Background and Objectives: Extracellular matrix (ECM) and collagen remodeling contribute to chronic kidney disease (CKD) progression and vascular access dysfunction. Conventional histological techniques rely on staining and provide limited sensitivity for detecting early or subtle ECM alterations. Nonlinear optical imaging modalities, including second-harmonic generation (SHG), third-harmonic generation (THG), and multiphoton fluorescence (MPF) microscopy, enable label-free, high-resolution visualization of fibrillar collagen and may offer additional structural information. This study aimed to evaluate the added value of nonlinear imaging beyond conventional histology for assessing ECM remodeling in renal and vascular tissues. Materials and Methods: A systematic literature review was conducted in accordance with the PRISMA 2020 guidelines. PubMed and Web of Science were searched for studies published between 1 January 2015, and 4 April 2025, investigating ECM or collagen remodeling in renal or vascular tissues using SHG, THG, or MPF microscopy. After screening 115 records, 10 studies were included in the qualitative synthesis. In addition, representative SHG, THG, and MPF images of excised human arteriovenous fistula (AVF) tissue were acquired as illustrative feasibility examples to demonstrate the application of these imaging modalities. The use of human tissue was approved by the Vilnius Regional Biomedical Research Ethics Committee (approval No. 2022/6-1443-917). Results: The included studies demonstrated that nonlinear microscopy enables label-free assessment of collagen density, organization, and fiber orientation. SHG imaging differentiated healthy from diseased tissues and has been reported to support fibrosis assessment and staging in preclinical and selected clinical studies and revealed microstructural remodeling patterns not readily detected by conventional histology. The illustrative AVF images demonstrated collagen disorganization consistent with patterns reported in the reviewed literature and are presented solely to demonstrate imaging feasibility, without implying disease phenotype or clinical outcome associations. Conclusions: Nonlinear optical microscopy provides complementary structural information on ECM organization that is not accessible with standard histological techniques. Further validation and methodological standardization are required to support its broader application in clinical nephrology and vascular medicine.

Characterization of myelin degradation in the white matter (WM) is important for understanding neurodegeneration. We demonstrate label-free in vivo imaging of myelin structure in the WM of mice, through intact cortex, using third harmonic generation (THG) microscopy at 1320-nm excitation. Longitudinal THG imaging of the same axons in the cuprizone mouse model of multiple sclerosis revealed dynamics of myelin blistering. Further, we measured intranodal distance at nodes of Ranvier in vivo and developed a novel metric of myelin structural change based on spatial concentration of the brightest THG signal. We also demonstrated compatibility with three-photon excited fluorescence microscopy by imaging GFP-labeled microglia in the WM in parallel with THG microscopy, thereby enabling detailed tracking of subcortical myelin and other cellular dynamics in neurodegenerative disease models.

Imaging subcellular structures deep within thick, turbid biological tissues remains fundamentally limited by light scattering, which distorts optical wavefronts and degrades contrast, resolution, and sensitivity. These limitations hinder quantitative interrogation of complex biological systems where resolving dynamic microenvironments at subcellular resolution is critical. Here, we introduce scattering-enabled epi-quantitative phase imaging (SEEQPI), a label-free method that leverages tissue scattering and provides subcellular spatial resolution, nanometer-scale spatiotemporal phase sensitivity, and millimeter-scale imaging depth in murine brains. SEEQPI is enabled by common-path phase-shifting confocal epi-interferometry with near-infrared illumination and the scattering-enabled phase reconstruction algorithm. SEEQPI requires low illumination power, minimizing tissue damage while enabling high-speed imaging of biological dynamics. We demonstrate simultaneous, colocalized imaging of subcellular structures with SEEQPI, third-harmonic generation, and three-photon fluorescence microscopy in liver cancer spheroids and in vivo mouse brains. SEEQPI enables quantitative, longitudinal studies of dry mass dynamics in intact, living biological systems.

