Nonlinear Optical Imaging Research Articles

Multimodal imaging to explore endogenous fluorescence of fresh and fixed human healthy and tumor brain tissues.

To complement a project toward label-free optical biopsy and enhanced resection which the overall goal is to develop a multimodal nonlinear endomicroscope, this multimodal approach aims to enhance the accuracy in classifying brain tissue into solid tumor, infiltration and normal tissue intraoperatively. Multiple optical measurements based on one- and two-photon spectral and lifetime autofluorescence, including second harmonic generation imaging, were acquired. As a prerequisite, studying the effect of the time of measurement postexcision on tissue's spectral/lifetime fluorescence properties was warranted, so spectral and lifetime fluorescences of fresh brain tissues were measured using a point-based linear endoscope. Additionally, a comparative study on tissue's optical properties obtained by multimodal nonlinear optical imaging microscope from fresh and fixed tissue was necessary to test whether clinical validation of the nonlinear endomicroscope is feasible by extracting optical signatures from fixed tissue rather than from freshly excised samples. The former is generally chosen for convenience. Results of this study suggest that an hour is necessary postexcision to have consistent fluorescence intensities\lifetimes. The fresh (a,b,c) vs fixed (d,e,f) tissue study indicates that while all optical signals differ after fixation. The characteristic features extracted from one- and two-photon excitation still discriminate normal brain (a,d) cortical tissue, glioblastoma (GBM) (b,e) and metastases (c,f).

Effect of refractive index mismatch on forward-to-backward ratios in SHG imaging.

Nonlinear optical imaging in the epi-direction is used to image subresolution features. We find that a refractive index mismatch between the object to be imaged and the background medium can change the far-field intensity image. As an example, we study second harmonic generation (SHG) microscopy where the forward-to-backward (F/B) ratio is used to quantify subresolution features. We show both theoretically and experimentally that the inhomogeneous refractive index in collagen tendon tissue creates near-field effects, which can change the F/B ratio by ∼20%-25%, even though the effect is negligible for most of the individual fibrils in the tissue. This is caused by the sensitivity of the backward signal on phase matching conditions.

Highly longitudinally polarized coherent anti-Stokes Raman scattering microscopy with improved spatial resolution

We proposed a method to enhance the longitudinally polarized component and improve the spatial resolution of radially polarized coherent anti-Stokes Raman scattering (CARS) microscopy by phase modulation. A specially designed phase pattern is applied onto the pump beam to suppress the radially polarized field component at the focal region. With this modulation, the calculated intensity ratio between the longitudinally and radially polarized CARS field is increased from 2.57 to 14.7, and the simulation of CARS imaging on a 120 nm polystyrene bead shows more than 3-fold spatial resolution improvement for both forward and backward detection. It is expected that this method could also be applied to other nonlinear optical imaging modalities for enhancing the longitudinally polarized component.

Understanding Dissolution and Crystallization with Imaging: A Surface Point of View.

The tendency for crystallization during storage and administration is the most considerable hurdle for poorly water-soluble drugs formulated in the amorphous form. There is a need to better detect often subtle and complex surface crystallization phenomena and understand their influence on the critical quality attribute of dissolution. In this study, the interplay between surface crystallization of the amorphous form during storage and dissolution testing, and its influence on dissolution behavior, is analyzed for the first time with multimodal nonlinear optical imaging (coherent anti-Stokes Raman scattering (CARS) and sum frequency generation (SFG)). Complementary analyses are provided with scanning electron microscopy, X-ray diffraction and infrared and Raman spectroscopies. Amorphous indomethacin tablets were prepared and subjected to two different storage conditions (30 °C/23% RH and 30 °C/75% RH) for various durations and then dissolution testing using a channel flow-through device. Trace levels of surface crystallinity previously imaged with nonlinear optics after 1 or 2 days of storage did not significantly decrease dissolution and supersaturation compared to the freshly prepared amorphous tablets while more extensive crystallization after longer storage times did. Multimodal nonlinear optical imaging of the tablet surfaces after 15 min of dissolution revealed complex crystallization behavior that was affected by both storage condition and time, with up to four crystalline polymorphs simultaneously observed. In addition to the well-known α- and γ-forms, the less reported metastable ε- and η-forms were also observed, with the ε-form being widely observed in samples that had retained significant surface amorphousness during storage. This form was also prepared in the pure form and further characterized. Overall, this study demonstrates the potential value of nonlinear optical imaging, together with more established solid-state analysis methods, to understand complex surface crystallization behavior and its influence on drug dissolution during the development of amorphous drugs and dosage forms.

