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     

Imaging of targeted lipid microbubbles to detect cancer cells using third harmonic generation microscopy

The use of receptor-targeted lipid microbubbles imaged by ultrasound is an innovative method of detecting and localizing disease. However, since ultrasound requires a medium between the transducer and the object being imaged, it is impractical to apply to an exposed surface in a surgical setting where sterile fields need be maintained and ultrasound gel may cause the bubbles to collapse. Multiphoton microscopy (MPM) is an emerging tool for accurate, label-free imaging of tissues and cells with high resolution and contrast. We have recently determined a novel application of MPM to be used for detecting targeted microbubble adherence to the upregulated plectin-receptor on pancreatic tumor cells. Specifically, the third-harmonic generation response can be used to detect bound microbubbles to various cell types presenting MPM as an alternative and useful imaging method. This is an interesting technique that can potentially be translated as a diagnostic tool for the early detection of cancer and inflammatory disorders.OCIS codes: (020.4180) Multiphoton processes, (170.1610) Clinical applications, (190.1900) Diagnostic applications of nonlinear optics, (320.7090) Ultrafast lasers     

Virtual optical biopsy of human adipocytes with third harmonic generation microscopy

Using the sectioning capability of third harmonic generation (THG) microscopy, we assessed the morphologic features of human adipocytes directly without fixation and labeling. At the plane of the largest cross-sectional area, both area-equivalent circular diameters (AECD) and perimeters of adipocytes were measured, and their statistical distributions were examined. We found, in patients with no cardiovascular risk factors, the average AECD of epicardial adipocytes were 70–90 μm with 11–17 μm standard deviations. In contrast, for patients with coronary artery disease, amounts of small-sized (AECD <40 µm) epicardial adipocytes were observed and the corresponding standard deviations of AECD were increased to 20–29 μm. Our results indicate that the THG tomography platform can be used to explore the histopathological features of adipocytes in clinical scenarios based on its superior resolution for virtual optical biopsy.OCIS codes: (170.6900) Three-dimensional microscopy, (190.1900) Diagnostic applications of nonlinear optics     

Imaging parameters on third harmonic transmit phasing for tissue harmonic generation

In third harmonic (3f0) transmit phasing, transmit waveforms comprising fundamental (f0) signal and 3f0 signal are used to generate both frequency-sum and frequency-difference components for manipulation of tissue harmonic amplitude. Nevertheless, the acoustic propagation of 3f0 transmit signal suffers from more severe attenuation and phase aberration than the f0 signal and hence degrades the performance of 3f0 transmit phasing. Besides, 3f0 transmit parameters such as aperture size and signal bandwidth are also influential in 3f0 transmit phasing. In this study, extensive simulations were performed to investigate the effects of these imaging parameters. Results indicate that the harmonic enhancement and suppression in 3f0 transmit phasing are compromised when the magnitude of frequency-difference component decreases in the presence of tissue attenuation and phase aberration. To compensate for the reduced frequency-difference component, a higher 3f0 transmit amplitude can be used. When the transmit parameters are concerned, a smaller 3f0 transmit aperture can provide more axially uniform harmonic enhancement and more effective suppression of harmonic amplitude. In addition, the spectral leakage signal also interferes with tissue harmonics and degrades the efficacy of 3f0 transmit phasing. Our results suggest that, in the method of 3f0 transmit phasing, the transmit amplitude, phase and aperture size of 3f0 signal should remain adjustable for optimization of clinical performance. Besides, multipulse sequences such as pulse inversion are also favorable for leakage removal in 3f0 transmit phasing.     

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.     

Imaging skeletal muscle using second harmonic generation and coherent anti-Stokes Raman scattering microscopy

We describe experimental results on label free imaging of striated skeletal muscle using second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy. The complementarity of the SHG and CARS data makes it possible to clearly identify the main sarcomere sub-structures such as actin, myosin, acto-myosin, and the intact T-tubular system as it emanates from the sarcolemma. Owing to sub-micron spatial resolution and the high sensitivity of the CARS microscopy technique we were able to resolve individual myofibrils. In addition, key organelles such as mitochondria, cell nuclei and their structural constituents were observed revealing the entire structure of the muscle functional units. There is a noticeable difference in the CARS response of the muscle structure within actin, myosin and t-tubule areas with respect to laser polarization. We attribute this to a preferential alignment of the probed molecular bonds along certain directions. The combined CARS and SHG microscopy approach yields more extensive and complementary information and has a potential to become an indispensable method for live skeletal muscle characterization.     

