Nonlinear Optical Imaging Research Articles

Chiral, Topological, and Knotted Colloids in Liquid Crystals

The geometric shape, symmetry, and topology of colloidal particles often allow for controlling colloidal phase behavior and physical properties of these soft matter systems. In liquid crystalline dispersions, colloidal particles with low symmetry and nontrivial topology of surface confinement are of particular interest, including surfaces shaped as handlebodies, spirals, knots, multi-component links, and so on. These types of colloidal surfaces induce topologically nontrivial three-dimensional director field configurations and topological defects. Director switching by electric fields, laser tweezing of defects, and local photo-thermal melting of the liquid crystal host medium promote transformations among many stable and metastable particle-induced director configurations that can be revealed by means of direct label-free three-dimensional nonlinear optical imaging. The interplay between topologies of colloidal surfaces, director fields, and defects is found to show a number of unexpected features, such as knotting and linking of line defects, often uniquely arising from the nonpolar nature of the nematic director field. This review article highlights fascinating examples of new physical behavior arising from the interplay of nematic molecular order and both chiral symmetry and topology of colloidal inclusions within the nematic host. Furthermore, the article concludes with a brief discussion of how these findings may lay the groundwork for new types of topology-dictated self-assembly in soft condensed matter leading to novel mesostructured composite materials, as well as for experimental insights into the pure-math aspects of low-dimensional topology.

Fourier Ptychographic Coherent Anti-Stokes Raman Scattering Microscopy with Point-Scanning for Super-Resolution Imaging.

Fourier ptychography (FP) is a high resolution wide-field imaging method based on the extended aperture in the Fourier space, which is synthesized from raw images with varying illumination angles. If FP is extended to coherent nonlinear optical imaging, the resolution could be further improved due to the increase of the cutoff frequency of the synthesized coherent optical transfer function (C-OTF) with respect to the order of nonlinear optical processes. However, there is a fundamental conflict between wide-field FP and nonlinear optical imaging, whereby the nonlinear optical imaging typically requires a focused excitation laser beam with high power density. To tackle the problem, in this work, a unique point-scanning FP (PS-FP) method is presented for super-resolution nonlinear optical imaging, in which the nonlinear optical signal is obtained by using focused laser beam, while the conventional FP algorithm can still be used to retrieve the super-resolution image. PS-FP coherent anti-Stokes Raman scattering (PS-FP-CARS) imaging on a variety of samples, where a 1.8-fold expansion of the OTF is achieved experimentally for enhancing vibrational imaging. Further theoretical calculation shows that the C-OTF of PS-FP higher-order CARS (PS-FP-HO-CARS) can be expanded up to ≈4.9-fold, thereby improving the spatial resolution by ≈3-fold in comparison with conventional point-scanning CARS with under tightly focused beams. The generality of PS-FP method developed in this work can be adapted to other coherent nonlinear optical imaging modalities for super-resolution imaging in tissue and cells.

Nonlinear Optical Imaging Modalities in Biomedicine

Nonlinear Optical Imaging Modalities in Biomedicine

Shot-Noise Limited Nonlinear Optical Imaging Excited With GHz Femtosecond Pulses and Denoised by Deep-Learning.

Multiphoton fluorescence microscopy excited with femtosecond pulses at high repetition rates, particularly in the range of 100's MHz to GHz, offers an alternative solution to suppress photoinduced damage to biological samples, for example,photobleaching. Here, we demonstrate the use of a U-Net-based deep-learning algorithm for suppressing the inherentshot noise of the two-photon fluorescence images excited with GHz femtosecond pulses. With the trained denoising neural network, the image quality of the representative two-photon fluorescence images of the biological samples is shownto be significantly improved. Moreover, for input raw images with even SNR reduced to -4.76 dB, the trained denoising networkcanrecover the main image structure from noise floor with acceptable fidelity and spatial resolution. It is anticipatedthat the combination of GHz femtosecond pulses and deep-learning denoising algorithm can be a promising solution for eliminating the trade-off between photoinduced damage and image quality in nonlinear optical imaging platforms.

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.