Super-resolution microscopy—imaging below the Abbe diffraction limit—was a resounding success in the development of optical far-field microscopy and revolutionized bioimaging. Yet most super-resolution techniques are based on fluorescence microscopy. However, fluorescent labels impose restrictions on implementing this technique in some fields of science. Label-free techniques such as third-harmonic generation (THG) microscopy are an alternative, but only provide limited resolution due to their infrared driver. In this work, we show how to optimize the point-spread function (PSF) of THG microscopes, first for a single-color harmonic driving laser, and then in a two-color field. For a single-color driver, a low input intensity leads to the smallest PSF. This occurs because an effective nonlinear order close to the order of the emitted harmonic leads to the maximum possible spot size reduction of the harmonic emission profile with respect to the incident intensity profile of the driver. In the two-color case, we utilize a second light pulse with a donut-shaped fluence profile in focus, which is generated by introducing orbital angular momentum. We show that this second pulse shrinks the PSF below the diffraction limit. Although there is no principal limit to this shrinkage, meaning that there is no principal limit to resolution in our approach, the current implementation is practically limited by sample damage. Currently, this promises a factor of 4 reduction of the PSF and a concomitant improvement of the resolution by a factor of 4 in a coherent harmonic microscopy imaging system. These findings open the pathway to implement super-resolution techniques in a broader scientific and industrial application, i.e., in condensed-matter physics and for semiconductor wafer metrology.

Lately the nonlinear optical third-harmonic generation (THG) microscopy is starting to emerge as a laboratory standard for label-free studies in biological samples. In this study, the THG signals produced from corn starch granules are investigated. In particular, the polarization-dependent THG (P-THG) signals emerging from the outer layer (shell) of the starch granules are compared with the P-THG signals originating from their inner portion (core). By rotating the linear polarization of the excitation beam, two distinct P-THG modulation patterns are revealed within single granules, corresponding to their shells and to their structurally different cores. These patterns are analyzed using a theoretical framework that describes THG from an orthorhombic crystal symmetry, characteristic of corn starch. This allows us to extract point-by-point in the granules the ratios of the χ(3) susceptibility tensor elements and the average molecular orientations. Then, the anisotropy ratio (AR = χxxxx(3)/χyyyy(3)) is defined and used as a quantitative descriptor of the local molecular arrangements. Our results show that the shells and cores exhibit distinct AR values, probing the anisotropy in the molecular arrangements between the two regions. This study establishes P-THG as a powerful contrast mechanism for probing structural anisotropy in biological samples beyond conventional THG intensity-only microscopy.

Metabolic coupling between neurons and glial cells plays a critical role in brain activity and myelin plasticity. Understanding its role in physiological and pathological contexts requires advanced methods to map metabolism and myelin in live tissue with high spatiotemporal resolution. Here, we present a label-free, multimodal, nonlinear optical microscopy platform integrated with an advanced image processing framework that simultaneously maps cellular metabolism and myelin distribution in organotypic cerebellar cultures. We combine third-harmonic generation microscopy for high-resolution myelin imaging with single axon precision with two-photon fluorescence lifetime microscopy of NAD(P)H metabolic biomarker to assess redox states with single-cell resolution. We introduce automated image analysis methods for cell segmentation and myelinated axon detection, enabling quantitative metabolic and myelin assessment in intact tissue during experimental myelination, demyelination and remyelination. Using this framework, we map the 3D myelin distribution in cerebellar folia and identify distinct metabolic signatures in neurons, oligodendrocytes, and microglia. Furthermore, we measure a metabolic shift in microglia along with myelin distribution changes during experimental demyelination. In conclusion, we establish label-free optical imaging as a powerful tool for the non-invasive characterization of neuro-glial metabolic coupling and myelin organization in living brain tissue, opening new perspectives for research in neuroinflammation and neurodegeneration.

