Third Harmonic Generation Microscopy 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.

Synthetic spatial aperture holographic third harmonic generation microscopy

Third harmonic generation (THG) provides a valuable, label-free approach to imaging biological systems. To date, THG microscopy has been performed using point-scanning methods that rely on intensity measurements lacking phase information of the complex field. We report the first demonstration, to the best of our knowledge, of THG holographic microscopy and the reconstruction of the complex THG signal field with spatial synthetic aperture imaging. Phase distortions arising from measurement-to-measurement fluctuations and imaging components cause optical aberrations in the reconstructed THG field. We have developed an aberration-correction algorithm that estimates and corrects these phase distortions to reconstruct the spatial synthetic aperture THG field without optical aberrations.

Label-free, whole-brain in vivo mapping in an adult vertebrate with third harmonic generation microscopy.

Comprehensive understanding of interconnected networks within the brain requires access to high resolution information within large field of views and over time. Currently, methods that enable mapping structural changes of the entire brain in vivo are extremely limited. Third harmonic generation (THG) can resolve myelinated structures, blood vessels, and cell bodies throughout the brain without the need for any exogenous labeling. Together with deep penetration of long wavelengths, this enables in vivo brain-mapping of large fractions of the brain in small animals and over time. Here, we demonstrate that THG microscopy allows non-invasive label-free mapping of the entire brain of an adult vertebrate, Danionella dracula, which is a miniature species of cyprinid fish. We show this capability in multiple brain regions and in particular the identification of major commissural fiber bundles in the midbrain and the hindbrain. These features provide readily discernable landmarks for navigation and identification of regional-specific neuronal groups and even single neurons during in vivo experiments. We further show how this label-free technique can easily be coupled with fluorescence microscopy and used as a comparative tool for studies of other species with similar body features to Danionella, such as zebrafish (Danio rerio) and tetras (Trochilocharax ornatus). This new evidence, building on previous studies, demonstrates how small size and relative transparency, combined with the unique capabilities of THG microscopy, can enable label-free access to the entire adult vertebrate brain.

Third harmonic generation microscopy of magnetic domains in garnet films

Nonlinear optical microscopy is a powerful non-destructive technique that allows to study a wide range of phenomena. Optical third harmonic generation (THG) is mostly known by its applications in investigation of biological structures, while magneto-optical effects in THG are not well recognized. Here we demonstrate high efficiency of the THG probe in studies of magnetic domain structure of epitaxial garnet films. We show that in spite of relatively small values of the magneto-optical effect at the THG wavelength, the THG microscopy may be efficient for the characterization of magnetic surface domains if the THG wavelength falls in the absorption band of a medium. Moreover, the in-depth sensitivity of this probe may be higher as compared to the case of the second harmonic generation microscopy.

Probing compositional engineering effects on lead-free perovskite-inspired nanocrystal thin films using correlative nonlinear optical microscopy.

We introduce the use of correlative third-harmonic generation and multiphoton-induced luminescence microscopy to investigate the impact of manganese (Mn) doping on bismuth (Bi)-based perovskite-inspired nanocrystal thin films. The technique was found to be extremely sensitive to the microscopic features of the perovskite film and its structural compositions, allowing the unambiguous detection of compositionally different emitters in the perovskite film and manipulation of their nonlinear optical responses. Our work unveils a new way to investigate, manipulate, and exploit perovskite-inspired functional materials for nonlinear optical conversion at the nanoscale.

All-fiberized 1840-nm femtosecond thulium fiber laser for label-free nonlinear microscopy.

We report an all-fiberized 1840-nm thulium-fiber-laser source, comprising a dissipative-soliton mode-locked seed laser and a chirped-pulse-amplification system for label-free biological imaging through nonlinear microscopy. The mode-locked thulium fiber laser generated dissipative-soliton pulses with a pre-chirped duration of 7 ps and pulse energy of 1 nJ. A chirped-pulse fiber-amplification system employing an in-house-fabricated, short-length, single-mode, high-absorption, thulium fiber delivered pulses with energies up to 105 nJ. The pulses were capable of being compressed to 416 fs by passing through a grating pair. Imaging of mouse tissue and human bone samples was demonstrated using this source via third-harmonic generation microscopy.