Preparation and characterization of multi-component tablets containing co-amorphous salts: Combining multimodal non-linear optical imaging with established analytical methods

Co-amorphous mixtures have rarely been formulated as oral dosage forms, even though they have been shown to stabilize amorphous drugs in the solid state and enhance the dissolution properties of poorly soluble drugs.In the present study we formulated tablets consisting of either spray dried co-amorphous ibuprofen-arginine or indomethacin-arginine, mannitol or xylitol and polyvinylpyrrolidone K30 (PVP). Experimental design was used for the selection of tablet compositions, and the effect of tablet composition on tablet characteristics was modelled. Multimodal non-linear imaging, including coherent anti-Stokes Raman scattering (CARS) and sum frequency/second harmonic generation (SFG/SHG) microscopies, as well as scanning electron microscopy, X-ray diffractometry and Fourier-transform infrared spectroscopy were utilized to characterize the tablets.The tablets possessed sufficient strength, but modelling produced no clear evidence about the compaction characteristics of co-amorphous salts. However, co-amorphous drug-arginine mixtures resulted in enhanced dissolution behaviour, and the PVP in the tableting mixture stabilized the supersaturation. The co-amorphous mixtures were physically stable during compaction, but the excipient selection affected the long term stability of the ibuprofen-arginine mixture. CARS and SFG/SHG proved feasible techniques in imaging the component distribution on the tablet surfaces, but possibly due to the limited imaging area, recrystallization detected with x-ray diffraction was not detected.

Spectro-temporal dispersion management of femtosecond pulses for fiber-optic two-photon endomicroscopy.

The emerging fiber-optic two-photon endomicroscopy technology holds a strong promise for enabling translational applications of nonlinear optical imaging. Effective femtosecond pulse dispersion management is critical for achieving high-quality imaging. Here we report systematic analyses and performance characterization of a dual-fiber spectro-temporal dispersion management scheme involving a grating pair as the pulse stretcher. Compared with conventional linear-only compensation, the grating-based spectro-temporal compensation also takes into account nonlinear effects and enhances the two-photon signal by ~3-fold as experimentally demonstrated. Numerical simulations were carried out to systematically investigate the influence of several key design parameters on the overall compensation efficacy. Furthermore, comprehensive performance comparison with an ideal grism-pair counterpart reveals that a grating-pair stretcher affords much higher power throughput and thus is preferable for portable endomicroscopy systems with limited laser source power.

Liquid Crystals under Confinement in Submicrometer Capsules.

Liquid crystal (LC) ordering and phase transition behavior under confined conditions have attracted extensive attention and enabled many applications. However, the ordering and phase transition behavior of LCs in submicrometer capsules have seldom been studied, primarily due to the lack of proper capsulizing and visualization approaches to such small LC microcapsules. Herein, we achieve submicrometer LC capsules with the sizes down to 100 nm by using emulsion-based interfacial sol-gel reaction. The behavior of LCs under the submicrometer confinement conditions is investigated while the sizes and chemical composition of the microcapsule shell surface are tuned in a controllable way. The phase transition temperatures of LCs in the submicrometer capsules shift from those of bulk LCs due to the surface-induced ordering of LCs under the strong confinement conditions, which causes formation of topological defects and alters the order parameter. Using nonlinear optical imaging technology, we explore the structures of director field of LCs that arise as a result of the competition between the surface boundary conditions and LC elasticity. The results show that the nanoscale encapsulation can significantly influence the structural configurations of the director and phase transitions of LCs under various confinement conditions.

