Imaging granularity of leukocytes with third harmonic generation microscopy

Using third harmonic generation (THG) microscopy, we demonstrate that granularity differences of leukocytes can be revealed without a label. Excited by a 1230 nm femtosecond laser, THG signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one order of magnitude smaller. Interestingly, the characteristic THG features can also be observed in vivo to track the newly recruited leukocytes following lipopolysaccharide (LPS) challenge. These results suggest that label-free THG imaging may provide timely tracking of leukocyte movement without disturbing the normal cellular or physiological status.OCIS codes: (180.4315) Nonlinear microscopy, (170.1530) Cell analysis     

Label-free multi-photon imaging using a compact femtosecond fiber laser mode-locked by carbon nanotube saturable absorber

We demonstrate label-free multi-photon imaging of biological samples using a compact Er³⁺-doped femtosecond fiber laser mode-locked by a single-walled carbon nanotube (CNT). These compact and low cost lasers have been developed by various groups but they have not been exploited for multiphoton microscopy. Here, it is shown that various multiphoton imaging modalities (e.g. second harmonic generation (SHG), third harmonic generation (THG), two-photon excitation fluorescence (TPEF), and three-photon excitation fluorescence (3PEF)) can be effectively performed on various biological samples using a compact handheld CNT mode-locked femtosecond fiber laser operating in the telecommunication window near 1560nm. We also show for the first time that chlorophyll fluorescence in plant leaves and diatoms can be observed using 1560nm laser excitation via three-photon absorption.OCIS codes: (170.5810) Scanning microscopy, (180.6900) Three-dimensional microscopy, (140.7090) Ultrafast lasers, (190.4180) Multiphoton processes     

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

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

Label-free imaging of age-related cardiac structural changes in non-human primates using multiphoton nonlinear microscopy

Heart failure is one of the most common causes of morbidity and mortality. Both maturational abnormalities and age-associated cardiac pathologies contribute to heart failure. Imaging-based assessment to discern detailed cardiac structure at various maturational stages is imperative for understanding mechanisms behind cardiac growth and aging. Using multiphoton nonlinear optical microscopy (NLOM) based label-free imaging, we investigated cardiac structural composition in a human-relevant aging model, the common marmoset monkey (Callithrix jacchus). Animals were divided into three different age groups including neonatal, young adult and old. By devising a unique strategy for segregating collagen and myosin emitted second harmonic generation (SHG) signals, we performed a volumetric assessment of collagen and total scattering tissue (collagen + myosin). Aged marmoset hearts exhibited an increase in collagen and total scattering tissue volume at the sites of severe tissue remodelling indicating age-related cardiac fibrosis. Significantly low scattering tissue volume in neonatal marmoset hearts was attributed to a lack of binding between the myofibrils in maturing cardiac tissue. Comprehensive quantitative assessment of structural composition during maturation and aging of marmoset hearts revealed significant differences in myofibril length, alignment, curvature and angular distribution. In conclusion, label-free high-resolution NLOM facilitates visualization and quantification of subcellular structural features for understanding vital age-related morphological alterations in the marmoset heart.     

Dual-wavelength multimodal multiphoton microscope with SMA-based depth scanning

We report on a multimodal multiphoton microscopy (MPM) system with depth scanning. The multimodal capability is realized by an Er-doped femtosecond fiber laser with dual output wavelengths of 1580 nm and 790 nm that are responsible for three-photon and two-photon excitation, respectively. A shape-memory-alloy (SMA) actuated miniaturized objective enables the depth scanning capability. Image stacks combined with two-photon excitation fluorescence (TPEF), second harmonic generation (SHG), and third harmonic generation (THG) signals have been acquired from animal, fungus, and plant tissue samples with a maximum depth range over 200 µm.     

