in a critical-sized calvarial defect; and evaluated bone regeneration using histological and µCT examination. The BMP2 and TGFß3 composite mRNA nanomedicine resulted in over 10-fold new bone volume (BV) regeneration in 8 weeks than the BMP2 mRNA nanomedicine administration alone, demonstrating that the TGFß3 mRNA nanomedicine synergistically enhances the bone's formation capability, which is induced by BMP2 mRNA nanomedicine. Our data demonstrated that mRNA-medicine-mediated endochondral ossification provides an alternative cell-free tissue engineering methodology for guiding craniofacial defect healing.

Revealing the Structural Organization of Gamma-irradiated Starch Granules Using Polarization-resolved Second Harmonic Generation Microscopy.

Starch is a semi-crystalline macromolecule with the presence of amorphous and crystalline components. The amorphous amylose and crystalline amylopectin regions in starch granules are susceptible to certain physical modifications, such as gamma irradiation. Polarization-resolved second harmonic generation (P-SHG) microscopy in conjunction with SHG-circular dichroism (CD) was used to assess the three-dimensional molecular order and inherent chirality of starch granules and their reaction to different dosages of gamma irradiation. For the first time, the relationship between starch achirality (χ21/χ16 and χ22/χ16) and chirality (χ14/χ16) determining susceptibility tensor ratios has been elucidated. The results showed that changes in the structure and orientation of long-chain amylopectin were supported by the decrease in the SHG anisotropy factor and the χ22/χ16 ratio. Furthermore, SHG-CD illustrated the molecular tilt angle by revealing the arrangement of amylopectin molecules pointing either upward or downward owing to molecular polarity.

Opioid-Modulated Receptor Localization and Erk1/2 Phosphorylation in Cells Coexpressing µ-Opioid and Nociceptin Receptors.

We attempted to examine the alterations elicited by opioids via coexpressed µ-opioid (MOP) and nociceptin/orphanin FQ (NOP) receptors for receptor localization and Erk1/2 (p44/42 MAPK) in human embryonic kidney (HEK) 293 cells. Through two-photon microscopy, the proximity of MOP and NOP receptors was verified by fluorescence resonance energy transfer (FRET), and morphine but not buprenorphine facilitated the process of MOP-NOP heterodimerization. Single-particle tracking (SPT) further revealed that morphine or buprenorphine hindered the movement of the MOP-NOP heterodimers. After exposure to morphine or buprenorphine, receptor localization on lipid rafts was detected by immunocytochemistry, and phosphorylation of Erk1/2 was determined by immunoblotting in HEK 293 cells expressing MOP, NOP, or MOP+NOP receptors. Colocalization of MOP and NOP on lipid rafts was enhanced by morphine but not buprenorphine. Morphine stimulated the phosphorylation of Erk1/2 with a similar potency in HEK 293 cells expressing MOP and MOP+NOP receptors, but buprenorphine appeared to activate Erk1/2 solely through NOP receptors. Our results suggest that opioids can fine-tune the cellular localization of opioid receptors and phosphorylation of Erk1/2 in MOP+NOP-expressing cells.

An insight into optical beam induced current microscopy: Concepts and applications.

Laser scanning optical beam induced current (OBIC) microscopy has become a powerful and nondestructive alternative to other complicated methods like electron beam induced current (EBIC) microscopy, for high resolution defect analysis of electronic devices. OBIC is based on the generation of electron-hole pairs in the sample due to the raster scanning of a focused laser beam with energy equal or greater than the band gap energy and synchronized detection of resultant current profile with respect to the beam positions. OBIC is particularly suitable to localize defect sites caused by metal-semiconductor interdiffusion or electrostatic discharge (ESD). OBIC signals, thus, are capable of revealing the parameters/factors directly related to the reliability and efficiency of the electronic device under test (DUT). In this review, the basic principles of OBIC microscopy strategies and their notable applications in semiconductor device characterization are elucidated. An overview on the developments of OBIC microscopy is also presented. Specifically, the recent progresses on the following three OBIC measurement strategies have been reviewed, which include continuous laser based single photon OBIC, pulsed laser based single photon OBIC, and multiphoton OBIC microscopy for three-dimensional mapping of photocurrent response of electronic devices at high spatiotemporal resolution. Challenges and future prospects of OBIC in characterizing complex electronic devices are also discussed. HIGHLIGHTS: Characterization of electronic device quality is of paramount importance. Optical beam induced current (OBIC) microscopy offers spatially resolved mapping of local electronic properties. This review presents the principle and notable applications of OBIC microscopy.