Label-free fluorescence microscopy: revisiting the opportunities with autofluorescent molecules and harmonic generations as biosensors and biomarkers for quantitative biology.

Over the past decade, the utilization of advanced fluorescence microscopy technologies has presented numerous opportunities to study or re-investigate autofluorescent molecules and harmonic generation signals as molecular biomarkers and biosensors for in vivo cell and tissue studies. The label-free approaches benefit from the endogenous fluorescent molecules within the cell and take advantage of their spectroscopy properties to address biological questions. Harmonic generation can be used as a tool to identify the occurrence of fibrillar or lipid deposits in tissues, by using second and third-harmonic generation microscopy. Combining autofluorescence with novel techniques and tools such as fluorescence lifetime imaging microscopy (FLIM) and hyperspectral imaging (HSI) with model-free analysis of phasor plots has revolutionized the understanding of molecular processes such as cellular metabolism. These tools provide quantitative information that is often hidden under classical intensity-based microscopy. In this short review, we aim to illustrate how some of these technologies and techniques may enable investigation without the need to add a foreign fluorescence molecule that can modify or affect the results. We address some of the most important autofluorescence molecules and their spectroscopic properties to illustrate the potential of these combined tools. We discuss using them as biomarkers and biosensors and, under the lens of this new technology, identify some of the challenges and potentials for future advances in the field.

Third-harmonic generation monitoring of femtosecond-laser-induced in-volume functional modifications

During the last two decades, ultrafast in-volume laser-based processing of transparent materials has emerged as a key 3D-printing method for manufacturing a variety of complex integrated photonic devices and micro-parts. Yet, identifying suitable laser process parameters for a given substrate remains a tedious, time-consuming task. Using a single laser source for both processing and monitoring, we demonstrate a method based on in situ full-field third-harmonic generation (THG) microscopy that exploits the properties of a low-noise CMOS imager to rapidly identify the entire processing space, discriminating different types of laser-induced modifications, and extracting incubation laws governing the laser exposure process. Furthermore, we show that full-field THG monitoring is capable of identifying parameters leading to enhanced functional properties, such as laser-enhanced etching selectivity. These findings enable accelerated implementations of laser processes of arbitrarily chosen transparent materials and, due to the rapid acquisition time (>100FPS) of the imager, closed-loop process control.

In vivo label-free measurement of blood flow velocity symmetry based on dual line scanning third-harmonic generation microscopy excited at the 1700 nm window

Measurement of blood flow velocity is key to understanding physiology and pathology in vivo. While most measurements are performed at the middle of the blood vessel, little research has been done on characterizing the instantaneous blood flow velocity distribution. This is mainly due to the lack of measurement technology with high spatial and temporal resolution. Here, we tackle this problem with our recently developed dual-wavelength line-scan third-harmonic generation (THG) imaging technology. Simultaneous acquisition of dual-wavelength THG line-scanning signals enables measurement of blood flow velocities at two radially symmetric positions in both venules and arterioles in mouse brain in vivo. Our results clearly show that the instantaneous blood flow velocity is not symmetric under general conditions.

Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi

We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus. THG microscopy images showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10−3 to 23 × 10−3 lipid droplets/µm2 after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5–0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.

Broadband‐Tunable Third‐Harmonic Generation Using Phase‐Change Chalcogenides

Despite remarkable progress in passive nonlinear nanophotonics, dynamically controlled ultracompact nonlinear optical sources with high efficiency remain elusive. Nonvolatility, large refractive index contrast between amorphous and crystalline phases, low thermal threshold for crystallization, and particularly high‐third‐order nonlinear optical susceptibility of phase‐change alloy Ge2Sb2Te5 (GST) make it a promising candidate for active subwavelength metaphotonic structures for realization of third‐harmonic generation (THG) devices. Herein, a GST‐based asymmetric Fabry–Perot cavity is numerically designed and experimentally demonstrated to show a dynamically reconfigurable structure with a large shift of the THG resonant band. In addition, continuous resonant spectral shifting bridged by a precisely controlled semicrystalline phase of GST is realized. Tunable THG with the fundamental wavelength ranging from 1150 to 1400 nm is presented, which corresponds to a broadband THG source in the violet‐blue visible wavelength range. Herein, the potential of GST subwavelength structures as a reliable platform for realization of the broadband‐tunable frequency‐conversion sources for applications such as THG microscopy and optical communication is indicated.