Second harmonic generation microscopy investigation of the crystalline ultrastructure of three barley starch lines affected by hydration

Second harmonic generation (SHG) microscopy is employed to study changes in crystalline organization due to altered gene expression and hydration in barley starch granules. SHG intensity and susceptibility ratio values (R’_SHG) are obtained using reduced Stokes-Mueller polarimetric microscopy. The maximum R’_SHG values occur at moderate moisture indicating the narrowest orientation distribution of nonlinear dipoles from the cylindrical axis of glucan helices. The maximum SHG intensity occurs at the highest moisture and amylopectin content. These results support the hypothesis that SHG is caused by ordered hydrogen and hydroxyl bond networks which increase with hydration of starch granules.OCIS codes: (170.3880) Medical and biological imaging, (170.4580) Optical diagnostics for medicine     

Video-rate high-resolution single-pixel nonscanning photoacoustic microscopy

Optical-resolution photoacoustic microscopy (OR-PAM) is widely utilized in biomedical applications because of its ability to noninvasively image biological tissues in vivo while providing high-resolution morphological and functional information. However, one drawback of conventional OR-PAM is its imaging speed, which is restricted by the scanning technique employed. To achieve a higher imaging frame rate, we present video-rate high-resolution single-pixel nonscanning photoacoustic microscopy (SPN-PAM), which utilizes Fourier orthogonal basis structured planar illumination to overcome the above-mentioned limitations. A 473 × 473 µm² imaging field of view (FOV) with 3.73 µm lateral resolution and video-rate imaging of 30 Hz were achieved. In addition, in both in vitro cell and in vivo mouse vascular hemodynamic imaging experiments, high-quality images were obtained at ultralow sampling rates. Thus, the proposed high-resolution SPN-PAM with video-rate imaging speed provides new insights into high-speed PA imaging and could be a powerful tool for rapid biological imaging.     

Widely-tunable synchronisation-free picosecond laser source for multimodal CARS,SHG, and two-photon microscopy

We demonstrate a continuous wave (CW) seeded synchronization-free optical parametric amplifier (OPA) pumped by a picosecond, 1 µm laser and show its performance when used as a simple yet powerful source for label-free coherent anti-Stokes Raman scattering (CARS), concurrent second harmonic generation (SHG), and two-photon fluorescence microscopy in an epi-detection geometry. The average power level of above 175 mW, spectral resolution of 8 cm⁻¹, and 2 ps pulse duration are well optimized for CARS microscopy in bio-science and bio-medical imaging systems. Our OPA is a much simpler setup than either the “gold-standard” laser and optical parametric oscillator (OPO) combination traditionally used for CARS imaging, or the more recently developed OPA systems pumped with femtosecond pulses [1]. Rapid and accurate tuning between resonances was achieved by changing the poled channels and temperature of the periodically-poled lithium niobate (PPLN) OPA crystal together with the OPA seed wavelength. The Pump-Stokes frequency detuning range fully covered the C-H stretching band used for the imaging of lipids. By enabling three multiphoton techniques using a compact, synchronization free laser source, our work paves the way for the translation of label-free multi-photon microscopy imaging from biomedical research to an imaging based diagnostic tool for use in the healthcare arena.     

Label-free multi-photon imaging of dysplasia in Barrett’s esophagus

Barrett’s esophagus (BE) is a metaplastic disorder where dysplastic and early cancerous changes are invisible to the naked eye and where the practice of blind biopsy is hampered by large sampling errors. Multi-photon microscopy (MPM) has emerged as an alternative solution for fast and label-free diagnostic capability for identifying the histological features with sub-micron accuracy. We developed a compact, inexpensive MPM system by using a handheld mode-locked fiber laser operating at 1560nm to study mucosal biopsies of BE. The combination of back-scattered THG, back-reflected forward THG and SHG signals generate images of cell nuclei and collagen, leading to label-free diagnosis in Barrett’s.OCIS codes: (020.4180) Multiphoton processes, (180.4315) Nonlinear microscopy, (180.5810) Scanning microscopy, (320.7090) Ultrafast lasers     

A novel approach for assessing cardiac fibrosis using label-free second harmonic generation