Time-of-flight resolved stimulated Raman scattering microscopy using counter-propagating ultraslow Bessel light bullets generation

We present a novel time-of-flight resolved Bessel light bullet-enabled stimulated Raman scattering (B2-SRS) microscopy for deeper tissue 3D chemical imaging with high resolution without a need for mechanical z-scanning. To accomplish the tasks, we conceive a unique method to enable optical sectioning by generating the counter-propagating pump and Stokes Bessel light bullets in the sample, in which the group velocities of the Bessel light bullets are made ultraslow (e.g., vg ≈ 0.1c) and tunable by introducing programmable angular dispersions with a spatial light modulator. We theoretically analyze the working principle of the collinear multicolor Bessel light bullet generations and velocity controls with the relative time-of-flight resolved detection for SRS 3D deep tissue imaging. We have also built the B2-SRS imaging system and present the first demonstration of B2-SRS microscopy with Bessel light bullets for 3D chemical imaging in a variety of samples (e.g., polymer bead phantoms, biological samples such as spring onion tissue and porcine brain) with high resolution. The B2-SRS technique provides a > 2-fold improvement in imaging depth in porcine brain tissue compared to conventional SRS microscopy. The method of optical sectioning in tissue using counter-propagating ultraslow Bessel light bullets developed in B2-SRS is generic and easy to perform and can be readily extended to other nonlinear optical imaging modalities to advance 3D microscopic imaging in biological and biomedical systems and beyond.

Intraoperative tumor mapping using pyridine-carbazole based multifunctional fluorescent probes for precise resection and photodynamic therapeutics

Precise intraoperative tumor localization plays a pivotal role in the quest for refined cancer therapeutics. Addressing this, we have developed a series of novel pyridine-carbazole-based probes (Mito-Pyca1 ∼ Mito-Pyca4) featuring viscosity-enhanced fluorescence and multiphoton absorption properties. Among them, Mito-Pyca4 showcases significant promise for two-photon activated photodynamic therapy. When subjected to near-infrared irradiation, it reliably produces copious singlet oxygen, crucial for targeted tumor treatment. In vitro and in vivo experiments validated the capacity of Mito-Pyca4 to effectively induce cell apoptosis and impede tumor growth with minimal toxicity, positioning it as a potential candidate for photodynamic therapy. Furthermore, the specificity of Mito-Pyca4 for cancer cells was confirmed in co-culture experiments, and the probe was utilized for in vivo fluorescence-guided surgery. Successfully distinguishing tumor tissue from normal tissue, Mito-Pyca4 facilitates precise tumor resection. In summary, this comprehensive study establishes Mito-Pyca4 as a versatile tool for nonlinear optical imaging, viscosity sensing, and fluorescence-guided cancer surgery and treatment, providing surgeons an unparalleled advantage in discerning tumor margins. Through this research, we underscore the transformative potential of integrating chemical innovation with surgical and therapeutic techniques.

Variable-Temperature Non-Linear Optical Imaging Witnesses Change in Crystalline Rotor Dynamics at Phase Transition

Variable-Temperature Non-Linear Optical Imaging Witnesses Change in Crystalline Rotor Dynamics at Phase Transition

Noninvasive Nonlinear Optical Computational Histology.

Cancer remains a global health challenge, demanding early detection and accurate diagnosis for improved patient outcomes. An intelligent paradigm is introduced that elevates label-free nonlinear optical imaging with contrastive patch-wise learning, yielding stain-free nonlinear optical computational histology (NOCH). NOCH enables swift, precise diagnostic analysis of fresh tissues, reducing patient anxiety and healthcare costs. Nonlinear modalities are evaluated, including stimulated Raman scattering and multiphoton imaging, for their ability to enhance tumor microenvironment sensitivity, pathological analysis, and cancer examination. Quantitative analysis confirmed that NOCH images accurately reproduce nuclear morphometric features across different cancer stages. Key diagnostic features, such as nuclear morphology, size, and nuclear-cytoplasmic contrast, are well preserved. NOCH models also demonstrate promising generalization when applied to other pathological tissues. The study unites label-free nonlinear optical imaging with histopathology using contrastive learning to establish stain-free computational histology. NOCH provides a rapid, non-invasive, and precise approach to surgical pathology, holding immense potential for revolutionizing cancer diagnosis and surgical interventions.