Runx1 Messenger RNA Delivered by Polyplex Nanomicelles Alleviate Spinal Disc Hydration Loss in a Rat Disc Degeneration Model.

Vertebral disc degenerative disease (DDD) affects millions of people worldwide and is a critical factor leading to low back and neck pain and consequent disability. Currently, no strategy has addressed curing DDD from fundamental aspects, because the pathological mechanism leading to DDD is still controversial. One possible mechanism points to the homeostatic status of extracellular matrix (ECM) anabolism, and catabolism in the disc may play a vital role in the disease's progression. If the damaged disc receives an abundant amount of cartilage, anabolic factors may stimulate the residual cells in the damaged disc to secrete the ECM and mitigate the degeneration process. To examine this hypothesis, a cartilage anabolic factor, Runx1, was expressed by mRNA through a sophisticated polyamine-based PEG-polyplex nanomicelle delivery system in the damaged disc in a rat model. The mRNA medicine and polyamine carrier have favorable safety characteristics and biocompatibility for regenerative medicine. The endocytosis of mRNA-loaded polyplex nanomicelles in vitro, mRNA delivery efficacy, hydration content, disc shrinkage, and ECM in the disc in vivo were also examined. The data revealed that the mRNA-loaded polyplex nanomicelle was promptly engulfed by cellular late endosome, then spread into the cytosol homogeneously at a rate of less than 20 min post-administration of the mRNA medicine. The mRNA expression persisted for at least 6-days post-injection in vivo. Furthermore, the Runx1 mRNA delivered by polyplex nanomicelles increased hydration content by ≈43% in the punctured disc at 4-weeks post-injection (wpi) compared with naked Runx1 mRNA administration. Meanwhile, the disc space and ECM production were also significantly ameliorated in the polyplex nanomicelle group. This study demonstrated that anabolic factor administration by polyplex nanomicelle-protected mRNA medicine, such as Runx1, plays a key role in alleviating the progress of DDD, which is an imbalance scenario of disc metabolism. This platform could be further developed as a promising strategy applied to regenerative medicine.

Label-Free Characterization of Collagen Crosslinking in Bone-Engineered Materials Using Nonlinear Optical Microscopy.

Engineered biomaterials provide unique functions to overcome the bottlenecks seen in biomedicine. Hence, a technique for rapid and routine tests of collagen is required, in which the test items commonly include molecular weight, crosslinking degree, purity, and sterilization induced structural change. Among them, the crosslinking degree mainly influences collagen properties. In this study, second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy are used in combination to explore the collagen structure at molecular and macromolecular scales. These measured parameters are applied for the classification and quantification among the different collagen scaffolds, which were verified by other conventional methods. It is demonstrated that the crosslinking status can be analyzed from SHG images and presented as the coherency of collagen organization that is correlated with the mechanical properties. Also, the comparative analyses of SHG signal and relative CARS signal of amide III band at 1,240 cm−1 to δCH2 band at 1,450 cm−1 of these samples provide information regarding the variation of the molecular structure during a crosslinking process, thus serving as nonlinear optical signatures to indicate a successful crosslinking.

Imaging of nanoscale birefringence using polarization-resolved chromatic confocal microscopy.

