Third Harmonic Generation Imaging Research Articles

Self-Supervised Image Denoising of Third Harmonic Generation Microscopic Images of Human Glioma Tissue by Transformer-based Blind Spot (TBS) Network.

Third harmonic generation (THG) microscopy shows great potential for instant pathology of brain tumor tissue during surgery. However, due to the maximal permitted exposure of laser intensity and inherent noise of the imaging system, the noise level of THG images is relatively high, which affects subsequent feature extraction analysis. Denoising THG images is challenging for modern deep-learning based methods because of the rich morphologies contained and the difficulty in obtaining the noise-free counterparts. To address this, in this work, we propose an unsupervised deep-learning network for denoising of THG images which combines a self-supervised blind spot method and a U-shape Transformer using a dynamic sparse attention mechanism. The experimental results on THG images of human glioma tissue show that our approach exhibits superior denoising performance qualitatively and quantitatively compared with previous methods. Our model achieves an improvement of 2.47-9.50 dB in SNR and 0.37-7.40 dB in CNR, compared to six recent state-of-the-art unsupervised learning models including Neighbor2Neighbor, Blind2Unblind, Self2Self+, ZS-N2N, Noise2Info and SDAP. To achieve an objective evaluation of our model, we also validate our model on public datasets including natural and microscopic images, and our model shows a better denoising performance than several recent unsupervised models such as Neighbor2Neighbor, Blind2Unblind and ZS-N2N. In addition, our model is nearly instant in denoising a THG image, which has the potential for real-time applications of THG microscopy.

Extreme Polarization Anisotropy in Resonant Third-Harmonic Generation from Aligned Carbon Nanotube Films.

Carbon nanotubes (CNTs) possess extremely anisotropic electronic, thermal, and optical properties owing to their 1D character. While their linear optical properties have been extensively studied, nonlinear optical processes, such as harmonic generation for frequency conversion, remain largely unexplored in CNTs, particularly in macroscopic CNT assemblies. In this work, macroscopic films of aligned and type-separated (semiconducting and metallic) CNTs are synthesized and polarization-dependent third-harmonic generation (THG) from the films with fundamental wavelengths ranging from 1.5 to 2.5µm is studied. Both films exhibited strongly wavelength-dependent, intense THG signals, enhanced through exciton resonances, and third-order nonlinear optical susceptibilities of 2.50×10-19 m2 V-2 (semiconducting CNTs) and 1.23×10-19 m2 V-2 (metallic CNTs), respectively are found, for 1.8µm excitation. Further, through systematic polarization-dependent THG measurements, the values of all elements of the susceptibility tensor are determined, verifying the macroscopically 1D nature of the films. Finally, polarized THG imaging is performed to demonstrate the nonlinear anisotropy in the large-size CNT film with good alignment. These findings promise applications of aligned CNT films in mid-infrared frequency conversion, nonlinear optical switching, polarized pulsed lasers, polarized long-wave detection, and high-performance anisotropic nonlinear photonic devices.

Resolving arteriolar wall structures in mouse brain in vivo with three-photon microscopy.

The brain arteriolar wall is a multilayered structure, whose integrity is of key significance to the brain function. However, resolving these different layers in anmial models in vivo is hampered by the lack of either labeling or imaging technology. Here, we demonstrate that three-photon microscopy (3PM) is an ideal solution. In mouse brain in vivo, excited at the 1700-nm window, label-free third-harmonic generation imaging and three-photon fluorescence (3PF) imaging with Alexa 633 labeling colocalize and resolve the internal elastic lamina. Furthermore, Alexa Fluor 594-conjugated Wheat Germ Agglutinin (WGA-594) shows time-dependent labeling behavior. As time lapses, WGA-594 first labels endothelium, and then vascular smooth muscle cells, which are readily captured and resolved with 3PF imaging. Our results show that 3PM, in combination with proper labeling, is a promising technology for investigating the structures of brain arteriolar wall in vivo.