Super‐Resolution Two‐Photon Fluorescence Tomography Through the Phase‐Shifted Optical Beatings of Bessel Beams for High‐Resolution Deeper Tissue 3D Imaging

AbstractA novel z‐scanning‐free epi‐detected super‐resolution two‐photon fluorescence tomography (TPFT) technique enabling super‐resolution deeper tissue 3D imaging is reported. To accomplish this, a unique method is conceived by generating the phase‐shifted optical beatings of Bessel beams (PS‐OB3) with a spatial light modulator (SLM) to break the diffraction limit for enhancing both the lateral and axial resolutions as well as improving the penetration depth in TPFT for super‐resolution deeper tissue imaging. By electronically varying the optical beating frequency and the phase shifts of the beating patterns through SLM, the depth‐resolved TPF signals about the volumetric tissue are encoded in the spatial frequency domain and hence, a series of depth‐resolved TPF images can be retrieved by implementing inverse fast Fourier transform without a need of mechanical depth‐scanning. PS‐OB3 TPFT provides ≈1.3‐ and 2‐fold improvements in lateral and axial resolutions in comparison with conventional point‐scan TPF imaging. It is also illustrated that the epi‐detected PS‐OB3 TPFT imaging with inherent scattering‐resilient properties of the Bessel beams employed gives over 2‐fold improvement in imaging depth in porcine brain tissue compared to conventional point‐scan Gaussian beam TPF imaging. The z‐scanning‐free optical sectioning ability of PS‐OB3 method developed in TPFT is universal, which can be readily extended to practically any other nonlinear optical imaging modalities for super‐resolution deeper 3D imaging in biological and biomedical tissues.

Exploring the effect of photobiomodulation and gamma visual stimulation induced by 808 nm and visible LED in Alzheimer's disease mouse model

Although photobiomodulation (PBM) and gamma visual stimulatqion (GVS) have been overwhelmingly explored in the recent time as a possible light stimulation (LS) means of Alzheimer's disease (AD) therapy, their effects have not been assessed at once. In our research, the AD mouse model was stimulated using light with various parameters [continuous wave (PBM) or 40 Hz pulsed visible LED (GVS) or 40 Hz pulsed 808 nm LED (PBM and GVS treatment)]]. The brain slices collected from the LS treated AD model mice were evaluated using (i) fluorescence microscopy to image thioflavine-S labeled amy-loid-β (Aβ) plaques (the main hallmark of AD), or (ii) two-photon excited fluorescence (TPEF) imaging of unlabeled Aβ plaques, showing that the amount of Aβ plaques was reduced after LS treatment. The imaging results correlated well with the results of Morris water maze (MWM) test, which demonstrated that the spatial learning and memory abilities of LS treated mice were noticeably higher than those of untreated mice. The LS effect was also assessed by in vivo nonlinear optical imaging, revealing that the cerebral amyloid angiopathy decreased spe-cifically as a result of 40 Hz pulsed 808 nm irradiation, on the contrary, the angiopathy reversed after visible 40 Hz pulsed light treatment. The obtained results provide useful reference for further optimization of the LS (PBM or GVS) parameters to achieve efficient phototherapy of AD.

Analysis of osteoporotic tissue using combination nonlinear optical imaging

Currently, a large number of stored tissue samples are unavailable for spectroscopic study without the time consuming and destructive process of paraffin removal. Instead, a structurally sensitive technique, sum frequency generation, and a chemically sensitive technique, coherent anti-Stokes Raman scattering enables imaging through the paraffin. This method is demonstrated by imaging collagen in mouse tibia. We introduce a statistical method for separating images by quality and, with the aid of machine learning, distinguish osteoporotic and healthy bone. This method has the potential to verify the results of previous studies and reduce new sample production by allowing retesting results with spectroscopy.