To determine whether second harmonic generation (SHG) can be used as a novel and improved label-free technique for detection of collagen deposition in the heart. To verify whether SHG will allow accurate quantification of altered collagen deposition in diseased hearts following hypertrophic remodelling. Minimally invasive transverse aortic banding (MTAB) of mouse hearts was used to generate a reproducible model of cardiac hypertrophy. Physiological and functional assessment of hypertrophic development was performed using echocardiography and post-mortem analysis of remodelled hearts. Cardiac fibroblasts were isolated from sham-operated and hypertrophied hearts and proliferation rates compared. Multi-photon laser scanning microscopy was used to capture both two-photon excited autofluorescence (TPEF) and SHG images simultaneously in two channels. TPEF images were subtracted from SHG images and the resulting signal intensities from ventricular tissue sections were calculated. Traditional picrosirius red staining was used to verify the suitability of the SHG application. MTAB surgery induced significant hypertrophic remodelling and increased cardiac fibroblast proliferation. A significant increase in the density of collagen fibres between hypertrophic and control tissues (p < 0.05) was evident using SHG. Similar increases and patterns of staining were observed using parallel traditional picrosirius red staining of collagen. Label-free SHG microscopy provides a new alternative method for quantifying collagen deposition in fibrotic hearts.     

Hypoperfusion in nucleus accumbens in chronic migraine using 3D pseudo-continuous arterial spin labeling imaging MRI

BackgroundNucleus accumbens (NAcc) played an important role in pain mediation, and presents changes of neuronal plasticity and functional connectivity. However, less is known about altered perfusion of NAcc in chronic migraine (CM). The aim of this study is to investigate the altered perfusion of the NAcc in CM using a MR three-dimensional pseudo-continuous arterial spin labeling (3D PCASL) imaging.MethodsThirteen CM patients and 15 normal controls (NC) were enrolled and underwent 3D PCASL and brain structural imaging. The cerebral blood flow (CBF) images were co-registered with the brain structural images, and the volume and CBF value of NAcc were extracted from the raw brain structural images and co-registered CBF images using an individual NAcc mask, which was obtained from the AAL3 template under transformation by the inverse deformation field generated from the segmentation of the brain structural images. The independent sample t test and receiver operating characteristic (ROC) curve was used to investigate the altered volume and perfusion of the NAcc in CM patients.ResultsThere was no significant difference for the volume of bilateral NAccs between CM and NC (p > 0.05). CM presented a lower CBF value (49.34 ± 6.09 ml/100 mg/min) compared with that of NC (55.83 ± 6.55 ml/100 mg/min) in left NAcc (p = 0.01), while right NAcc showed no significant difference between CM and NC (p = 0.11). ROC analysis identified that the area under the curve was 0.73 (95CI% 0.53–0.88) with cut-off value 48.63 ml/100 mg/min with sensitivity 50.00% and specificity 93.33%. The correlation analysis found a negative correlation between the CBF value of the left NAcc and VAS score (r = -0.61, p = 0.04).ConclusionHypoperfusion of the left NAcc was observed in CM, which could be considered as a potential diagnostic imaging biomarker in CM.     

Imaging morphodynamics of human blood cells in vivo with video-rate third harmonic generation microscopy

With a video-rate third harmonic generation (THG) microscopy system, we imaged the micro-circulation beneath the human skin without labeling. Not only the speed of circulation but also the morpho-hydrodynamics of blood cells can be analyzed. Lacking of nuclei, red blood cells (RBCs) shows typical parachute-like and hollow-core morphology under THG microscopy. Quite different from RBCs, every now and then, round and granule rich blood cells with strong THG contrast appear in circulation. The corresponding volume densities in blood, evaluated from their frequencies of appearance and the velocity of circulation, fall within the physiological range of human white blood cell counts.OCIS codes: (170.6900) Three-dimensional microscopy, (190.1900) Diagnostic applications of nonlinear optics     

Ultrastructural features of collagen in thyroid carcinoma tissue observed by polarization second harmonic generation microscopy