Collagen fiber crimping following in vivo UVA-induced corneal crosslinking

The purpose of this study was to measure collagen fiber crimping (CFC) using nonlinear optical imaging of second harmonic generated (SHG) signals to determine the effects of UVA-riboflavin induced corneal collagen crosslinking (UVA CXL) on collagen structure. Two groups, four rabbits each, were treated in the right eye with standard UVA CXL. In vivo confocal microscopy was performed at 1, 2, and 4 weeks after treatment for the first group and up to three months for the second group to measure epithelial/stromal thickness and corneal haze during recovery. Rabbits were sacrificed at one and three months, respectively, and their corneas fixed under pressure. Regions of crosslinking were identified by the presence of collagen autofluorescence (CAF) and then collagen structure was imaged using SHG microscopy. The degree of CFC was determined by measuring the percentage difference between the length of the collagen fiber and the linear distance traveled. CFC was measured in the central anterior and posterior CXL region, the peripheral non-crosslinked region in the same cornea, and the central cornea of the non-crosslinked contralateral eye. No change in corneal thickness was detected after one month, however the stromal thickness surpassed its original baseline thickness at three months by 25.9 μm. Corneal haze peaked at one month and then began to clear. Increased CAF was detected in all CXL corneas, localized to the anterior stroma and extending to 42.4 ± 3.4% and 47.7 ± 7.6% of the corneal thickness at one and three months. There was a significant (P < 0.05) reduction in CFC in the CAF region in all eyes averaging 1.007 ± 0.006 and 1.009 ± 0.005 in one and three month samples compared to 1.017 ± 0.04 and 1.016 ± 0.06 for controls. These results indicate that there is a significant reduction in collagen crimping following UVA CXL of approximately 1%. One possible explanation for this loss of crimping could be shortening of the collagen fibers over the CXL region.

The Impact of Compressed Femtosecond Laser Pulse Durations on Neuronal Tissue Used for Two-Photon Excitation Through an Endoscope

Accurate intraoperative tumour margin assessment is a major challenge in neurooncology, where sparse tumours beyond the bulk tumour are left undetected under conventional resection. Non-linear optical imaging can diagnose tissue at the sub-micron level and provide functional label-free histopathology in vivo. For this reason, a non-linear endomicroscope is being developed to characterize brain tissue intraoperatively based on multiple endogenous optical contrasts such as spectrally- and temporally-resolved fluorescence. To produce highly sensitive optical signatures that are specific to a given tissue type, short femtosecond pulsed lasers are required for efficient two-photon excitation. Yet, the potential of causing bio-damage has not been studied on neuronal tissue. Therefore, as a prerequisite to clinically testing the non-linear endomicroscope in vivo, the effect of short laser pulse durations (40–340 fs) on ex vivo brain tissue was investigated by monitoring the intensity, the spectral, and the lifetime properties of endogenous fluorophores under 800 and 890 nm two-photon excitation using a bi-modal non-linear endoscope. These properties were also validated by imaging samples on a benchtop multiphoton microscope. Our results show that under a constant mean laser power, excitation pulses as short as 40 fs do not negatively alter the biochemical/ biophysical properties of tissue even for prolonged irradiation.

Application of Nonlinear Optical Microscopic Imaging Technology for Quality Assessment of Donor Kidneys