Fast interferometric second harmonic generation microscopy

We report the implementation of fast Interferometric Second Harmonic Generation (I-SHG) microscopy to study the polarity of non-centrosymmetric structures in biological tissues. Using a sample quartz plate, we calibrate the spatially varying phase shift introduced by the laser scanning system. Compensating this phase shift allows us to retrieve the correct phase distribution in periodically poled lithium niobate, used as a model sample. Finally, we used fast interferometric second harmonic generation microscopy to acquire phase images in tendon. Our results show that the method exposed here, using a laser scanning system, allows to recover the polarity of collagen fibrils, similarly to standard I-SHG (using a sample scanning system), but with an imaging time about 40 times shorter.OCIS codes: (180.4315) Nonlinear microscopy, (190.2620) Harmonic generation and mixing, (170.6935) Tissue characterization, (190.4180) Multiphoton processes, (190.4710) Optical nonlinearities in organic materials     

Spectroscopic second and third harmonic generation microscopy using a femtosecond laser source in the third near-infrared (NIR-III) optical window

In this study, second harmonic generation (SHG) and third harmonic generation (THG) spectroscopic imaging were performed on biological samples using a femtosecond laser source in the third near-infrared (NIR) optical window (NIR-III). Using a visible-NIR spectrometer, the SHG and THG signals were simultaneously detected and were extracted using spectral analysis. Visualization of biological samples such as cultured cells (HEK293 T), mouse brain slices, and the nematode Caenorhabditis elegans was performed in a label-free manner. In particular, in an SHG image of an entire coronal brain section (8 × 6 mm²), we observed mesh-like and filamentous structures in the arachnoid mater and wall of the cerebral ventricle, probably corresponding to the collagen fibers, cilia, and rootlet. Moreover, the THG images clearly depicted the densely packed axons in the white matter and cell nuclei at the cortex of the mouse brain slice sample and lipid-rich granules such as lipid droplets inside the nematode. The observations and conclusions drawn from this technique confirm that it can be utilized for various biological applications, including in vivo label-free imaging of living animals.     

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     

Second harmonic generation double stokes Mueller polarimetric microscopy of myofilaments

The experimental implementation of double Stokes Mueller polarimetric microscopy is presented. This technique enables a model-independent and complete polarimetric characterization of second harmonic generating samples using 36 Stokes parameter measurements at different combinations of incoming and outgoing polarizations. The degree of second harmonic polarization and the molecular nonlinear susceptibility ratio are extracted for individual focal volumes of a fruit fly larva wall muscle.OCIS codes: (180.4315) Nonlinear microscopy, (110.5405) Polarimetric imaging, (170.3880) Medical and biological imaging     

In vivo optical virtual biopsy of human oral mucosa with harmonic generation microscopy

Recent clinical studies on human skin indicated that in vivo multi-harmonic generation microscopy (HGM) can achieve sub-micron resolution for histopathological analysis with a high penetration depth and leave no energy or photodamages in the interacted tissues. It is thus highly desired to apply HGM for in vivo mucosa histopathological diagnosis. In this paper, the first in vivo optical virtual biopsy of human oral mucosa by using epi-HGM is demonstrated. We modified an upright microscope to rotate the angle of objective for in vivo observation. Our clinical study reveals the capability of HGM to in vivo image cell distributions in human oral mucosa, including epithelium and lamina propria with a high penetration depth greater than 280 μm and a high spatial resolution better than 500 nm. We also found that the third-harmonic-generation (THG) contrast on nucleus depends strongly on its thicknesses, in agreement with a numerical simulation. Besides, 4% acetic acid was found to be able to enhance the THG contrast of nucleus in oral mucosa, while such enhancement was found to decay due to the metabolic clearance of the contrast enhancer by the oral mucosa. Our clinical study indicated that, the combined epi-THG and epi-second-harmonic-generation (SHG) microscopy is a promising imaging tool for in vivo noninvasive optical virtual biopsy and disease diagnosis in human mucosa.     

Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo

Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other.OCIS codes: (170.0170) Medical optics and biotechnology, (170.1790) Confocal microscopy, (170.2520) Fluorescence microscopy, (170.4470) Ophthalmology     

Determination of chronological aging parameters in epidermal keratinocytes by in vivo harmonic generation microscopy

Skin aging is an important issue in geriatric and cosmetic dermatology. To quantitatively analyze changes in keratinocytes related to intrinsic aging, we exploited a 1230 nm-based in vivo harmonic generation microscopy, combining second- and third-harmonic generation modalities. 52 individuals (21 men and 31 women, age range 19–79) were examined on the sun-protected volar forearm. Through quantitative analysis by the standard algorithm provided, we found that the cellular and nuclear size of basal keratinocytes, but not that of granular cells, was significantly increased with advancing age. The cellular and nuclear areas, which have an increase of 0.51 μm² and 0.15 μm² per year, respectively, can serve as scoring indices for intrinsic skin aging.OCIS codes: (110.0180) Microscopy, (170.1530) Cell analysis, (170.1610) Clinical applications, (170.1870) Dermatology, (170.3880) Medical and biological imaging, (190.4160) Multiharmonic generation     