Deep-skin third-harmonic generation (THG) imagingin vivoexcited at the 2200 nm window

The skin is heterogeneous and exerts strong scattering and aberration onto excitation light in multiphoton microscopy (MPM). Shifting to longer excitation wavelengths may help reduce skin scattering and aberration, potentially enabling larger imaging depths. However, previous demonstrations of skin MPM employ excitation wavelengths only up to the 1700[Formula: see text]nm window, leaving an open question as to whether longer excitation wavelengths are suitable for deep-skin MPM. Here, in order to explore the longer-wavelength territory, first, we demonstrate characterization of the broadband transmittance of excised mouse skin, revealing a high transmittance window at 2200[Formula: see text]nm. Then, we demonstrate third-harmonic generation (THG) imaging in mouse skin in vivo excited at this window. With 9[Formula: see text]mW optical power on the skin surface operating at 1[Formula: see text]MHz repetition rate, we can get THG signals of 250[Formula: see text][Formula: see text]m below the skin surface. Comparative THG imaging excited at the 1700[Formula: see text]nm window shows that as imaging depth increases, THG signals decay even faster than those excited at 2200[Formula: see text]nm. Our results thus uncover the 2200[Formula: see text]nm window as a new, promising excitation window potential for deep-skin MPM.

Application of a Deep-Learning Technique to Non-Linear Images From Human Tissue Biopsies for Shedding New Light on Breast Cancer Diagnosis.

The development of label-free non-destructive techniques to be used as diagnostic tools in cancer research is of great importance for improving the quality of life for millions of patients. Previous studies have demonstrated that Third Harmonic Generation (THG) imaging could differentiate malignant from benign unlabeled human breast biopsies and distinguish the different grades of cancer. Towards the application of such technologies to clinic, in the present report, a deep learning technique was applied to THG images recorded from breast cancer tissues of grades 0, I, II, and III. By the implementation of a convolutional neural network (CNN) model, the differentiation of malignant from benign breast tissue samples and the discrimination of the different grades of cancer in a fast and accurate way were achieved. The obtained results provide a step ahead towards the use of optical diagnostic tools in conjunction with the CNN image classifier for the reliable and rapid malignancy diagnosis in clinic.

Spectroscopic second and third harmonic generation microscopy using a femtosecond laser source in the third near-infrared (NIR-III) optical window.

In this study, second harmonic generation (SHG) and third harmonic generation (THG) spectroscopic imaging were performed on biological samples using a femtosecond laser source in the third near-infrared (NIR) optical window (NIR-III). Using a visible-NIR spectrometer, the SHG and THG signals were simultaneously detected and were extracted using spectral analysis. Visualization of biological samples such as cultured cells (HEK293 T), mouse brain slices, and the nematode Caenorhabditis elegans was performed in a label-free manner. In particular, in an SHG image of an entire coronal brain section (8 × 6 mm2), we observed mesh-like and filamentous structures in the arachnoid mater and wall of the cerebral ventricle, probably corresponding to the collagen fibers, cilia, and rootlet. Moreover, the THG images clearly depicted the densely packed axons in the white matter and cell nuclei at the cortex of the mouse brain slice sample and lipid-rich granules such as lipid droplets inside the nematode. The observations and conclusions drawn from this technique confirm that it can be utilized for various biological applications, including in vivo label-free imaging of living animals.

3-photon fluorescence and third-harmonic generation imaging of myelin sheaths in mouse digital skin in vivo: A comparative study

Myelin sheaths wrapping axons are key structures that help maintain the propagation speed of action potentials in both central and peripheral nervous systems (CNS and PNS). However, noninvasive, deep imaging technologies visualizing myelin sheaths in the digital skin in vivo are lacking in animal models. 3-photon fluorescence (3PF) imaging excited at the 1700-nm window enables deep imaging of myelin sheaths, but necessitates labeling by exogenous fluorescent dyes. Since myelin sheaths are lipid-rich structures which generate strong third-harmonic signals, in this paper, we perform a detailed comparative experimental study of both third-harmonic generation (THG) and 3PF imaging in the mouse digital skin in vivo. Our results show that THG imaging also enables visualization of myelin sheaths deep in the mouse digital skin, which shows colocalization with 3PF signals from labeled myelin sheaths. Besides its superior label-free advantage, THG does not suffer from photobleaching due to its 3PF property.

Deep-skin multiphoton microscopy of lymphatic vessels excited at the 1700-nm window in vivo.

Visualization of lymphatic vessels is key to the understanding of their structure, function, and dynamics. Multiphoton microscopy (MPM) is a potential technology for imaging lymphatic vessels, but tissue scattering prevents its deep penetration in skin. Here we demonstrate deep-skin MPM of the lymphatic vessels in mouse hindlimb in vivo, excited at the 1700 nm window. Our results show that with contrast provided by indocyanine green (ICG), 2-photon fluorescence (2PF) imaging enables noninvasive imaging of lymphatic vessels 300 μm below the skin surface, visualizing both its structure and contraction dynamics. Simultaneously acquired second-harmonic generation (SHG) and third-harmonic generation (THG) images visualize the local environment in which the lymphatic vessels reside. After removing the surface skin layer, 2PF and THG imaging visualize finer structures of the lymphatic vessels: most notably, the label-free THG imaging visualizes lymphatic valves and their open-and-close dynamics in real time. MPM excited at the 1700-nm window thus provides a promising technology for the study of lymphatic vessels.