We demonstrate a homebuilt confocal microscope with ∼60 nm axial resolution to visualize the optical path length (OPL) of liquid crystals (LCs) inside a 2-domain alignment LC cell. Since the microscope is sensitive to light polarization, it is capable of determining LC orientation by accounting for the OPL variation, ΔOPL. The resolution of birefringence depends on the measured ΔOPL from two cross-polarized channel detections, of which the concept is different from other polarization-resolved optical imaging techniques, but is relatively simple in optical layout and analysis. The different orientations of LCs and the voltage-dependent LC rotation properties in the 2-domain LC cell are monitored and analyzed. Additionally, the complicated LC orientation distribution at the junction of the two domains with different alignments can be clearly observed. It shows great possibilities of examining tissue birefringence related to disease progression and tiny birefringence variation of electro-optical materials under an external field, which are hardly resolved by conventional optical imaging techniques.

Wave-vector-encoded nonlinear endomicroscopy.

Based on a rigid square fiber for wave vector delivery, we present a novel (to the best of our knowledge) wave-vector-encoded nonlinear-optical endomicroscopy (WENE). WENE overcomes three tangled issues, including femtosecond pulse broadening induced signal degradation, complexity of packaging miniaturized scanners in the distal end, and pixel-like images, which cannot be fully addressed by current distal scanning nonlinear endomicroscopy (NE) or fiber-bundle-based proximal scanning NE. Due to the advantages of its simplicity in overall configuration and package in the distal end, the capability of addressing the issue of pulse broadening, and offering continuous wave vector delivery, the demonstrated WENE shows great promise for future basic research on biomedical processes and minimally invasive utilization for clinical diagnosis.

Label-free characterization of collagen fibers in cancerous esophagus tissues using ratiometric nonlinear optical microscopy.

IMPACT STATEMENT: The issue of classifying esophageal cancer at various developmental stages is crucial for determining the optimized treatment protocol for the patients, as well as the prognosis. Precision improvement in staging esophageal cancer keeps seeking quantitative and analytical imaging methods that could augment histopathological techniques. In this work, we used nonlinear optical microscopy for ratiometric analysis on the intrinsic signal of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) from single collagen fibers only in submucosa of esophageal squamous cell carcinoma (ESCC). The blind tests of TPEF/SHG and forward (F)/backward (B) SHG were demonstrated to compare with the histology conclusion. The discussion of sensitivity and specificity was provided via statistical comparison between the four stages of esophageal cancer. To the best of our knowledge, this is the first study of using these two ratios in combination for staging ESCC.

Microscopic and spectroscopic characterization of rice and corn starch.

Starch granules from rice and corn were isolated, and their molecular mechanism on interaction with α-amylase was characterized through biochemical test, microscopic imaging, and spectroscopic measurements. The micro-scale structure of starch granules were observed under an optical microscope and their average size was in the range 1-100 µm. The surface topological structures of starch with micro-holes due to the effect of α- amylase were also visualized under scanning electron microscope. The crystallinity was confirmed by X-ray diffraction patterns as well as second-harmonic generation microscopy. The change in chemical bonds before and after hydrolysis of the starch granules by α- amylase was determined by Fourier transform infrared spectroscopy. Combination of microscopy and spectroscopy techniques relates structural and chemical features that explain starch enzymatic hydrolysis which will provide a valid basis for future studies in food science and insights into the energy transformation dynamics.

Spontaneous loss versus stimulation gain in pump-probe microscopy: a proof of concept demonstration.