Automated Detection of Corneal Edema With Deep Learning-Assisted Second Harmonic Generation Microscopy

Second Harmonic Generation Microscopy (SHG) is widely acknowledged as a valuable non-linear optical imaging tool, its contrast mechanism providing the premises to non-invasively identify, characterize, and monitor changes in the collagen architecture of tissues. However, the interpretation of SHG data can pose difficulties even for experts histopathologists, which represents a bottleneck for the translation of SHG-based diagnostic frameworks to clinical settings. The use of artificial intelligence methods for automated SHG analysis is still in an early stage, with only few studies having been reported to date, none addressing ocular tissues yet. In this work we explore the use of three Deep Learning models, the highly popular InceptionV3 and ResNet50, alongside FLIMBA, a custom developed architecture, requiring no pre-training, to automatically detect corneal edema in SHG images of porcine cornea. We observe that Deep Learning models building on different architectures provide complementary results for the classification of cornea SHG images and demonstrate an AU-ROC = 0.98 for their joint use. These results have potential to be extrapolated to other diagnostics scenarios, such as automated extraction of hydration level of cornea, or identification of corneal edema causes, and thus pave the way for novel methods for precision diagnostics of the cornea with Deep-Learning assisted SHG imaging.

Insights into solid dosage forms with nonlinear optical imaging

Microscopic chemical and solid-state structures and their changes in solid drugs and dosage forms can profoundly affect pharmaceutical performance and patient safety. Despite this, their detailed spatially-resolved analysis can be difficult or impossible with established analytical technologies. Multimodal non-linear optical imaging represents opportunities for sensitive and specific chemical and solid-state pharmaceutical imaging. Non-linear optical imaging encompasses several nonlinear optical phenomena, including coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS), and sum frequency/second harmonic generation (SFG/SHG). Imaging in 3D with (sub)micron resolution is rapid, non-destructive, possible in situ in aqueous media, and generally does not require prior sample preparation. This mini-review explores several applications of non-linear optical imaging for solid drug and dosage form analysis.

Simultaneous 3D Construction and Imaging of Plant Cells Using Plasmonic Nanoprobe-Assisted Multimodal Nonlinear Optical Microscopy.

Nonlinear optical (NLO) imaging has emerged as a promising plant cell imaging technique due to its large optical penetration, inherent 3D spatial resolution, and reduced photodamage; exogenous nanoprobes are usually needed for nonsignal target cell analysis. Here, we report in vivo, simultaneous 3D labeling and imaging of potato cell structures using plasmonic nanoprobe-assisted multimodal NLO microscopy. Experimental results show that the complete cell structure can be imaged via the combination of second-harmonic generation (SHG) and two-photon luminescence (TPL) when noble metal silver or gold ions are added. In contrast, without the noble metal ion solution, no NLO signals from the cell wall were acquired. The mechanism can be attributed to noble metal nanoprobes with strong nonlinear optical responses formed along the cell walls via a femtosecond laser scan. During the SHG-TPL imaging process, noble metal ions that crossed the cell wall were rapidly reduced to plasmonic nanoparticles with the fs laser and selectively anchored onto both sides of the cell wall, thereby leading to simultaneous 3D labeling and imaging of the potato cells. Compared with the traditional labeling technique that needs in vitro nanoprobe fabrication and cell labeling, our approach allows for one-step, in vivo labeling of plant cells, thus providing a rapid, cost-effective method for cellular structure construction and imaging.

Imaging and component analysis of pumpkin stem tissue with simultaneous SF-CARS and TPEF microscopy.

A multimodal nonlinear optical imaging platform based on a single femtosecond oscillator is built for simultaneous TPEF and SF-CARS imaging. TPEF microscopy and SF-CARS microscopy is utilized for mapping the distribution of the lignin component and the polysaccharide component, respectively. Visualization of vessel structure is realized. And the relative distribution of lignin and polysaccharide of vessel structure is mapped. Two pumpkin stem tissue areas with different degrees of lignification are observed with simultaneous TPEF and SF-CARS imaging, and two types of cell walls are identified. The different distribution patterns of lignin and polysaccharide in these two types of cell walls, induced by different degrees of lignification, are analyzed in detail.

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.