Investigating the optical clearing effects of 50% glycerol in ex vivo human skin by harmonic generation microscopy

Imaging depth and quality of optical microscopy can be enhanced by optical clearing. Here we investigate the optical clearing of the ex vivo human skin by 50% glycerol topical application, which is allowed for cosmetic usage. Harmonic generation microscopy, by combining second and third harmonic generation (THG) modalities, was utilized to examine the clearing effect. The THG image intensity is sensitive to the improved optical homogeneity after optical clearing, and the second harmonic generation (SHG) image intensity in the dermis could serve as a beacon to confirm the reduction of the scattering in the epidermis layer. As a result, our study supports the OC effect through 50% glycerol topical application. Our study further indicates the critical role of stratum corneum shrinkage for the observed SHG and THG signal recovery.

Quantitative third-harmonic generation imaging of mouse visual cortex areas reveals correlations between functional maps and structural substrates.

The structure of brain regions is assumed to correlate with their function, but there are very few instances in which the relationship has been demonstrated in the live brain. This is due to the difficulty of simultaneously measuring functional and structural properties of brain areas, particularly at cellular resolution. Here, we performed label-free, third-harmonic generation (THG) microscopy to obtain a key structural signature of cortical areas, their effective attenuation lengths (EAL), in the vertical columns of functionally defined primary visual cortex and five adjacent visual areas in awake mice. EALs measured by THG microscopy in the cortex and white matter showed remarkable correspondence with the functional retinotopic sign map of each area. Structural features such as cytoarchitecture, myeloarchitecture and blood vessel architecture were correlated with areal EAL values, suggesting that EAL is a function of these structural features as an optical property of these areas. These results demonstrate for the first time a strong relationship between structural substrates of visual cortical areas and their functional representation maps in vivo. This study may also help in understanding the coupling between structure and function in other animal models as well as in humans.

Nonlinear Optics with Near-Infrared Excitation Enable Real-Time Quantitative Diagnosis of Human Cervical Cancers.

Histopathologic analysis through biopsy has been one of the most useful methods for the assessment of malignant neoplasms. However, some aspects of the analysis such as invasiveness, evaluation range, and turnaround time from biopsy to report could be improved. Here, we report a novel method for visualizing human cervical tissue three-dimensionally, without biopsy, fixation, or staining, and with sufficient quality for histologic diagnosis. Near-infrared excitation and nonlinear optics were employed to visualize unstained human epithelial tissues of the cervix uteri by constructing images with third-harmonic generation (THG) and second-harmonic generation (SHG). THG images enabled evaluation of nuclear morphology in a quantitative manner with six parameters after image analysis using deep learning. It was also possible to quantitatively assess intraepithelial fibrotic changes based on SHG images and another deep learning analysis. Using each analytical procedure alone, normal and cancerous tissue were classified quantitatively with an AUC ≥0.92. Moreover, a combinatory analysis of THG and SHG images with a machine learning algorithm allowed accurate classification of three-dimensional image files of normal tissue, intraepithelial neoplasia, and invasive carcinoma with a weighted kappa coefficient of 0.86. Our method enables real-time noninvasive diagnosis of cervical lesions, thus constituting a potential tool to dramatically change early detection. SIGNIFICANCE: This study proposes a novel method for diagnosing cancer using nonlinear optics, which enables visualization of histologic features of living tissues without the need for any biopsy or staining dye.

Third Harmonic Generation microscopy distinguishes malignant cell grade in human breast tissue biopsies

The ability to distinguish and grade malignant cells during surgical procedures in a fast, non-invasive and staining-free manner is of high importance in tumor management. To this extend, Third Harmonic Generation (THG), Second Harmonic Generation (SHG) and Fourier-Transform Infrared (FTIR) spectroscopy were applied to discriminate malignant from healthy cells in human breast tissue biopsies. Indeed, integration of non-linear processes into a single, unified microscopy platform offered complementary structural information within individual cells at the submicron level. Using a single laser beam, label-free THG imaging techniques provided important morphological information as to the mean nuclear and cytoplasmic area, cell volume and tissue intensity, which upon quantification could not only distinguish cancerous from benign breast tissues but also define disease severity. Simultaneously, collagen fibers that could be detected by SHG imaging showed a well structured continuity in benign tumor tissues, which were gradually disoriented along with disease severity. Combination of THG imaging with FTIR spectroscopy could provide a clearer distinction among the different grades of breast cancer, since FTIR analysis showed increased lipid concentrations in malignant tissues. Thus, the use of non-linear optical microscopy can be considered as powerful and harmless tool for tumor cell diagnostics even during real time surgery procedures.