BackgroundNonlinear optical microscopic (NLOM) imaging technique shows its high resolution imaging features in histocytology. The purpose of this study was to investigate NLOM imaging technique as a useful tool for a donor kidney quality assessment. Materials and MethodsEighty-three pretransplant kidney biopsies from adult donors were analyzed retrospectively. Each specimen was paraffin-embedded and sectioned into 2 consecutive 5-μm thick sections. One section was stained with Masson trichrome, and the other was left unstained for NLOM imaging using second harmonic generation combined with two-photon excited fluorescence (SHG/TPEF). The pretransplant kidney quality was assessed by an experienced pathologist using the Remuzzi scoring system, which characterizes renal tissue morphology into 4 aspects: tubular atrophy, interstitial fibrosis, glomerulosclerosis, and vascular injury. The K coefficient was used to measure the consistency of the Remuzzi scores between conventional Masson trichrome stained images and SHG/TPEF images. ResultsNLOM imaging technology can capture high-resolution tissue images from unstained renal tissue, is easy to operate, and shortens time-consuming histological processing procedures. No significant differences (P > .05) were found between the Remuzzi scores of the SHG/TPEF images and the Masson trichrome stained images. The high κ coefficients (0.804–0.895) showed a good consistency between these 2 techniques. ConclusionThe NLOM technique is suitable for renal tissue imaging and could potentially be used for routine pretransplant kidney evaluation in clinical settings.

Porphyrin Dyes for Nonlinear Optical Imaging of Live Cells

SummarySecond harmonic generation (SHG)-based probes are useful for nonlinear optical imaging of biological structures, such as the plasma membrane. Several amphiphilic porphyrin-based dyes with high SHG coefficients have been synthesized with different hydrophilic head groups, and their cellular targeting has been studied. The probes with cationic head groups localize better at the plasma membrane than the neutral probes with zwitterionic or non-charged ethylene glycol-based head groups. Porphyrin dyes with only dications as hydrophilic head groups localize inside HEK293T cells to give SHG, whereas tricationic dyes localize robustly at the plasma membrane of cells, including neurons, in vitro and ex vivo. The copper(II) complex of the tricationic dye with negligible fluorescence quantum yield works as an SHG-only dye. The free-base tricationic dye has been demonstrated for two-photon fluorescence and SHG-based multimodal imaging. This study demonstrates the importance of a balance between the hydrophobicity and hydrophilicity of amphiphilic dyes for effective plasma membrane localization.

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing.

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP-labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full-thickness cutaneous wound site in streptozotocin-induced diabetic mice. Wounds treated with MSC-ADM demonstrated an increased percentage of wound closure. The treatment of MSC-ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col-I) fibers synthesis via second harmonic generation imaging. The synthesis of Col-I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP-labeled MSCs during wound healing was simultaneously traced via two-photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo-multiplier tube.

Axial point source localization using variable displacement–change point detection

A three-dimensional point-source localization technique is demonstrated using two-photon photoluminescence and four-wave mixing nonlinear optical signals from plasmonic gold nanorods (AuNRs), imaged at the single-particle level. Introduction of position-dependent latitudinal astigmatisms into the imaging system, in combination with a change point detection (CPD) algorithm, resulted in localization of single particles with high precision in three dimensions. Astigmatisms were generated using axial sample-position displacements spanning the range from ±10 to ±90 nm with a minimum step-size resolution of ±3 nm. Based on the current data, 20 nm point source localization was achieved in the axial dimension using a single imaging objective. This technique is named variable displacement–change point detection (VD-CPD). The influence of plasmon enhancement on achievable axial localization was also quantified. Two AuNR systems with different length-to-diameter aspect ratios (AR, where AR=1.86 and 3.90) were selected for this purpose; the AR=1.86 and AR=3.90 had nonresonant and resonant longitudinal surface plasmon resonances (LSPR) energies, respectively, with the laser fundamental. Matching the fundamental wave LSPR energies resulted in increased axial localizations. Power-dependent analysis of the LSPR-mediated nonlinear optical images revealed that resonantly excited AuNRs results in third-order signals. The axial localization provided by VD-CPD exceeds what could be obtained using astigmatic imaging alone by a factor of 2.5. This advance will facilitate the in-depth study of photonic materials and complex biological environments that can benefit from increased axial position determinations.