Changes in collagen ultrastructure between malignant and normal human thyroid tissue were investigated ex vivo using polarization second harmonic generation (SHG) microscopy. The second-order nonlinear optical susceptibility tensor component ratio and the degree of linear polarization (DOLP) of the SHG signal were measured. The ratio values are related to the collagen ultrastructure, while DOLP indicates the relative amount of coherent signal and incoherent scattering of SHG. Increase in ratio values and decrease in DOLP were observed for tumor tissue compared to normal thyroid, indicating higher ultrastructural disorder in tumor collagen.OCIS codes: (180.4315) Nonlinear microscopy, (170.3880) Medical and biological imaging, (170.4730) Optical pathology, (170.6935) Tissue characterization     

Quantitative SHG imaging in osteoarthritis model mice,implying a diagnostic application

Osteoarthritis (OA) restricts the daily activities of patients and significantly decreases their quality of life. The development of non-invasive quantitative methods for properly diagnosing and evaluating the process of degeneration of articular cartilage due to OA is essential. Second harmonic generation (SHG) imaging enables the observation of collagen fibrils in live tissues or organs without staining. In the present study, we employed SHG imaging of the articular cartilage in OA model mice ex vivo. Consequently, three-dimensional SHG imaging with successive image processing and statistical analyses allowed us to successfully characterize histopathological changes in the articular cartilage consistently confirmed on histological analyses. The quantitative SHG imaging technique presented in this study constitutes a diagnostic application of this technology in the setting of OA.OCIS codes: (170.3880) Medical and biological imaging, (190.0190) Nonlinear optics, (180.4315) Nonlinear microscopy, (100.6890) Three-dimensional image processing, (190.1900) Diagnostic applications of nonlinear optics, (170.6935) Tissue characterization, (170.4580) Optical diagnostics for medicine     

Functional second harmonic generation microscopy probes molecular dynamics with high temporal resolution

Second harmonic generation (SHG) microscopy is a powerful tool for label free ex vivo or in vivo imaging, widely used to investigate structure and organization of endogenous SHG emitting proteins such as myosin or collagen. Polarization resolved SHG microscopy renders supplementary information and is used to probe different molecular states. This development towards functional SHG microscopy is calling for new methods for high speed functional imaging of dynamic processes. In this work we present two approaches with linear polarized light and demonstrate high speed line scan measurements of the molecular dynamics of the motor protein myosin with a time resolution of 1 ms in mammalian muscle cells. Such a high speed functional SHG microscopy has high potential to deliver new insights into structural and temporal molecular dynamics under ex vivo or in vivo conditions.OCIS codes: (180.4315) Nonlinear microscopy, (190.2620) Harmonic generation and mixing, (170.2655) Functional monitoring and imaging     

Maxillary third molars with horizontal impaction: A cross-sectional study using computed tomography in young Japanese patients

ObjectiveTo examine the anatomical characteristics of impacted maxillary third molars to help predict and prevent possible adverse events during extraction.MethodsThis retrospective study enrolled young Japanese patients that were assessed for third molar extraction. Patients with maxillary third molar impaction presenting with no space between the maxillary second molar and the occlusal surface of the maxillary third molar were analysed using computed tomography data.ResultsIn this study, a total of 663 patients aged 20–29 years were examined for third molar extraction and 26 teeth in 23 patients were analysed. The mean ± SD angle between the second and third molar axes was 54.2° ± 7.5° and the mean ± SD length of the maxillary third molar was 16.1 ± 1.9 mm. The maxillary third molars showed close or extensive contact with the maxillary sinus and computed tomography did not show any bone from the maxillary sinus floor. Furthermore, in all patients, the crowns of the third molars were not completely covered by the alveolar bone.ConclusionsMaxillary third molars with horizontal impaction showed close or extensive contact with the maxillary sinus and their crowns were not completely covered by the alveolar bone.     