SIGNIFICANCE: The large background, narrow dynamic range, and detector saturation have been the common limiting factors in stimulated emission (SE)-based pump-probe microscopy, attributed to the very small signal overriding the very intense laser probe beam. To better differentiate the signal of interest from the background, lock-in detection is used to measure the fluorescence quenching, which is termed spontaneous loss (SL). The advantages are manifold. The spontaneous fluorescence signal can be well separated from both the pump and the probe beams with filters, thus eliminating the background, enlarging the dynamic range, and avoiding the saturation of the detector. AIM: We propose and demonstrate an integrated pump-probe microscopy technique based on lock-in detection for background removal and dynamic range enhancement through SL detection. APPROACH: The experimental setup is configured with a pulsed diode laser at a wavelength λpu = 635 nm, acting as a pump (excitation) and a mode-locked Ti:sapphire laser at a central wavelength λpr = 780 nm, serving as the probe beam (stimulation). Both pulse trains are temporally synchronized through high precision delay control by adjusting the length of the triggering cables. The pump and probe beams are alternatively modulated at different frequencies f1 and f2 to extract the stimulated gain (SG) and SL signal. RESULTS: SG signal shows saturation due to the irradiation of the intense probe beam onto the photodetector. However, the detector saturation does not occur at high probe beam power for SL detection. The fluorescence lifetime images are acquired with reduced background. The theoretical signal-to-noise ratios for SG and SL are also estimated by photon statistics. CONCLUSION: We have confirmed that the detection of SL allows the elimination of the large background without photodetector saturation, which commonly exists in SG configuration. This modality would allow unprecedented manipulation and investigation of fluorophores in fluorescence imaging.

Polarimetric Measurements of Surface Chirality Based on Linear and Nonlinear Light Scattering.

A molecule, molecular aggregate, or protein that cannot be superimposed on its mirror image presents chirality. Most living systems are organized by chiral building blocks, such as amino acids, peptides, and carbohydrates, and any change in their molecular structure (i.e., handedness or helicity) alters the biochemical and pharmacological functions of the molecules, many of which take place at surfaces. Therefore, studying surface chirogenesis at the nanoscale is fundamentally important and derives various applications. For example, since proteins contain highly ordered secondary structures, the intrinsic chirality can be served as a signature to measure the dynamics of protein adsorption and protein conformational changes at biological surfaces. Furthermore, a better understanding of chiral recognition and separation at bio-nanointerfaces is helpful to standardize chiral drugs and monitor the synthesis of adsorbents with high precision. Thus, exploring the changes in surface chirality with polarized excitations would provide structural and biochemical information of the adsorbed molecules, which has led to the development of label-free and noninvasive measurement tools based on linear and nonlinear optical effects. In this review, the principles and selected applications of linear and nonlinear optical methods for quantifying surface chirality are introduced and compared, aiming to conceptualize new ideas to address critical issues in surface biochemistry.

Enantiomeric Recognition and Separation by Chiral Nanoparticles.

Chiral molecules are stereoselective with regard to specific biological functions. Enantiomers differ considerably in their physiological reactions with the human body. Safeguarding the quality and safety of drugs requires an efficient analytical platform by which to selectively probe chiral compounds to ensure the extraction of single enantiomers. Asymmetric synthesis is a mature approach to the production of single enantiomers; however, it is poorly suited to mass production and allows for only specific enantioselective reactions. Furthermore, it is too expensive and time-consuming for the evaluation of therapeutic drugs in the early stages of development. These limitations have prompted the development of surface-modified nanoparticles using amino acids, chiral organic ligands, or functional groups as chiral selectors applicable to a racemic mixture of chiral molecules. The fact that these combinations can be optimized in terms of sensitivity, specificity, and enantioselectivity makes them ideal for enantiomeric recognition and separation. In chiral resolution, molecules bond selectively to particle surfaces according to homochiral interactions, whereupon an enantiopure compound is extracted from the solution through a simple filtration process. In this review article, we discuss the fabrication of chiral nanoparticles and look at the ways their distinctive surface properties have been adopted in enantiomeric recognition and separation.

In situ high-resolution thermal microscopy on integrated circuits.

The miniaturization of metal tracks in integrated circuits (ICs) can cause abnormal heat dissipation, resulting in electrostatic discharge, overvoltage breakdown, and other unwanted issues. Unfortunately, locating areas of abnormal heat dissipation is limited either by the spatial resolution or imaging acquisition speed of current thermal analytical techniques. A rapid, non-contact approach to the thermal imaging of ICs with sub-µm resolution could help to alleviate this issue. In this work, based on the intensity of the temperature-dependent two-photon fluorescence (TPF) of Rhodamine 6G (R6G) material, we developed a novel fast and non-invasive thermal microscopy with a sub-µm resolution. Its application to the location of hotspots that may evolve into thermally induced defects in ICs was also demonstrated. To the best of our knowledge, this is the first study to present high-resolution 2D thermal microscopic images of ICs, showing the generation, propagation, and distribution of heat during its operation. According to the demonstrated results, this scheme has considerable potential for future in situ hotspot analysis during the optimization stage of IC development.