In vivo deep-brain blood flow speed measurement through third-harmonic generation imaging excited at the 1700-nm window.

Measurement of the hemodynamic physical parameter blood flow speed in the brain in vivo is key to understanding brain physiology and pathology. 2-photon fluorescence microscopy with single blood vessel resolution is typically used, which necessitates injection of toxic fluorescent dyes. Here we demonstrate a label-free nonlinear optical technique, third-harmonic generation microscopy excited at the 1700-nm window, that is promising for such measurement. Using a simple femtosecond laser system based on soliton self-frequency shift, we can measure blood flow speed through the whole cortical grey matter, even down to the white matter layer. Together with 3-photon fluorescence microscopy, we further demonstrate that the blood vessel walls generate strong THG signals, and that plasma and circulating blood cells are mutually exclusive in space. This technique can be readily applied to brain research.

Miniaturized multimodal multiphoton microscope for simultaneous two-photon and three-photon imaging with a dual-wavelength Er-doped fiber laser.

A multimodal multiphoton microscopy (MPM) is developed to acquire both two-photon microscopy (2PM) and three-photon microscopy (3PM) signals. A dual-wavelength Er-doped fiber laser is used as the light source, which provides the fundamental pulse at 1580 nm to excite third harmonic generation (THG) and the frequency-doubled pulse at 790 nm to excite intrinsic two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG). Due to their different contrast mechanisms, the TPEF, SHG, and THG images can acquire complementary information about tissues, including cells, collagen fibers, lipids, and interfaces, all label-free. The compact MPM imaging probe is developed using miniature objective lens and a micro-electro-mechanical scanner. Furthermore, the femtosecond laser pulses are delivered by a single mode fiber and the signals are collected by a multimode fiber, which makes the miniaturized MPM directly fiber-coupled, compact, and portable. Design considerations on using the dual excitation wavelengths are discussed. Multimodal and label-free imaging by TPEF, SHG, and THG are demonstrated on biological samples. The miniaturized multimodal MPM is shown to have great potential for label-free imaging of thick and live tissues.

Third-order nonlinear optical properties of phycobiliproteins from cyanobacteria and red algae

Second hyperpolarizability (γ) measurements as well as third harmonic generation (THG) imaging was performed to determine if phycobiliproteins contribute to cyanobacteria THG, and to establish if phycobiliproteins are suitable dyes for THG microscopy. Three phycobiliproteins and one phycobilin were investigated via THG ratio γ measurements, revealing phycocyanobilin has ~7× higher γ than β-carotene, a standard THG dye. Phycobiliproteins also had large γ values, showing they are promising THG dyes. Stoichiometric summations of phycobilin γ values revealed they are the dominant source of THG signal from phycobiliproteins. THG imaging was performed to demonstrate phycobiliproteins and phycobilins can be useful THG dyes.

A Method to Measure Myeloarchitecture of the Murine Cerebral Cortex in vivo and ex vivo by Intrinsic Third-Harmonic Generation.

A new label-free method is presented for measuring myeloarchitecture of the murine cerebral cortex in vivo and ex vivo. Growing evidence suggests that cortical myelination plays significant roles in neuronal plasticity and pathologies, such as multiple sclerosis (MS), but illuminating the mechanism requires longitudinal imaging of the same brains. Here we demonstrate imaging unlabeled myelinated fibers in a live mouse brain by third-harmonic generation (THG). Contrary to other label-free microscopies based on reflectance, fibers of all orientations could be visualized, i.e., radial and tangential to the pia, which is suitable for revealing the three-dimensional connectivity. The depth of THG imaging in an intact brain was approximately 200 μm, so the network of myelinated fibers could be captured into layers 2/3 in vivo. THG provides a novel base for reconstruction of morphology. Semi-automatic tracing of THG-positive axons unraveled the depth-dependent distribution of the myelin lattice. Finally, a unique light property of THG was exploited for the estimation of the g-ratio. The demonstrated THG morphometry of the length density, orientation, and sheath thickness of cortical myelin could be useful for elucidating its function and how it is modulated during learning and disease.

