Third Harmonic Generation Microscopy Research Articles

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.

In vivo third-harmonic generation microscopy study on vitiligo patients.

.Melanin is known to provide strong third-harmonic generation (THG) contrast in human skin. With a high concentration in basal cell cytoplasm, THG contrast provided by melanin overshadows other THG sources in human skin studies. For better understanding of the THG signals in keratinocytes without the influence of melanin, an in vivo THG microscopy (THGM) study was first conducted on vitiliginous skin. As a result, the THG-brightness ratio between the melanin-lacking cytoplasm of basal cells and collagen fibers is about 1.106 at the dermal–epidermal junctions of vitiliginous skin, indicating high sensitivity of THGM for the presence of melanin. We further applied the in vivo THGM to assist evaluating the therapeutic outcome from the histopathological point of view for those showed no improvement under narrowband ultraviolet B therapy based on the seven-point Physician Global Assessment score. Our clinical study indicates the high potential of THGM to assist the histopathological assessment of the therapeutic efficacy of vitiligo treatments.

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.

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.

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.

New Templated Ostwald Ripening Process of Mesostructured FeOOH for Third-Harmonic Generation Bioimaging.

Emerging advances in iron oxide nanoparticles exploit their high magnetization for various applications, such as bioseparation, hyperthermia, and magnetic resonance imaging. In contrast to their excellent magnetic performance, the harmonic generation and luminescence properties of iron oxide nanoparticles have not been thoroughly explored, thus limiting their development as a tool in photomedicine. In this work, a seed/growth-inspired synthesis is developed combined with primary mineralization and a ligand-assisted secondary growth strategy to prepare mesostructured α-FeOOH nanorods (NRs). The sub-wavelength heterogeneity of the refractive index leads to enhanced third-harmonic generation (THG) signals under near-infrared excited wavelengths at 1230 nm. The as-prepared NRs exhibit an 11-fold stronger THG intensity compared to bare α-FeOOH NRs. Using these unique nonlinear optical properties, it is demonstrated that mesostructured α-FeOOH NRs can serve as biocompatible and nonbleaching contrast agents in THG microscopy for long-term labeling of cells as well as in angiography in vivo by modifying lectin to enhance the binding efficiency to the glycocalyx layers on the wall of blood vessels. These results provide a new insight into Fe-based nanoplatforms capable of emitting coherent light as molecular probes in optical microscopy, thus establishing a complementary microscopic imaging method for macroscopic magnetic imaging systems.

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.

Third-harmonic generation modulation achieved through ground-state depletion.

Without real-state transition, third-harmonic generation (THG) cannot be modulated simply by fluorescence-based methods. However, utilizing the absorption-enhancement property of THG, the modulation of THG intensity has been demonstrated through ground-state depletion (GSD) in this Letter. By suppressing the absorption of materials with GSD, the THG intensity can be reduced due to a decreasing of absorption enhancement. The ability to modulate THG intensity can benefit from applying super-resolution techniques to THG microscopy.

Third harmonic generation imaging and analysis of the effect of low gravity on the lacuno-canalicular network of mouse bone.

The lacuno-canalicular network (LCN) hosting the osteocytes in bone tissue represents a biological signature of the mechanotransduction activity in response to external biomechanical loading. Using third-harmonic generation (THG) microscopy with sub-micrometer resolution, we investigate the impact of microgravity on the 3D LCN structure in mice following space flight. A specific analytical procedure to extract the LCN characteristics from THG images is described for ex vivo studies of bone sections. The analysis conducted in different anatomical quadrants of femoral cortical bone didn’t reveal any statistical differences between the control, habitat control and flight groups, suggesting that the LCN connectivity is not affected by one month space flight. However, significant variations are systematically observed within each sample. We show that our current lack of understanding of the extent of the LCN heterogeneity at the organ level hinders the interpretation of such investigations based on a limited number of samples and we discuss the implications for future biomedical studies.

Label-free imaging of atherosclerotic plaques using third-harmonic generation microscopy.

Multiphoton microscopy using laser sources in the mid-infrared range (MIR, 1,300 nm and 1,700 nm) was used to image atherosclerotic plaques from murine and human samples. Third harmonic generation (THG) from atherosclerotic plaques revealed morphological details of cellular and extracellular lipid deposits. Simultaneous nonlinear optical signals from the same laser source, including second harmonic generation and endogenous fluorescence, resulted in label-free images of various layers within the diseased vessel wall. The THG signal adds an endogenous contrast mechanism with a practical degree of specificity for atherosclerotic plaques that complements current nonlinear optical methods for the investigation of cardiovascular disease. Our use of whole-mount tissue and backward scattered epi-detection suggests THG could potentially be used in the future as a clinical tool.

Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy.

Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning.

Examination of Drosophila eye development with third harmonic generation microscopy.

Third harmonic generation (THG) microscopy can exploit endogenous harmonophores such as pigment macromolecules for enhanced image contrast, and therefore can be used without exogenous contrast agents. Previous studies have established that carotenoid compounds are ideal harmonophores for THG microscopy; we therefore sought to determine whether THG from endogenous carotenoid-derived compounds, such as retinal in photoreceptor cells, could serve as a new label-free method for developmental studies. Here we study the development of the pupal eye in Drosophila melanogaster and determine the localization of rhodopsin using THG microscopy technique. Additionally, by altering the chromophore or the opsin protein we were able to detect changes in both the retinal distribution morphology and in THG intensity age-dependent profiles. These results demonstrate that THG microscopy can be used to detect altered photoreceptor development and may be useful in clinically relevant conditions associated with photoreceptor degeneration.

Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy

Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration. Here we address this issue using label-free third-harmonic generation (THG) microscopy. We find that the porous lacuno-canalicular network (LCN), revealing the geometry of osteocytes in the bone matrix, can be directly visualized in 3D with submicron precision over millimetric fields of view compatible with histology. THG also reveals interfaces delineating volumes formed at successive remodeling stages. Finally, we show that the structure of the LCN can be analyzed in relation with that of the extracellular matrix and larger-scale structures by simultaneously recording THG and second-harmonic generation (SHG) signals relating to the collagen organization.

Rapid visualization of grain boundaries in monolayer MoS2 by multiphoton microscopy

Grain boundaries have a major effect on the physical properties of two-dimensional layered materials. Therefore, it is important to develop simple, fast and sensitive characterization methods to visualize grain boundaries. Conventional Raman and photoluminescence methods have been used for detecting grain boundaries; however, these techniques are better suited for detection of grain boundaries with a large crystal axis rotation between neighbouring grains. Here we show rapid visualization of grain boundaries in chemical vapour deposited monolayer MoS2 samples with multiphoton microscopy. In contrast to Raman and photoluminescence imaging, third-harmonic generation microscopy provides excellent sensitivity and high speed for grain boundary visualization regardless of the degree of crystal axis rotation. We find that the contrast associated with grain boundaries in the third-harmonic imaging is considerably enhanced by the solvents commonly used in the transfer process of two-dimensional materials. Our results demonstrate that multiphoton imaging can be used for fast and sensitive characterization of two-dimensional materials.

Rapid and Large-Area Characterization of Exfoliated Black Phosphorus Using Third-Harmonic Generation Microscopy.

Black phosphorus (BP) is a layered semiconductor that recently has been the subject of intense research due to its novel electrical and optical properties, which compare favorably to those of graphene and the transition metal dichalcogenides. In particular, BP has a direct bandgap that is thickness-dependent and highly anisotropic, making BP an interesting material for nanoscale optical and optoelectronic applications. Here, we present a study of the anisotropic third-harmonic generation (THG) in exfoliated BP using a fast scanning multiphoton characterization method. We find that the anisotropic THG arises directly from the crystal structure of BP. We calculate the effective third-order susceptibility of BP to be ∼1.64 × 10-19 m2 V-2. Further, we demonstrate that multiphoton microscopy can be used for rapid, large-area characterization indexing of the crystallographic orientations of many exfoliated BP flakes from one set of multiphoton images. This method is therefore beneficial for samples of areas ∼1 cm2 in future investigations of the properties and growth of BP.

