Multimodal Nonlinear Optical Microscopy Research Articles

Review on Multimodal Nonlinear Optical Microscopy Imaging Technology

Review on Multimodal Nonlinear Optical Microscopy Imaging Technology

A multimodal nonlinear optical microscopy study of the responses of Pseudomonas aeruginosa to blue light and antibiotic treatment.

Pseudomonas aeruginosa (P. aeruginosa) is a multidrug-resistant human pathogen involved in numerous infections. Understanding the response of P. aeruginosa to various treatments is critical to developing new ways for the antimicrobial susceptibly test and more effective treatment methods. Conventional antimicrobial susceptibility tests lack molecular information at the single bacterium level. In this study, we used label-free multimodal nonlinear optical microscopy to identify an autofluorescence signal from pyoverdine, a siderophore of the bacteria, for quantification of P. aeruginosa responses to antibiotics and blue light treatment. We also discovered that the bleaching of the pyoverdine autofluorescence signals is correlated with the inactivation of P. aeruginosa and is perhaps one of the mechanisms involved in the blue light inactivation of P. aeruginosa.

Intravital microscopy of satellite cell dynamics and their interaction with myeloid cells during skeletal muscle regeneration.

Skeletal muscle regeneration requires the highly coordinated cooperation of muscle satellite cells (MuSCs) with other cellular components. Upon injury, myeloid cells populate the wound site, concomitant with MuSC activation. However, detailed analysis of MuSC-myeloid cell interaction is hindered by the lack of suitable live animal imaging technology. Here, we developed a dual-laser multimodal nonlinear optical microscope platform to study the dynamics of MuSCs and their interaction with nonmyogenic cells during muscle regeneration. Using three-dimensional time-lapse imaging on live reporter mice and taking advantages of the autofluorescence of reduced nicotinamide adenine dinucleotide (NADH), we studied the spatiotemporal interaction between nonmyogenic cells and muscle stem/progenitor cells during MuSC activation and proliferation. We discovered that their cell-cell contact was transient in nature. Moreover, MuSCs could activate with notably reduced infiltration of neutrophils and macrophages, and their proliferation, although dependent on macrophages, did not require constant contact with them. These findings provide a fresh perspective on myeloid cells' role during muscle regeneration.

In vivo label-free optical signatures of chemotherapy response in human pancreatic ductal adenocarcinoma patient-derived xenografts

Pancreatic cancer is a devastating disease often detected at later stages, necessitating swift and effective chemotherapy treatment. However, chemoresistance is common and its mechanisms are poorly understood. Here, label-free multi-modal nonlinear optical microscopy was applied to study microstructural and functional features of pancreatic tumors in vivo to monitor inter- and intra-tumor heterogeneity and treatment response. Patient-derived xenografts with human pancreatic ductal adenocarcinoma were implanted into mice and characterized over five weeks of intraperitoneal chemotherapy (FIRINOX or Gem/NabP) with known responsiveness/resistance. Resistant and responsive tumors exhibited a similar initial metabolic response, but by week 5 the resistant tumor deviated significantly from the responsive tumor, indicating that a representative response may take up to five weeks to appear. This biphasic metabolic response in a chemoresistant tumor reveals the possibility of intra-tumor spatiotemporal heterogeneity of drug responsiveness. These results, though limited by small sample size, suggest the possibility for further work characterizing chemoresistance mechanisms using nonlinear optical microscopy.

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.

Deep ensemble learning and transfer learning methods for classification of senescent cells from nonlinear optical microscopy images.

The success of chemotherapy and radiotherapy anti-cancer treatments can result in tumor suppression or senescence induction. Senescence was previously considered a favorable therapeutic outcome, until recent advancements in oncology research evidenced senescence as one of the culprits of cancer recurrence. Its detection requires multiple assays, and nonlinear optical (NLO) microscopy provides a solution for fast, non-invasive, and label-free detection of therapy-induced senescent cells. Here, we develop several deep learning architectures to perform binary classification between senescent and proliferating human cancer cells using NLO microscopy images and we compare their performances. As a result of our work, we demonstrate that the most performing approach is the one based on an ensemble classifier, that uses seven different pre-trained classification networks, taken from literature, with the addition of fully connected layers on top of their architectures. This approach achieves a classification accuracy of over 90%, showing the possibility of building an automatic, unbiased senescent cells image classifier starting from multimodal NLO microscopy data. Our results open the way to a deeper investigation of senescence classification via deep learning techniques with a potential application in clinical diagnosis.