Root biology is pivotal in addressing global challenges including sustainable agriculture and climate change. However, roots have been relatively understudied among plant organs, partly due to the difficulties in imaging root structures in their natural environment. Here we used microfabricated ecosystems (EcoFABs) to establish growing environments with optical access and employed nonlinear multimodal microscopy of third-harmonic generation (THG) and three-photon fluorescence (3PF) to achieve label-free, in situ imaging of live roots and microbes at high spatiotemporal resolution. THG enabled us to observe key plant root structures including the vasculature, Casparian strips, dividing meristematic cells, and root cap cells, as well as subcellular features including nuclear envelopes, nucleoli, starch granules, and putative stress granules. THG from the cell walls of bacteria and fungi also provides label-free contrast for visualizing these microbes in the root rhizosphere. With simultaneously recorded 3PF signal, we demonstrated our ability to investigate root-microbe interactions by achieving single-bacterium tracking and subcellular imaging of fungal spores and hyphae in the rhizosphere.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-20030-9.

Structural organization of harmonophores used in hematoxylin (H) and eosin (E) staining is studied with polarimetric multimodal second-harmonic generation (SHG), third-harmonic generation (THG), and multiphoton excitation fluorescence (MPF) microscopy in rat tail tendon histology sections. The polarimetric microscopy imaging reveals that hemalums (complexes of hematoxylin and aluminum) are well aligned with C6h symmetry along the collagen fibers in H-stained tissue, while eosin Y is partially aligned along the fibers in E-stained tissue and also follows the organization of C6h symmetry. When both hemalum and eosin are used for H&E staining, the dye molecules interact and align noncentrosymmetrically with C6 symmetry along the collagen fibers, while the stained nuclei appear isotropically organized. The polar alignment of the hemalum and eosin complexes increases the achiral second-order susceptibility tensor component ratio / in H&E-stained tissue. The alignment of hemalum and eosin molecules, and their complexes in collagenous tissue, must be considered in nonlinear microscopy and polarimetric analysis of H&E-stained histopathology.

There were two issues in the original article [Opt. Express16(19), 14703 (2008)10.1364/OE.16.014703]. The expression for the third harmonic tensor for isotropic media contained an error, and the x - y figures were rotated by 90 degrees. One conclusion must be reversed (the angular dependence in Figure6), but the other results remain qualitatively unchanged.

Myelin is essential for axonal conduction and metabolic support. To better understand its role in health and disease, it is necessary to establish accurate methods for in situ mapping of myelin at scales ranging from submicrometer to centimeters. Third-harmonic generation (THG) microscopy has recently been proposed as an efficient label-free method to visualize myelin in thick and living tissue. However, the contrast mechanism of THG from myelinated axons is complex and poorly described, which has limited the development of THG as a quantitative probe of myelin distribution. Here, we present a systematic characterization and modeling of polarization-resolved THG (pTHG) signals from individual axons as a function of their diameter and myelin thickness, and we show that pTHG can be used to derive myelin scores in several biological systems. First, we confirm the sensitivity and specificity of the THG contrast for myelinated axons in mouse brain tissue and its ability to detect isolated micrometer-sized axons oriented both in-plane and out-of-plane. We then present a detailed characterization of the pTHG contrast of small and large axons in live zebrafish larvae at different developmental stages, and we demonstrate that pTHG detects early axon development in vivo. We show that classical models of coherent multiphoton microscopy fail to reproduce pTHG profiles of axons because they neglect myelin-induced optical aberrations, and we establish a numerical strategy based on finite-difference time-domain calculations that can accurately relate pTHG signal profiles to axonal diameter and myelin thickness. Finally, we illustrate the relevance of pTHG microscopy for characterizing myelin distribution at different scales in fixed mouse and human brain tissue.

