Actomyosin contractility drives bile regurgitation as an early response during obstructive cholestasis.

BACKGROUND & AIMS: A wide range of liver diseases manifest as biliary obstruction, or cholestasis. However, the sequence of molecular events triggered as part of the early hepatocellular homeostatic response in obstructive cholestasis is poorly elucidated. Pericanalicular actin is known to accumulate during obstructive cholestasis. Therefore, we hypothesized that the pericanalicular actin cortex undergoes significant remodeling as a regulatory response to obstructive cholestasis. METHODS: In vivo investigations were performed in a bile duct-ligated mouse model. Actomyosin contractility was assessed using sandwich-cultured rat hepatocytes transfected with various fluorescently labeled proteins and pharmacological inhibitors of actomyosin contractility. RESULTS: Actomyosin contractility induces transient deformations along the canalicular membrane, a process we have termed inward blebbing. We show that these membrane intrusions are initiated by local ruptures in the pericanalicular actin cortex; and they typically retract following repair by actin polymerization and actomyosin contraction. However, above a certain osmotic pressure threshold, these inward blebs pinch away from the canalicular membrane into the hepatocyte cytoplasm as large vesicles (2-8µm). Importantly, we show that these vesicles aid in the regurgitation of bile from the bile canaliculi. CONCLUSION: Actomyosin contractility induces the formation of bile-regurgitative vesicles, thus serving as an early homeostatic mechanism against increased biliary pressure during cholestasis. LAY SUMMARY: Bile canaliculi expand and contract in response to the amount of secreted bile, and resistance from the surrounding actin bundles. Further expansion due to bile duct blockade leads to the formation of inward blebs, which carry away excess bile to prevent bile build up in the canaliculi.

Multiphoton microscopy imaging of developing tooth germs.

BACKGROUND/PURPOSE: Traditionally, tooth germ is observed by histological investigation with hematoxylin and eosin stain and information may loss during the process. The purpose of this study is to use multiphoton laser fluorescence microscopy to observe the developing tooth germs of mice for building up the database of the images of tooth germs and compare with those from conventional histological analysis. METHODS: Tooth germs were isolated from embryonic and newborn mice with age of Embryonic Day 14.5 and Postnatal Days 1, 3, 5, and 7. RESULTS: Comparison of the images of tooth germ sections in multiphoton microscopy with the images of histology was performed for investigating the molar tooth germs. It was found that various signals arose from different structures of tooth germs. Pre-dentin and dentin have strong second-harmonic generation signals, while ameloblasts and enamel tissues were shown with strong autofluorescence signals. CONCLUSION: In this study, a novel multiphoton microscopy database of images from developing tooth germs in mice was set up. We confirmed that multiphoton laser microscopy is a powerful tool for investigating the development of tooth germ and is worthy for further application in the study of tooth regeneration.

Multiphoton imaging of the monosachharide induced formation of fluorescent advanced glycation end products in tissues.

We studied the in vitro rate of fluorescent advanced glycation end products (fAGEs) formation with multiphoton microscopy in different porcine tissues (aorta, cornea, kidney, dermis, and tendon). These tissues were treated with d-glucose, d-galactose, and d-fructose, three primary monosaccharides found in human diets. We found that the use of d-fructose resulted in the highest glycation rate, followed by d-galactose and then d-glucose. Moreover, compared to non-collagen tissue constituents such as elastic fibers and cells, the rate of tissue glycation was consistently higher in collagen, suggesting that collagen is a more sensitive target for fAGE formation. However, we also found that collagen in different tissues exhibits different rates of fAGE formation, with slower rates observed in tightly packed tissues such as cornea and tendon. Our study suggests that for fAGE to be developed into a long-term glycemic biomarker, loosely organized collagen tissues located in the proximity of vasculature may be the best targets.

Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning.