Assessment of Cerebral Tumors and Metastases by Two-Photon Excitation Microscopy

Objective: The study subject approaches a topic of cerebral pathology, namely the development of a pathological diagnosis that is as accurate as possible for the cerebral tumors and the metastases, using a new method that complements the optical microscopic examination and immunohistochemistry to correctly diagnose cerebral malignancies. Methods: Two-photon excitation (TPE) microscopy techniques are non-linear optical imaging methods that are gaining momentum in the investigation of fixed tissue sections, fresh tissue, or even for in vivo experiments. This method can be used for offering complementary information on the tissue architecture. We argue that using non-linear optical microscopy (which includes TPE microscopy) images collected on this category of brain tissue samples, can facilitate the interpretation of brain tumors and metastases, thus it is a solution worthy to explore. Scanning large areas of the lesions is important to avoid false-negative diagnoses because lesions are often non-uniform. Finding potential invasion sites is also restricted when using a narrow field-of-view imaging method. Results: Our study demonstrated that TPE microscopy can detect specific cellular features of cerebral tumors and metastases in good correlation with histopathological results. This approach can improve the accuracy of the cerebral tumors diagnosis and possibly other neoplasms. Conclusion: TPE microscopy is very important for future research because it could prevent the false or inadequate diagnosis of specific lesions and differentiate a primary tumor from a metastasis. The significance of the findings is attributable to cross-disciplinary cooperation and the constant use of a working standard across all of the experiments in this study.

Topological solitonic macromolecules

Being ubiquitous, solitons have particle-like properties, exhibiting behaviour often associated with atoms. Bound solitons emulate dynamics of molecules, though solitonic analogues of polymeric materials have not been considered yet. Here we experimentally create and model soliton polymers, which we call “polyskyrmionomers”, built of atom-like individual solitons characterized by the topological invariant representing the skyrmion number. With the help of nonlinear optical imaging and numerical modelling based on minimizing the free energy, we reveal how topological point defects bind the solitonic quasi-atoms into polyskyrmionomers, featuring linear, branched, and other macromolecule-resembling architectures, as well as allowing for encoding data by spatial distributions of the skyrmion number. Application of oscillating electric fields activates diverse modes of locomotion and internal vibrations of these self-assembled soliton structures, which depend on symmetry of the solitonic macromolecules. Our findings suggest new designs of soliton meta matter, with a potential for the use in fundamental research and technology.

A Single-Cell Atlas of Small Airway Disease in Chronic Obstructive Pulmonary Disease: A Cross-Sectional Study.

Rationale: Emerging data demonstrate that the smallest conducting airways, terminal bronchioles, are the early site of tissue destruction in chronic obstructive pulmonary disease (COPD) and are reduced by as much as 41% by the time someone is diagnosed with mild (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 1) COPD. Objectives: To develop a single-cell atlas that describes the structural, cellular, and extracellular matrix alterations underlying terminal bronchiole loss in COPD. Methods: This cross-sectional study of 262 lung samples derived from 34 ex-smokers with normal lung function (n = 10) or GOLD stage 1 (n = 10), stage 2 (n = 8), or stage 4 (n = 6) COPD was performed to assess the morphology, extracellular matrix, single-cell atlas, and genes associated with terminal bronchiole reduction using stereology, micro-computed tomography, nonlinear optical microscopy, imaging mass spectrometry, and transcriptomics. Measurements and Main Results: The lumen area of terminal bronchioles progressively narrows with COPD severity as a result of the loss of elastin fibers within alveolar attachments, which was observed before microscopic emphysematous tissue destruction in GOLD stage 1 and 2 COPD. The single-cell atlas of terminal bronchioles in COPD demonstrated M1-like macrophages and neutrophils located within alveolar attachments and associated with the pathobiology of elastin fiber loss, whereas adaptive immune cells (naive, CD4, and CD8 T cells, and B cells) are associated with terminal bronchiole wall remodeling. Terminal bronchiole pathology was associated with the upregulation of genes involved in innate and adaptive immune responses, the interferon response, and the degranulation of neutrophils. Conclusions: This comprehensive single-cell atlas highlights terminal bronchiole alveolar attachments as the initial site of tissue destruction in centrilobular emphysema and an attractive target for disease modification.