The role of altered lipid composition and distribution in liver fibrosis revealed by multimodal nonlinear optical microscopy.

Intracellular lipid accumulation is commonly seen in fibrotic livers, but its exact role in liver fibrosis remains elusive. Here, we established a multimodal nonlinear optical microscopy to quantitatively map distribution of biomolecules in fibrotic livers. Our data revealed that unsaturated triglycerides were predominantly accumulated in central vein area during liver fibrosis but not in portal vein area. Moreover, the lipid homeostasis was remarkably dysregulated in the late-stage compared to the early-stage fibrosis, including increased unsaturated triglycerides with decreased lipid unsaturation degree and decreased membrane fluidity. Such alterations were likely due to up-regulated lipogenesis, desaturation, and peroxidation, which consequently led to endoplasmic reticulum stress and cell death. Inspiringly, injured hepatocyte could be rescued by remodeling lipid homeostasis via either supply of unsaturated fatty acids or enhancement of membrane fluidity. Collectively, our study improves current understanding of the role of lipid homeostasis in fibrosis and open opportunities for treatment.

Label-free multimodal non-linear optical imaging of three-dimensional cell cultures

3D cell cultures recapitulate tissue-specific diversity and better mimic the in vivo conditions compared to 2D cell cultures. Although confocal fluorescence microscopy is probably the most commonly used optical imaging technique to characterize 3D cell cultures, the limited imaging depth greatly hinders its application. Moreover, due to difficult diffusion of fluorescent probes into thick 3D cultures, the optical imaging techniques that rely on exogeneous fluorescent probes are not desirable. To address these problems, non-linear optical (NLO) microscopy uses near infrared light excitation to achieve significant larger imaging depth and gains molecular selectivity in a label-free manner. Taking advantages of 3D optical sectioning capability, submicron resolution, and high speed, label-free multimodal NLO microscopy has offered great promise for regenerative medicine, tumor microenvironment research, and drug discovery. This article overviews and discusses the latest applications of label-free multimodal NLO microscopy as an emerging platform to facilitate 3D cell culture research.

Label-free multimodal nonlinear optical microscopy reveals features of bone composition in pathophysiological conditions.

Bone tissue features a complex microarchitecture and biomolecular composition, which determine biomechanical properties. In addition to state-of-the-art technologies, innovative optical approaches allowing the characterization of the bone in native, label-free conditions can provide new, multi-level insight into this inherently challenging tissue. Here, we exploited multimodal nonlinear optical (NLO) microscopy, including co-registered stimulated Raman scattering, two-photon excited fluorescence, and second-harmonic generation, to image entire vertebrae of murine spine sections. The quantitative nature of these nonlinear interactions allowed us to extract accurate biochemical, morphological, and topological information on the bone tissue and to highlight differences between normal and pathologic samples. Indeed, in a murine model showing bone loss, we observed increased collagen and lipid content as compared to the wild type, along with a decreased craniocaudal alignment of bone collagen fibres. We propose that NLO microscopy can be implemented in standard histopathological analysis of bone in preclinical studies, with the ambitious future perspective to introduce this technique in the clinical practice for the analysis of larger tissue sections.

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

Accurate evaluation of the treatment effects of immunotherapy on subcutaneous ovarian cancer in mice with nonlinear optical imaging and algorithmic analysis.

Immunotherapy and its evaluation have shown great promise for cancer treatment. Here, a mouse subcutaneous transplantable tumor model was applied to testing therapeutic strategies. The mouse model was treated by regulating anti-PD-L1, anti-CTLA-4, cisplatin and their combined therapy. Biochemistry experiments have found that after immunotherapy, mice have more immune responses, which were manifested by an increase in the content of growth factors and the activation of T cells. Meanwhile, multimodal nonlinear optical microscopy imaging combined with algorithms was used to evaluate the treatment's effectiveness. By detecting the metabolism rate and microstructure in tissue, it was proved that combined therapies including immune checkpoint inhibitors do have a better effect on ovarian tumors. Our discovery of valid treatments for mice with ovarian tumor and provides an evaluation tool via nonlinear optics combined with algorithms offers new insights into therapeutic effect.