Stain-free subcellular-resolution astrocyte imaging using third-harmonic generation

We demonstrate stain-free, high-contrast, subcellular-resolution imaging of astroglial cells using epi-detected third-harmonic generation (THG). The astrocyte-imaging capability of THG is verified by colocalizing THG images with fluorescence images of astrocytes expressing a genetically encodable fluorescent reporter. We show that THG imaging with an optimized point-spread function can reliably detect significant subcellular features of astrocytes, including cell nuclei, as well as the soma shape and boundaries.

Quantitative Third Harmonic Generation Microscopy for Assessment of Glioma in Human Brain Tissue.

Distinguishing tumors from normal brain cells is important but challenging in glioma surgery due to the lack of clear interfaces between the two. The ability of label‐free third harmonic generation (THG) microscopy in combination with automated image analysis to quantitatively detect glioma infiltration in fresh, unprocessed tissue in real time is assessed. The THG images reveal increased cellularity in grades II–IV glioma samples from 23 patients, as confirmed by subsequent hematoxylin and eosin histology. An automated image quantification workflow is presented for quantitative assessment of the imaged cellularity as a reflection of the degree of glioma invasion. The cellularity is validated in three ways: 1) Quantitative comparison of THG imaging with fluorescence microscopy of nucleus‐stained samples demonstrates that THG reflects the true tissue cellularity. 2) Thresholding of THG cellularity differentiates normal brain from glioma infiltration, with 96.6% sensitivity and 95.5% specificity, in nearly perfect (93%) agreement with pathologists. 3) In one patient, a good correlation between THG cellularity and preoperative magnetic resonance and positron emission tomography imaging is demonstrated. In conclusion, quantitative real‐time THG microscopy accurately assesses glioma infiltration in ex vivo human brain samples, and therefore holds strong potential for improving the accuracy of surgical resection.

Deep‐brain three‐photon microscopy excited at 1600 nm with silicone oil immersion

Three-photon microscopy excited at the 1700-nm window (roughly covering 1600-1840 nm) is especially suitable for deep-brain imaging in living animals. To match the brain refractive index, D2 O has been exclusively used as the immersion medium. However, the hygroscopic property of D2 O leads to a decrease of transmittance of the excitation light and as a result a decrease in three-photon signals over time. Solutions such as replacing D2 O from time to time, wrapping both the objective lens and the immersion D2 O, and sealing D2 O with paraffin liquid have all been demonstrated, which add to the system complexity. Based on our recent characterization of immersion oils, we propose using silicone oil as a potential alternative to D2 O for deep-brain imaging. Excited at 1600 nm, our comparative deep-brain imaging using both D2 O and silicone oil immersion show that silicone oil immersion yields 17% higher three-photon signal in third-harmonic generation imaging within the white matter. Besides, silicone oil immersion also enables three-photon fluorescence imaging of vasculature up to 1460 μm (mechanical depth) into the mouse brain in vivo acquired at 2 seconds/frame. Together with the nonhygroscopic physical property, silicone oil is promising for long-span three-photon brain imaging excited at the 1700-nm window.

Third Harmonic Generation Imaging using Common Histological Dyes

Histology has long relied on optical microscopy of tissue stained with colorful absorptive dyes, which enables easy visual differentiation between cell nuclei and various extracellular structures. Histological stains are presently the gold standard in diagnoses by medical pathologists. While these stains provide an effective way to visualize tissue components, they still require thin sectioning which results in only two dimensional data that cannot be easily stitched in three dimensions. As tissue inherently exists as a three dimensional structure, it is important to develop histological methods to investigate it in such form while maintaining the high spatial resolution of light microscopy. Third harmonic generation (THG) imaging is a nonlinear microscopy technique that is gaining attention in the biomedical community. In this technique, three incident photons are combined into one by light-matter interactions with the sample, resulting in high contrast and inherent three dimensional optical sectioning. While in practice THG only typically occurs at optical heterogeneities and interfaces within the biological sample, some materials present enhanced THG emission due to resonance effects. These materials have also been called harmoniphores. Hematoxylin (H), a dye that is often used in combination with eosin (E) as part of the H&E stain, has previously been reported as an effective harmoniphore for distinguishing cell nuclei by 3D THG imaging. In this work, we apply trans THG imaging to skin and liver sections to show that in addition to H&E, other commonly used histological dyes in elastic and collagen staining can serve as effective THG harmoniphores. We find that elastic fibers can be easily visualized by THG microscopy using Verhoeff Van Gieson (VVG) staining while collagen is directly highlighted by THG imaging of picrosirius red staining. By combining THG imaging with common histological dyes, 3D histological imaging could be achieved.