Assessment of the corneal collagen organization after chemical burn using second harmonic generation microscopy

The organization of the corneal stoma is modified due to different factors, including pathology, surgery or external damage. Here the changes in the organization of the corneal collagen fibers during natural healing after chemical burn are investigated using second harmonic generation (SHG) imaging. Moreover, the structure tensor (ST) was used as an objective tool for morphological analyses at different time points after burn (up to 6 months). Unlike control corneas that showed a regular distribution, the collagen pattern at 1 month of burn presented a non-organized arrangement. SHG signal levels noticeably decreased and individual fibers were hardly visible. Over time, the healing process led to a progressive re-organization of the fibers that could be quantified through the ST. At 6 months, the stroma distribution reached values similar to those of control eyes and a dominant direction of the fibers re-appeared. The present results show that SHG microscopy imaging combined with the ST method is able to objectively monitor the temporal regeneration of the corneal organization after chemical burn. Future implementations of this approach into clinically adapted devices would help to diagnose and quantify corneal changes, not only due to chemical damages, but also as a result of disease or surgical procedures.     

Correlation between polarization sensitive optical coherence tomography and second harmonic generation microscopy in skin

Both polarization sensitive optical coherence tomography (PS-OCT) and second harmonic generation (SHG) microscopy are 3D optical imaging methods providing information related to collagen in the skin. PS-OCT provides birefringence information which is due to the collagen composition of the skin. SHG microscopy visualizes collagen fibers in the skin based on their SHG property. These two modalities have been applied to the same skin pathologies associated with collagen changes, but their relationship has not been examined. In this study, we tried to find the relationship by imaging the same skin samples with both modalities. Various parts of the normal rat skin and burn damaged skin were imaged ex vivo, and their images were analyzed both qualitatively and quantitatively. PS-OCT images were analyzed to obtain tissue birefringence. SHG images were analyzed to obtain collagen orientation indices by applying 2D Fourier transform. The skin samples having higher birefringence values had higher collagen orientation indices, and a linear correlation was found between them. Burn damaged skin showed decreases in both parameters compared to the control skins. This relationship between the bulk and microscopic properties of skin may be useful for further skin studies.OCIS codes: (260.1440) Birefringence, (170.3890) Medical optics instrumentation, (180.4315) Nonlinear microscopy, (170.4500) Optical coherence tomography, (170.2655) Functional monitoring and imaging     

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     

Blood flow estimation with harmonic Flash Echo Imaging

Blood volume flow estimation remains an important task for the functional evaluation of internal organs. In vitro and in vivo flow estimation has been carried out using Flash Echo Imaging in harmonic mode after the administration of microbubble-based ultrasound contrast agents. The in vitro flow volume rate correlated with the constant replenishment level but less so with the video intensity decay rate. The in vivo volume flow rates obtained in four dogs agreed well with CVI-Q (time domain correlation) based flow measurement. Our experimental results indicate that the volume flow rate can be accurately estimated in vitro and in vivo using harmonic Flash Echo imaging.     

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     

In vivo harmonic generation microscopy for monitoring the height of basal keratinocytes in solar lentigines after laser depigmentation treatment

The development of solar lentigines (SLs) is related to chronic ultraviolet exposure-induced cell senescence. We have previously demonstrated that basal keratinocyte enlargement is a morphological hallmark of skin senescence correlated to the process of skin aging, while clinical studies on the long-term monitoring of the cellular morphological changes in SLs after laser treatment are lacking. In this study, we have developed the harmonic generation microscopy (HGM) for in vivo monitoring the height of basal keratinocytes (HBK) and had administered Q-switched ruby laser or picosecond 532-nm Nd:YAG laser treatment on each side of the face of 25 Asian patients with facial SLs, respectively. In vivo HGM imaging was conducted to longitudinally analyze HBK and the horizontal cell size (HCS). Before treatment, the HBK was significantly higher in the SLs lesional area than that in the adjacent normal region, whereas there was no significant difference in the HCS. After treatment, the lesional HBK remained significantly higher than normal skin regardless of the laser treatment used. Our study indicates that the basal keratinocytes remain abnormal after laser treatment and demonstrates the capability of in vivo HGM for longitudinal, quantitative monitoring of cell senescence and therapeutic effect in SLs.