Simultaneous label-free autofluorescence-multiharmonic microscopy driven by femtosecond sources based on self-phase modulation enabled spectral selection

Nonlinear optical microscopy technique has unique advantages in tissue imaging, such as enhanced contrast, high resolution, and label-free deep optical sectioning capabilities. Nonlinear optical microscopy also has multiple imaging modalities, corresponding to various components in biological tissues. Unfortunately, its wide applications are hindered due to the lack of broadly tunable femtosecond sources designed for driving multimodalities simultaneously. To solve this challenge, we propose a new wavelength conversion approach—self-phase modulation (SPM) enabled spectral selection, dubbed as SESS. The SESS employs SPM to broaden the input spectrum in a short fiber, and the broadened spectrum features well-isolated spectral lobes. Using the suitable optical filters to select the outermost spectral lobes produces nearly transform-limited femtosecond pulses. In this work, we demonstrate a fiber-optic SESS source for multimodal nonlinear optical microscopy. Based on a 43-MHz Yb-fiber laser, this SESS source can emit 990-nm, 84-fs pulses with >5-nJ energy and ~84-fs pulse duration; it can also produce 1110-nm, 48-fs pulses with 15-nJ energy. The 990-nm pulses are used to drive two-photon excitation fluorescence of many important fluorophores and second-harmonic generation microscopy, which, combined with image splicing technology, enables us to obtain a large field of view image of the gastric tissue. We also employ the 1110-nm pulses to drive simultaneous label-free autofluorescence-multiharmonic microscopy for multimodal imaging of gastric tissue. Two-photon excitation fluorescence, three-photon excitation fluorescence, second-harmonic generation and third-harmonic generation signals of gastric tissue are simultaneously excited efficiently. Such a multimodal nonlinear optical microscopy driven by SESS sources becomes a powerful tool in biomedical imaging.

Differential characterization of lumbar spine associated tissue histology with nonlinear optical microscopy.

Percutaneous endoscopic lumbar discectomy (PELD) is the major effective treatment for lumbar disc herniation, and rapid histological identification of dissected tissue is critical to guide the discectomy. In this work, we revealed the histological features of different types of peridural tissues of the lumbar spine by label-free multi-modal nonlinear optical microscopy. Stimulated Raman scattering (SRS) was used to extract lipid and protein distributions, while second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) signals were applied to image the collagen and elastin fibers at the same time. Our results demonstrated that the nonlinear optical features of the dura and adjacent soft tissues were significantly different, showing the potentials of our method for intraoperative differentiation of these critical tissues and improving the surgical outcome of PELD.

Label-free multimodal nonlinear optical microscopy for biomedical applications

This paper addresses the application of multimodal nonlinear optical (MNLO) microscopy to clinical research within the context of label-free non-invasive molecular imaging. Here, a compact MNLO microscope based on a laser scanning microscope, a femtosecond laser, a time-correlated single-photon counting system, and a photonic crystal fiber are introduced for biomedical applications. By integrating two-photon fluorescence, two-photon fluorescence lifetime imaging, second-harmonic generation, and coherent anti-Stokes Raman scattering microscopy, the proposed scheme provides profound insights into the physicochemical properties related to 3D molecular orientation distribution, inter- and intra-molecular interactions, and disease progression in biological systems and organs. The high peak power and the low average intensity of near-infrared laser pulses allow for deep-penetration imaging without compromising sample vitality. Linking nonlinear optical phenomena with time/spectral/polarization-resolved imaging also makes it possible to obtain multidimensional information to address complex biomedical questions.

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.

Visualizing intra-medulla lipids in human hair using ultra-multiplex CARS, SHG, and THG microscopy.

We performed label-free imaging of human-hair medulla using multi-modal nonlinear optical microscopy. Intra-medulla lipids (IMLs) were clearly visualized by ultra-multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging. Two groups of IMLs were found: second harmonic generation (SHG) active and inactive. By combining SHG analysis with CARS, the two groups were identified as free fatty acids and wax esters, respectively.