Myelin pathology is known to play a central role in disorders such as multiple sclerosis (MS) among others. Despite this, the pathological mechanisms underlying these conditions are often difficult to unravel. Conventional techniques like immunohistochemistry or dye-based approaches, do not provide a temporal characterization of the pathophysiological aberrations responsible for myelin changes in human specimens. Here, to circumvent this curb, we present a label-free, live-cell imaging approach of myelin using recent advancements in nonlinear harmonic generation microscopy applied to physiologically viable human brain tissue from post-mortem donors. Gray and white matter brain tissue from epilepsy surgery and post-mortem donors was excised. To sustain viability of the specimens for several hours, they were subjected to either acute or organotypic slice culture protocols in artificial cerebral spinal fluid. Imaging was performed using a femtosecond pulsed 1050 nm laser to generate second harmonic generation (SHG) and third harmonic generation (THG) signals directly from myelin and axon-like structures without the need to add any labels. Experiments on acute human brain slices and post-mortem human slice cultures reveal that myelin, along with lipid bodies, are the prime sources of THG signal. We show that tissue viability is maintained over extended periods during THG microscopy, and that prolonged THG imaging is able to detect experimentally induced subtle alterations in myelin morphology. Finally, we provide practical evidence that live-cell imaging of myelin with THG microscopy is a sensitive tool to investigate subtle changes in white matter of neurological donors. Overall, our findings support that nonlinear live-cell imaging is a suitable setup for researching myelin morphology in neurological conditions like MS.

Microadditive manufacturing has revolutionized the production of complex, nano- to microscale components across various fields. This work investigates two-photon (2P) and three-photon (3P) fluorescence imaging, as well as third-harmonic generation (THG) microscopy, to examine periodic microarchitected lattice structures fabricated using multiphoton lithography (MPL). By immersing the structures in refractive index matching fluids, we demonstrate high-fidelity 3D reconstructions of both fluorescent structures using 2P and 3P microscopy as well as low-fluorescence structures using THG microscopy. These results show that multiphoton fluorescence (MPF) imaging offers reduced signal decay with respect to depth compared to single-photon techniques in the examined structures. We further demonstrate the ability to nondestructively identify intentional internal modifications of the structure that are not immediately visible with scanning electron microscope (SEM) images and compression-induced fractures, highlighting the potential of these techniques for quality control and defect detection in microadditively manufactured components.

The third-order nonlinear optical properties of a series of novel pyrrole-containing compounds were explored. A custom nonlinear optical microscope was used to measure the third harmonic generation (THG) intensity ratio from two interfaces of an ultrasmall cuvette: a glass-solution interface incorporating the pyrrole-containing compound, and an air-glass interface. The THG intensity ratio, along with refractive index measurements of the solution at the laser wavelength (1030 nm) and the third harmonic wavelength (343 nm), were used to calculate χ(3) values of each pyrrole-containing solution. The second hyperpolarisability γ was then extracted from concentration-dependent measurements of χ(3). Trends in the magnitude of γ for the studied pyrrole-containing compounds became apparent, including that several formyl-substituted dipyrrole derivatives exhibited elevated γ values. Further, an enhancement in γ was observed with increasing conjugation length when formyl-substituted dipyrroles with differing internal linkers were studied. Lastly, the complexation of ruthenium by a formyl-substituted dipyrrole enhanced the magnitude of the γ value by ∼3 to ∼300 times, depending on the dipyrrole derivative. These trends can be used to understand the chemical features needed for the emergent development of dyes for THG microscopy.

Traumatic peripheral nerve injuries represent a spectrum of conditions and remain challenging to diagnose and prognosticate. High-resolution ultrasonography and magnetic resonance neurography have emerged as useful diagnostic modalities in the evaluation of traumatic peripheral nerve and brachial plexus injuries. Ultrasonography is noninvasive, is able to rapidly interrogate large areas and multiple nerves, allows for a dynamic assessment of nerves and their surrounding anatomy, and is cost-effective. It allows for the prompt differentiation of neurotmetic versus axonotmetic traumatic nerve injuries, which informs surgical decision making. Magnetic resonance neurography yields images of peripheral nerves with high structural resolution down to the level of fascicles, is able to evaluate difficult anatomic areas that may not be reached by ultrasonography, and can detect signs of acute and chronic muscle denervation. Advanced preclinical techniques, such as second-harmonic generation microscopy, use multiphoton microscopy to visualize the internal collagenous structure of peripheral nerves, while third-harmonic generation microscopy can image myelin. One potential future application for multiphoton microscopy is the in vivo real-time assessment of nervous tissue. Near-infrared fluorescence also has the potential to assist with intraoperative peripheral nerve identification and assessment of pathology, but many challenges remain in identifying or designing the ideal contrast agent.