Polarization resolved second harmonic microscopy.

Second harmonic (SH) microscopy has proven to be a powerful imaging modality over the past years due to its intrinsic advantages as a multiphoton process with endogenous contrast specificity, which allows pinhole-less optical sectioning, non-invasive observation, deep tissue penetration, and the possibility of easier signal detection at visible wavelengths. Depending on the relative orientation between the polarization of the incoming light and the second-order susceptibility of non-centrosymmetric structures, SH microscopy provides the unique capacity to probe the absolute molecular structure of a broad variety of biological tissues without the necessity for additional labeling. In addition, SH microscopy, when working with polarimetry, provides clear and in-depth insights on the details of molecular orientation and structural symmetry. In this review, the working principles of the polarization resolving techniques and the corresponding implements of SH microscopy are elucidated, with focus on Stokes vector based polarimetry. An overview of the advancements on SH anisotropy measurements are also presented. Specifically, the recent progresses on the following three topics in polarization resolved SH microscopy will be elucidated, which include Stokes vector resolving for imaging molecular structure and orientation, 3-D structural chirality by SH circular dichroism, and correlation with fluorescence lifetime imaging (FLIM) for in vivo wound healing diagnosis. The potentials and challenges for future researches in exploring complex biological tissues are also discussed.

Label-free, ultrahigh-speed, 3D observation of bidirectional and correlated intracellular cargo transport by coherent brightfield microscopy.

The investigation of intracellular transport at the molecular scale requires measurements at high spatial and temporal resolutions. We demonstrate the label-free, direct imaging and tracking of native cell vesicles in live cells at an ultrahigh spatiotemporal resolution. Using coherent brightfield (COBRI) microscopy, we monitor individual cell vesicles traveling inside the cell with nanometer spatial precision in 3D at 30 000 frames per second. The stepwise directional motion of the vesicle on the cytoskeletal track is clearly resolved. We also observe the repeated switching of the transport direction of the vesicle in a continuous trajectory. Our high-resolution measurement unveils the transient pausing and subtle bidirectional motion of the vesicle, taking place over tens of nanometers in tens of milliseconds. By tracking multiple particles simultaneously, we found strong correlations between the motions of two neighboring vesicles. Our label-free ultrahigh-speed optical imaging provides the opportunity to visualize intracellular cargo transport at the nanoscale in the microsecond timescale with minimal perturbation.

Coherent Brightfield Microscopy Provides the Spatiotemporal Resolution To Study Early Stage Viral Infection in Live Cells.

Viral infection starts with a virus particle landing on a cell surface followed by penetration of the plasma membrane. Due to the difficulty of measuring the rapid motion of small-sized virus particles on the membrane, little is known about how a virus particle reaches an endocytic site after landing at a random location. Here, we use coherent brightfield (COBRI) microscopy to investigate early stage viral infection with ultrahigh spatiotemporal resolution. By detecting intrinsic scattered light via imaging-based interferometry, COBRI microscopy allows us to track the motion of a single vaccinia virus particle with nanometer spatial precision (<3 nm) in 3D and microsecond temporal resolution (up to 100,000 frames per second). We explore the possibility of differentiating the virus signal from cell background based on their distinct spatial and temporal behaviors via digital image processing. Through image postprocessing, relatively stationary background scattering of cellular structures is effectively removed, generating a background-free image of the diffusive virus particle for precise localization. Using our method, we unveil single virus particles exploring cell plasma membranes after attachment. We found that immediately after attaching to the membrane (within a second), the virus particle is locally confined within hundreds of nanometers where the virus particle diffuses laterally with a very high diffusion coefficient (∼1 µm2/s) at microsecond time scales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.