Second harmonic generation microscopy of otoconia

The origin of second harmonic generation (SHG) signal in otoconia was investigated. SHG signal intensity from otoconia was compared to pure calcite crystals, given calcite is the primary component of otoconia and is known to emit surface SHG. The SHG intensity from calcite was found to be ∼41× weaker than the SHG intensity from otoconia signifying that the SHG signal from otoconia is likely generated from the organic matrix. Furthermore, the SHG intensity from otoconia increased when treated with a chelating agent known to dissolve calcite which confirms that calcite is not the source of SHG. Additionally, polarization-resolved SHG microscopy imaging revealed that the arrangement of the SHG emitters is radial and can form highly ordered domains.     

Predictive value on diffusion weighted imaging scores for basilar artery occlusion after endovascular treatment

PurposeTo assess the predictive value of three scoring systems based on diffusion weighted imaging in basilar artery occlusion patients after endovascular treatment.MethodsWe analyzed clinical and radiological data of patients with basilar artery occlusion from January 2010 to June 2019, with modified Rankin Scale of 0–2 and 3–6 defined as favorable outcome and unfavorable outcome at three months. Diffusion weighted imaging posterior circulation ASPECTS Score (DWI pc-ASPECT Score), Renard diffusion weighted imaging Score, and diffusion weighted imaging Brainstem Score were used to evaluate the early ischemic changes.ResultsThere were a total of 88 basilar artery occlusion patients enrolled in the study after endovascular treatment, with 33 of them getting a favorable outcome. According to the analysis, the time from onset to puncture within 12 h (odds ratio: 4.34; 95% confidence interval: 1.55–12.16; P = 0.01), presence of collateral flow via PCoA (odds ratio: 0.31; 95%CI: 0.12–0.79; P = 0.01) or between PICA and SCA (odds ratio: 0.18; 95%CI: 0.07–0.47; P = 0.00), equal or less than 15 points on baseline NIHSS (area under the curve 0.79, 95% CI 0.69–0.89; sensitivity = 69.1%, specificity = 81.8%; P = 0.00), and equal or less than 1.5 points on diffusion weighted imaging Renard score (area under the curve 0.63, 95% CI 0.51–0.75; sensitivity = 83.6%, specificity = 39.4%; P = 0.046) were independently associated with favorable outcome.ConclusionsRenard diffusion weighted imaging score may be an independent predictor of functional outcome in basilar artery occlusion patients after endovascular treatment.     

High-speed multifocus phase imaging in thick tissue

Phase microscopy is widely used to image unstained biological samples. However, most phase imaging techniques require transmission geometries, making them unsuited for thick sample applications. Moreover, when applied to volumetric imaging, phase imaging generally requires large numbers of measurements, often making it too slow to capture live biological processes with fast 3D index-of-refraction variations. By combining oblique back-illumination microscopy and a z-splitter prism, we perform phase imaging that is both epi-mode and multifocus, enabling high-speed 3D phase imaging in thick, scattering tissues with a single camera. We demonstrate here 3D qualitative phase imaging of blood flow in chick embryos over a field of view of 546 × 546 × 137 µm³ at speeds up to 47 Hz.     

Imaging and modeling collagen architecture from the nano to micro scale

The collagen meshwork plays a central role in the functioning of a range of tissues including cartilage, tendon, arteries, skin, bone and ligament. Because of its importance in function, it is of considerable interest for studying development, disease and regeneration processes. Here, we have used second harmonic generation (SHG) to image human tissues on the hundreds of micron scale, and developed a numerical model to quantitatively interpret the images in terms of the underlying collagen structure on the tens to hundreds of nanometer scale. Focusing on osteoarthritic changes in cartilage, we have demonstrated that this combination of polarized SHG imaging and numerical modeling can estimate fibril diameter, filling fraction, orientation and bundling. This extends SHG microscopy from a qualitative to quantitative imaging technique, providing a label-free and non-destructive platform for characterizing the extracellular matrix that can expand our understanding of the structural mechanisms in disease.OCIS codes: (170.0170) Medical optics and biotechnology, (190.0190) Nonlinear optics