Single-shot femtosecond bulk micromachining of silicon with mid-IR tightly focused beams

Being the second most abundant element on earth after oxygen, silicon remains the working horse for key technologies for the years. Novel photonics platform for high-speed data transfer and optical memory demands higher flexibility of the silicon modification, including on-chip and in-bulk inscription regimes. These are deepness, three-dimensionality, controllability of sizes and morphology of created modifications. Mid-IR (beyond 4 µm) ultrafast lasers provide the required control for all these parameters not only on the surface (as in the case of the lithographic techniques), but also inside the bulk of the semiconductor, paving the way to an unprecedented variety of properties that can be encoded via such an excitation. We estimated the deposited energy density as 6 kJ cm−3 inside silicon under tight focusing of mid-IR femtosecond laser radiation, which exceeds the threshold value determined by the specific heat of fusion (~ 4 kJ cm−3). In such a regime, we successfully performed single-pulse silicon microstructuring. Using third-harmonic and near-IR microscopy, and molecular dynamics, we demonstrated that there is a low-density region in the center of a micromodification, surrounded by a “ring” with higher density, that could be an evidence of its micro-void structure. The formation of created micromodification could be controlled in situ using third-harmonic generation microscopy. The numerical simulation indicates that single-shot damage becomes possible due to electrons heating in the conduction band up to 8 eV (mean thermal energy) and the subsequent generation of microplasma with an overcritical density of 8.5 × 1021 cm−3. These results promise to be the foundation of a new approach of deep three-dimensional single-shot bulk micromachining of silicon.

Structure and principles of self-assembly of giant “sea urchin” type sulfonatophenyl porphine aggregates

Principles of molecular self-assembly into giant hierarchical structures of hundreds of micrometers in size are studied in aggregates of meso-tetra(4-sulfonatophenyl)porphine (TPPS4). The aggregates form a central tubular core, which is covered with radially protruding filamentous non-branching aggregates. The filaments cluster and orient at varying angles from the core surface and some filaments form bundles. Due to shape resemblance, the structures are termed giant sea urchin (GSU) aggregates. Spectrally resolved fluorescence microscopy reveals J- and H-bands of TPPS4 aggregates in both the central core and the filaments. The fluorescence of the core is quenched while filaments exhibit strong fluorescence. Upon drying, the filament fluorescence gets quenched while the core is less affected, showing stronger relative fluorescence. Fluorescence-detected linear dichroism (FDLD) microscopy reveals that absorption dipoles corresponding to J-bands are oriented along the filament axis. The comparison of FDLD with scanning electron microscopy (SEM) reveals the structure of central core comprised of multilayer ribbons, which wind around the core axis forming a tube. Polarimetric second-harmonic generation (SHG) and third-harmonic generation microscopy exhibits strong signal from the filaments with nonlinear dipoles oriented close to the filament axis, while central core displays very low SHG due to close to centrosymmetric organization. Large chiral nonlinear susceptibility points to helical arrangement of the filaments. The investigation shows that TPPS4 molecules form distinct aggregate types, including chiral nanotubes and nanogranular aggregates that associate into the hierarchical GSU structure, prototypical to complex biological structures. The chiral TPPS4 aggregates can serve as harmonophores for nonlinear microscopy.

Boundary-Preserved Deep Denoising of Stochastic Resonance Enhanced Multiphoton Images.

Objective: With the rapid growth of high-speed deep-tissue imaging in biomedical research, there is an urgent need to develop a robust and effective denoising method to retain morphological features for further texture analysis and segmentation. Conventional denoising filters and models can easily suppress the perturbative noise in high-contrast images; however, for low photon budget multiphoton images, a high detector gain will not only boost the signals but also bring significant background noise. In such a stochastic resonance imaging regime, subthreshold signals may be detectable with the help of noise, meaning that a denoising filter capable of removing noise without sacrificing important cellular features, such as cell boundaries, is desirable. Method: We propose a convolutional neural network-based denoising autoencoder method — a fully convolutional deep denoising autoencoder (DDAE) — to improve the quality of three-photon fluorescence (3PF) and third-harmonic generation (THG) microscopy images. Results: The average of 200 acquired images of a given location served as the low-noise answer for the DDAE training. Compared with other conventional denoising methods, our DDAE model shows a better signal-to-noise ratio (28.86 and 21.66 for 3PF and THG, respectively), structural similarity (0.89 and 0.70 for 3PF and THG, respectively), and preservation of the nuclear or cellular boundaries (F1-score of 0.662 and 0.736 for 3PF and THG, respectively). It shows that DDAE is a better trade-off approach between structural similarity and preserving signal regions. Conclusions: The results of this study validate the effectiveness of the DDAE system in boundary-preserved image denoising. Clinical Impact: The proposed deep denoising system can enhance the quality of microscopic images and effectively support clinical evaluation and assessment.