Triple Modal Coherent Nonlinear Imaging with Vibrational Contrast

By combining spectroscopic contrast with imaging speed, coherent Raman scattering (CRS) microscopy techniques have reiterated the usefulness of vibrational microscopy. In the nonlinear optical (NLO) microscope, CRS is often combined with other nonlinear optical signals, such as two-photon excited fluorescence or second-harmonic generation, providing images with multimodal contrast. However, it is also possible to expand the vibrational capabilities of the NLO microscope, namely through incorporation of other vibrationally selective nonlinear optical signals beyond CRS. Recently, it has been shown that vibrationally resonant sum-frequency generation (SFG) microscopy can be integrated into the imaging platform as well, offering additional vibrational spectroscopic probing capabilities. In this contribution, we highlight the concept of triple-modal vibrational microscopy, which combines CRS, SFG, and third-order sum-frequency generation on a single laser-scanning microscope. These three vibrationally sensitive NLO imaging techniques provide complementary information about the sample, allowing for more detailed molecular spectroscopic studies of biological specimens. We review the principles of the three light-matter interactions that underlie the techniques and discuss some of the imaging properties of the triple-modal vibrational microscope. We provide several imaging examples and conclude with discussing some of the outstanding challenges.

Nonlinear QR code based optical image encryption using spiral phase transform, equal modulus decomposition and singular value decomposition

In this study, we propose a quick response (QR) code based nonlinear optical image encryption technique using spiral phase transform (SPT), equal modulus decomposition (EMD) and singular value decomposition (SVD). First, the primary image is converted into a QR code and then multiplied with a spiral phase mask (SPM). Next, the product is spiral phase transformed with particular spiral phase function, and further, the EMD is performed on the output of SPT, which results into two complex images, Z1 and Z2. Among these, Z1 is further Fresnel propagated with distance d, and Z2 is reserved as a decryption key. Afterwards, SVD is performed on Fresnel propagated output to get three decomposed matrices i.e. one diagonal matrix and two unitary matrices. The two unitary matrices are modulated with two different SPMs and then, the inverse SVD is performed using the diagonal matrix and modulated unitary matrices to get the final encrypted image. Numerical simulation results confirm the validity and effectiveness of the proposed technique. The proposed technique is robust against noise attack, specific attack, and brutal force attack. Simulation results are presented in support of the proposed idea.

Nonlinear optical imaging of extracellular matrix proteins.

Over the last 2 decades, nonlinear imaging methods such as multiharmonic imaging microscopy (MHIM) have become powerful approaches for the label-free visualization of biological structures. Multiharmonic signals are generated when an intense electromagnetic field propagates through a sample that either has a specific molecular orientation or exhibits certain physical properties. It can provide complementary morphological information when integrated with other nonlinear optical imaging techniques such as two-photon excitation (TPE). Here, we present the necessary methodology to implement an integrated approach for multiharmonic and TPE imaging of the mouse aorta using a commercial two-photon microscope. This approach illustrates how to differentiate the microstructure of the mouse aorta that are due to collagen fibrils and elastic laminae under 820 and 1230nm excitation. Our method also demonstrates how to perform multiharmonic generation by reflectance of the forwardly propagating emission signal. The ability to visualize biological samples without additional genetically targeted or chemical stains makes MHIM well suited for studying the morphology of the mouse aorta and has the potential to be applied to other collagen and elastin-rich tissues.

Multimodal nonlinear optical imaging of unstained retinas in the epi-direction with a sub-40 fs Yb-fiber laser.

Ultrafast lasers have potential use in ophthalmology for diagnoses through non-invasive imaging as well as for surgical therapies or for evaluating pharmacological therapies. New ultrafast laser sources, operating at 1.07 μm and sub-40 fs pulse durations, offer exciting possibilities in multiphoton imagining of the retina as the bulk of the eye is relatively transparent to this wavelength, three-photon excitation is not absorbed by DNA, and this wavelength has a greater penetration depth compared to the commonly used 800 nm Ti:Sapphire laser. In this work, we present the first epi-direction detected cross-section and depth-resolved images of unstained isolated retinas obtained using multiphoton microscopy with an ultrafast fiber laser centered at 1.07 μm and a ~38 fs pulse duration. Spectral and temporal characterization of the autofluorescence signals show two distinct regions; the first one from the nerve fiber layer to the inner receptor layer, and the second being the retinal pigmented epithelium and choroid.