Multimodal nonlinear optical imaging of Caenorhabditis elegans with multiplex coherent anti-Stokes Raman scattering, third-harmonic generation, second-harmonic generation, and two-photon excitation fluorescence

Multimodal nonlinear optical microscopy was applied to the nematode Caenorhabditis elegans. Transgenic worms expressing the yellow fluorescent protein variant Venus underwent analyses. The lipid and protein distribution was clearly visualized using multiplex coherent anti-Stokes Raman scattering, second-harmonic generation (SHG), third-harmonic generation, and two-photon excitation fluorescence (TPEF). The TPEF and SHG signals were found to overlap, which allowed the identification of the ventral nerve cord. These findings demonstrate the powerful potential of multimodal nonlinear optical microscopy to reveal C. elegans microstructures in vivo.

Label-Free Non-linear Multimodal Optical Microscopy—Basics, Development, and Applications

Nonlinear optical (NLO) microscopy has proven to be a powerful tool especially for tissue imaging with sub-cellular resolution, high penetration depth, endogenous contrast specificity, pinhole-less optical sectioning capability. In this review, we discuss label-free nonlinear optical microscopes including the two-photon fluorescence (TPF), fluorescence lifetime imaging microscopy (FLIM), polarization-resolved second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) techniques with various samples. The nonlinear signals are generated from collagen in tissue (SHG), amylopectin from starch granules (SHG), sarcomere structure of fresh muscle (SHG), elastin in skin (TPF), nicotinamide adenine dinucleotide (NADH) in cells (TPF) and lipid droplets in cells (CARS). Again, the nonlinear signals are very specific to the molecular structure of the sample and its relative orientation to the polarization of the incident light. Thus, polarization-resolved nonlinear optical microscopy provides high image contrast and quantitative estimate of sample orientation. An overview of the advancements on polarization-resolved SHG microscopy including Stokes vector based polarimetry, circular dichroism, and susceptibility are also presented in this review article. The working principles and corresponding implements of above-mentioned microscopy techniques are elucidated. The potential of time-resolved TPF lifetime imaging microscopy (TP-FLIM) is explored by imaging endogenous fluorescence of NAD(P)H, a key coenzyme in cellular metabolic processes. We also discuss single laser source time-resolved multimodal CARS-FLIM microscopy using time-correlated single-photon counting (TCSPC) in combination with continuum generation from photonic crystal fiber (PCF). Using examples, we demonstrate that the multimodal NLO microscopy is a powerful tool to assess the molecular specificity with high resolution.

Aggregation-Induced Nonlinear Optical Effects of AIEgen Nanocrystals for Ultradeep In Vivo Bioimaging.

Nonlinear optical microscopy has become a powerful tool in bioimaging research due to its unique capabilities of deep optical sectioning, high-spatial-resolution imaging, and 3D reconstruction of biological specimens. Developing organic fluorescent probes with strong nonlinear optical effects, in particular third-harmonic generation (THG), is promising for exploiting nonlinear microscopic imaging for biomedical applications. Herein, a simple method for preparing organic nanocrystals based on an aggregation-induced emission (AIE) luminogen (DCCN) with bright near-infrared emission is successfully demonstrated. Aggregation-induced nonlinear optical effects, including two-photon fluorescence (2PF), three-photon fluorescence (3PF), and THG, of DCCN are observed in nanoparticles, especially for crystalline nanoparticles. The nanocrystals of DCCN are successfully applied for 2PF microscopy at 1040 nm NIR-II excitation and THG microscopy at 1560 nm NIR-II excitation, respectively, to reconstruct the 3D vasculature of the mouse cerebral vasculature. Impressively, the THG microscopy provides much higher spatial resolution and brightness than the 2PF microscopy and can visualize small vessels with diameters of ≈2.7 µm at the deepest depth of 800 µm in a mouse brain. Thus, this is expected to inspire new insights into the development of advanced AIE materials with multiple nonlinearity, in particular THG, for multimodal nonlinear optical microscopy.

Observation of Cell Division in a Fertilized Egg of a Zebrafish by Using a Multimodal Nonlinear Optical Microscope

We have developed a high-speed and high-resolution multimodal nonlinear optical microscope, which can acquire in vivo label and label-free 3D images. Using this microscope, we could successfully observe dynamic images of the zebrafish heartbeat with 33-millisecond intervals without staining process, indicating 1.68 beats per second. In addition, we show that the label-free, high-speed, real-time images can be obtained during somatic cell divisions in the zebrafish’s embryo.