Abstract Myelin pathology is known to play a central role in disorders such as multiple sclerosis (MS) among others. Despite this, the pathological mechanisms underlying these conditions are often difficult to unravel. Conventional techniques like immunohistochemistry or dye-based approaches, do not provide a temporal characterization of the pathophysiological aberrations responsible for myelin changes in human specimens. Here, to circumvent this curb, we present a label-free, live-cell imaging approach of myelin using recent advancements in nonlinear harmonic generation microscopy applied to physiologically viable human brain tissue from post-mortem donors. Gray and white matter brain tissue from epilepsy surgery and post-mortem donors was excised. To sustain viability of the specimens for several hours, they were subjected to either acute or organotypic slice culture protocols in artificial cerebral spinal fluid. Imaging was performed using a femtosecond pulsed 1060 nm laser to generate second harmonic generation (SHG) and third harmonic generation (THG) signals directly from myelin and axon-like structures without the need to add any labels. Experiments on acute human brain slices and post-mortem human slice cultures reveal that myelin, along with lipid bodies, are the prime sources of THG signal. We show that tissue viability is maintained over extended periods during THG microscopy, and that prolonged THG imaging is able to detect experimentally induced subtle alterations in myelin morphology. Finally, we provide practical evidence that live-cell imaging of myelin with THG microscopy is a sensitive tool to investigate subtle changes in white matter of neurological donors. Overall, our findings support that nonlinear live-cell imaging is a suitable setup for researching myelin morphology in neurological conditions like MS.

ABSTRACTEffective interplay between the uterus and the embryo is essential for pregnancy establishment; however, convenient methods to screen embryo implantation success and maternal uterine response in experimental mouse models are currently lacking. Here, we report 3DMOUSEneST, a groundbreaking method for analyzing mouse implantation sites based on label-free higher harmonic generation microscopy, providing unprecedented insights into the embryo–uterine dynamics during early pregnancy. The 3DMOUSEneST method incorporates second-harmonic generation microscopy to image the three-dimensional structure formed by decidual fibrillar collagen, named ‘decidual nest’, and third-harmonic generation microscopy to evaluate early conceptus (defined as the embryo and extra-embryonic tissues) growth. We demonstrate that decidual nest volume is a measurable indicator of decidualization efficacy and correlates with the probability of early pregnancy progression based on a logistic regression analysis using Smad1/5 and Smad2/3 conditional knockout mice with known implantation defects. 3DMOUSEneST has great potential to become a principal method for studying decidual fibrillar collagen and characterizing mouse models associated with early embryonic lethality and fertility issues.

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.

Third-harmonic generation microscopy is a powerful label-free nonlinear imaging technique, providing essential information about structural characteristics of cells and tissues without requiring external labelling agents. In this work, we integrated a recently developed compact adaptive optics module into a third-harmonic generation microscope, to measure and correct for optical aberrations in complex tissues. Taking advantage of the high sensitivity of the third-harmonic generation process to material interfaces and thin membranes, along with the 1,300-nm excitation wavelength used here, our adaptive optical third-harmonic generation microscope enabled high-resolution in vivo imaging within highly scattering biological model systems. Examples include imaging of myelinated axons and vascular structures within the mouse spinal cord and deep cortical layers of the mouse brain, along with imaging of key anatomical features in the roots of the model plant Brachypodium distachyon. In all instances, aberration correction led to enhancements in image quality.

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.

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.