Multimodal Nonlinear Optical Imaging for Sensitive Detection of Multiple Pharmaceutical Solid-State Forms and Surface Transformations.

Two nonlinear imaging modalities, coherent anti-Stokes Raman scattering (CARS) and sum-frequency generation (SFG), were successfully combined for sensitive multimodal imaging of multiple solid-state forms and their changes on drug tablet surfaces. Two imaging approaches were used and compared: (i) hyperspectral CARS combined with principal component analysis (PCA) and SFG imaging and (ii) simultaneous narrowband CARS and SFG imaging. Three different solid-state forms of indomethacin-the crystalline gamma and alpha forms, as well as the amorphous form-were clearly distinguished using both approaches. Simultaneous narrowband CARS and SFG imaging was faster, but hyperspectral CARS and SFG imaging has the potential to be applied to a wider variety of more complex samples. These methodologies were further used to follow crystallization of indomethacin on tablet surfaces under two storage conditions: 30 °C/23% RH and 30 °C/75% RH. Imaging with (sub)micron resolution showed that the approach allowed detection of very early stage surface crystallization. The surfaces progressively crystallized to predominantly (but not exclusively) the gamma form at lower humidity and the alpha form at higher humidity. Overall, this study suggests that multimodal nonlinear imaging is a highly sensitive, solid-state (and chemically) specific, rapid, and versatile imaging technique for understanding and hence controlling (surface) solid-state forms and their complex changes in pharmaceuticals.

Polarization and angular sensibility in the natural hyperbolic hexagonal boron nitride arrays

This paper demonstrates a tunable and flexible metamaterial with the cuboid hexagonal boron nitride (h-BN) arrays on a dielectric substrate. We numerically investigated the effect of the polarization together with the incident angle of the probe light on the transmission spectra of metamaterials and designed different geometric structures tuning the resonant frequency near the Reststrahlen bands of the h-BN in the mid-infrared. We further simulated the electric field distribution and calculated charge density associated to the specific responses that respectively exhibit the strong electric dipole resonances between the adjacent unit cells and inside the h-BN cuboids. The results show that both the positions of the resonance and the coupling strength strongly depend on geometric construction and corresponding external excitation condition. Hence the amplitude of the resonance can be actively modulated via altering excitation conditions and the metamaterial arrays can be designed to control the resonance in different spectra positions. Such metamaterials are important for its strong anisotropy and the extraordinary sensitivity which may find numerous applications in the design of metamaterials with specific sensing, nonlinear optical response and imaging capabilities creating new avenues for the development of devices in the mid-infrared.

Synthesis, nonlinear optical properties and cellular imaging of hybrid ZnS nanoparticles capped with conjugated terpyridine derivatives

A novel organic/inorganic nanohybrids which consisted of ligand L (L = 2-[(2-hydroxy-ethyl)-(4-[2,2′;6′,2″] terpyridin-4′-yl-phenyl)-amino]-ethanol), an optical terpyridine derivative and ZnS nanoparticles (NPs), had been prepared through a solution-phase synthesis technique. The intermolecular interactions at the interface between ZnS and the ligand components were analyzed by FT-IR, far-IR, UV–Vis absorption spectroscopy, XRD and TEM. Particular properties had been shown by fluorescence spectra, fluorescence lifetime, Raman spectrum, aggregation emission and non-linear optical response. The results indicated that the nano-composite L–ZnS NPs had an obvious aggregation-induced emission in ethanol/n-hexane mixtures, and had larger two-photon absorption (TPA) when compared to the free ligand L. The data for the TPA cross-section value (σ = 16,247.8 GM), nonlinear refractive index (γ = 4.46 × 10−13 cm2 W−1) and the third order nonlinear polarizability [Imχ (3) (esu) = 1.13 × 10−14] were measured and discussed. Meanwhile, due to the laser irradiation induced charge transfer from the ligand to ZnS NPs, the composite could be potentially applied in vitro and in vivo cellular imaging.