Lead-Free Cesium Titanium Bromide Double Perovskite Nanocrystals.

Double perovskites are a promising family of lead-free materials that not only replace lead but also enable new optoelectronic applications beyond photovoltaics. Recently, a titanium (Ti)-based vacancy-ordered double perovskite, Cs2TiBr6, has been reported as an example of truly sustainable and earth-abundant perovskite with controversial results in terms of photoluminescence and environmental stability. Our work looks at this material from a new perspective, i.e., at the nanoscale. We demonstrate the first colloidal synthesis of Cs2TiX6 nanocrystals (X = Br, Cl) and observe tunable morphology and size of the nanocrystals according to the set reaction temperature. The Cs2TiBr6 nanocrystals synthesized at 185 °C show a bandgap of 1.9 eV and are relatively stable up to 8 weeks in suspensions. However, they do not display notable photoluminescence. The centrosymmetric crystal structure of Cs2TiBr6 suggests that this material could enable third-harmonic generation (THG) responses. Indeed, we provide a clear evidence of THG signals detected by the THG microscopy technique. As only a few THG-active halide perovskite materials are known to date and they are all lead-based, our findings promote future research on Cs2TiBr6 as well as on other lead-free double perovskites, with stronger focus on currently unexplored nonlinear optical applications.

Recent Advancements in Optical Harmonic Generation Microscopy: Applications and Perspectives.

Second harmonic generation (SHG) and third harmonic generation (THG) microscopies have emerged as powerful imaging modalities to examine structural properties of a wide range of biological tissues. Although SHG and THG arise from very different contrast mechanisms, the two are complimentary and can often be collected simultaneously using a modified multiphoton microscope. In this review, we discuss the needed instrumentation for these modalities as well as the underlying theoretical principles of SHG and THG in tissue and describe how these can be leveraged to extract unique structural information. We provide an overview of recent advances showing how SHG microscopy has been used to evaluate collagen alterations in the extracellular matrix and how this has been used to advance our knowledge of cancers, fibroses, and the cornea, as well as in tissue engineering applications. Specific examples using polarization-resolved approaches and machine learning algorithms are highlighted. Similarly, we review how THG has enabled developmental biology and skin cancer studies due to its sensitivity to changes in refractive index, which are ubiquitous in all cell and tissue assemblies. Lastly, we offer perspectives and outlooks on future directions of SHG and THG microscopies and present unresolved questions, especially in terms of overall miniaturization and the development of microendoscopy instrumentation.

Third-harmonic generation microscopy of undeveloped photopolymerized structures

Third-harmonic generation (THG) microscopy is demonstrated as a powerful technique to visualize undeveloped photopolymerized microstructures within a negative photoresist film. By comparing the THG microscopy images of developed and undeveloped single-photon polymerized structures in a SU-8 film, THG was found to provide sufficient contrast for distinguishing polymerized and unpolymerized regions. This also suggests that the technique can be used as a complementary technique to visualize the effect of photoresist development where microstructure shrinkage could occur. In addition, we applied the technique to visualize a three-photon polymerized microstructure that was fabricated in the same microscopy setup. This demonstrates the potential of the technique for in situ microscopy of photopolymerized microstructures in three dimensions.

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.

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.

Multiphoton microscopy: a personal historical review, with some future predictions.

.The historical development of multiphoton microscopy is described, starting with a review of two-photon absorption, and including two- and three-photon fluorescence microscopies, and second- and third-harmonic generation microscopies. The effects of pulse length on signal strength and breakdown are considered. Different contrast mechanisms, including use of nanoparticles, are discussed. Two new promising techniques that can be applied to multiphoton microscopy are described.