Special Section Guest Editorial:Laser Damage III

Laser damage of optical materials, first reported in 1964, continues to limit the output energy and power of pulsed and continuous-wave laser systems. In spite of some 48 years of research in this area, interest from the international laser community to laser damage issues remains at a very high level and does not show any sign of decreasing. Moreover, it grows with the development of novel laser systems, for example, ultrafast and short-wavelength lasers that involve new damage effects and specific mechanisms not studied before. This interest is evident from the high level of attendance and presentations at the annual SPIE Laser Damage Symposium (aka, Boulder Damage Symposium) that has been held in Boulder, Colorado, since 1969. This special section of Optical Engineering is the first one devoted to the entire field of laser damage rather than to a specific part. It is prepared in response to growing interest from the international laser-damage community. Some papers in this special section were presented at the Laser Damage Symposium; others were submitted in response to the general call for papers for this special section. The 18 papers compiled into this special section represent many sides of the broad field of laser-damage research.more » They consider theoretical studies of the fundamental mechanisms of laser damage including laser-driven electron dynamics in solids (O. Brenk and B. Rethfeld; A. Nikiforov, A. Epifanov, and S. Garnov; T. Apostolova et al.), modeling of propagation effects for ultrashort high-intensity laser pulses (J. Gulley), an overview of mechanisms of inclusion-induced damage (M. Koldunov and A. Manenkov), the formation of specific periodic ripples on a metal surface by femtosecond laser pulses (M. Ahsan and M. Lee), and the laser-plasma effects on damage in glass (Y. Li et al). Material characterization is represented by the papers devoted to accurate and reliable measurements of absorption with special emphasis on thin films (C. Muhlig and S. Bublitz; B. Cho, E. Danielewicz, and J. Rudisill; W. Palm et al; and J. Lu et al.). Statistical treatment of measurements of the laser-damage threshold (J. Arenberg) and the relationship to damage mechanisms (F. Wagner et al.) represent the large subfield of laser-damage measurements. Various aspects of multilayer coating and thin-film characterization are considered in papers by B. Cho, J. Rudisill, and E. Danielewicz (spectral shift in multilayer mirrors) and R. Weber et al. (novel approach to damage studies based on third-harmonic generation microscopy). Of special interest for readers is the paper by C. Stolz that summarizes the results of four “thin-film damage competitions” organized as a part of the Laser Damage Symposium. Another paper is devoted to thermal annealing of damage precursors (N. Shen et al.). Finally, the influence of nano-size contamination on initiation of laser damage by ultrashort pulses is considered in paper of V. Komolov et al.« less

Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood.

Red blood cells (RBC) in two-photon excited fluorescence (TPEF) microscopy usually appear as dark disks because of their low fluorescent signal. Here we use 15fs 800nm pulses for TPEF, 45fs 1060nm pulses for three-photon excited fluorescence, and third harmonic generation (THG) imaging. We find sufficient fluorescent signal that we attribute to hemoglobin fluorescence after comparing time and wavelength resolved spectra of other expected RBC endogenous fluorophores: NADH, FAD, biliverdin, and bilirubin. We find that both TPEF and THG microscopy can be used to examine erythrocyte morphology non-invasively without breaching a blood storage bag.

Differentiating intratumoral melanocytes from Langerhans cells in nonmelanocytic pigmented skin tumors in vivo by label-free third-harmonic generation microscopy.

Morphology and distribution of melanocytes are critical imaging information for the diagnosis of melanocytic lesions. However, how to image intratumoral melanocytes noninvasively in pigmented skin tumors is seldom investigated. Third-harmonic generation (THG) is shown to be enhanced by melanin, whereas high accuracy has been demonstrated using THG microscopy for in vivo differential diagnosis of nonmelanocytic pigmented skin tumors. It is thus desirable to investigate if label-free THG microscopy was capable to in vivo identify intratumoral melanocytes. In this study, histopathological correlations of label-free THG images with the immunohistochemical images stained with human melanoma black (HMB)-45 and cluster of differentiation 1a (CD1a) were made. The correlation results indicated that the intratumoral THG-bright dendritic-cell-like signals were endogenously derived from melanocytes rather than Langerhans cells (LCs). The consistency between THG-bright dendritic-cell-like signals and HMB-45 melanocyte staining showed a kappa coefficient of 0.807, 84.6% sensitivity, and 95% specificity. In contrast, a kappa coefficient of −0.37, 21.7% sensitivity, and 30% specificity were noted between the THG-bright dendritic-cell-like signals and CD1a staining for LCs. Our study indicates the capability of noninvasive label-free THG microscopy to differentiate intratumoral melanocytes from LCs, which is not feasible in previous in vivo label-free clinical-imaging modalities.

Third-harmonic generation imaging of breast tissue biopsies.

We demonstrate for the first time the imaging of unstained breast tissue biopsies using third-harmonic generation (THG) microscopy. As a label-free imaging technique, THG microscopy is compared to phase contrast and polarized light microscopy which are standard imaging methods for breast tissues. A simple feature detection algorithm is applied to detect tumour-associated lymphocyte rich regions in unstained breast biopsy tissue and compared with corresponding regions identified by a pathologist from bright-field images of hematoxylin and eosin stained breast tissue. Our results suggest that THG imaging holds potential as a complementary technique for analysing breast tissue biopsies.