In this study, a microscope based on spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. Key features of this microscope are the integrations of a 10 kHz repetition rate ultrafast amplifier featuring high instantaneous peak power (maximum 400 µJ/pulse at a 90 fs pulse width) and a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled camera into a spatiotemporal focusing microscope. This configuration can produce multiphoton images with an excitation area larger than 200 × 100 µm² at a frame rate greater than 100 Hz (current maximum of 200 Hz). Brownian motions of fluorescent microbeads as small as 0.5 µm were observed in real-time with a lateral spatial resolution of less than 0.5 µm and an axial resolution of approximately 3.5 µm. Furthermore, second harmonic images of chicken tendons demonstrate that the developed widefield multiphoton microscope can provide high resolution z-sectioning for bioimaging.

Investigation of two-photon excited fluorescence increment via crosslinked bovine serum albumin.

The two-photon excited fluorescence (TPEF) increments of two dyes via bovine serum albumin (BSA) microstructures fabricated by the two-photon crosslinking technique were investigated. One is Rose Bengal (RB) with a high non-radiative decay rate, while the other is Eosin Y with a low non-radiative decay rate. Experimental results demonstrate that the quantum yield and lifetime of RB are both augmented via crosslinked BSA microstructures. Compared with theoretical analysis, this result indicates that the non-radiative decay rate of RB is decreased; hence, the quenched effect induced by BSA solution is suppressed. However, the fluorescence lifetime of Eosin Y is acutely abated despite the augmented quantum yield for the two-photon crosslinking processing from BSA solution. This result deduces that the radiative decay rate increased. Furthermore, the increased TPEF intensity and lifetime of RB correlated with the concentration of fabricated crosslinked BSA microstructures through pulse selection of the employed femtosecond laser is demonstrated and capable of developing a zone-plate-like BSA microstructure.

Dynamic development of the extracellular matrix in chick cornea.

Understanding the mechanisms of structural organization of corneal stroma is scientifically interesting and may lead to advances in corneal tissue engineering. In the past, we used the combination of second harmonic generation and fast Fourier transform to study a number of topics including the structural alteration of human keratoconus. Recently, we applied this approach to studying changes of chick embryonic cornea during development. As numerous studies were performed with ex vivo specimens, a thorough understanding of the parameters associated with the development process cannot be achieved until experiments are performed in living chick embryos.

Determination of collagen nanostructure from second-order susceptibility tensor analysis.

A model is proposed to describe the polarization dependence of second harmonic generation (SHG) from type I collagen fibrils. The model is based on sum-frequency vibrational spectrum experiments that attribute the molecular origins of collagen second-order susceptibility to the peptide groups in the backbone of the collagen α-helix and the methylene groups in the pyrrolidine rings. Applying our model to a polarization SHG (P-SHG) experiment leads to a predicted collagen I peptide pitch-angle of 45.82° ± 0.46° and methylene pitch-angle of 94.80° ± 0.97°. Compared to a previous model that accounts for only the peptide contribution, our results are more consistent with the x-ray diffraction determination of collagen-like peptide. Application of our model to type II collagen from rat trachea cartilage leads to similar results. The peptide pitch-angle of 45.72° ± 1.17° is similar to that of type I collagen, but a different methylene pitch-angle of 97.87° ± 1.79° was found. Our work demonstrates that far-field P-SHG measurements can be used to extract molecular structural information of collagen fibers.

Optimizing silver film for surface plasmon-coupled emission induced two-photon excited fluorescence imaging.

In this study, the optimal condition of a silver (Ag) film deposited on a cover slip for surface plasmon-coupled emission (SPCE) induced two-photon excited fluorescence (TPEF) based on an objective-based, total internal reflection (TIR) microscope was investigated. According to the theoretical simulations of local electric field enhancement and fluorescence coupled emission efficiency, the thickness of the Ag film should be about 40 nm in order to maximize the TPEF collection efficiency by the objective. The deposited Ag film with a germanium seed layer on a cover slip exhibits additional improvement in surface smoothness by reducing variations in surface roughness to below 1.0 nm, thereby reduces local hot spots which degrade the image uniformity. Moreover, an Ag film with a 20 nm-thick SiO2 spacer not only prevents damage caused through interaction with the aqueous solution under high laser power irradiance, but also reduces the fluorescence quenching effect by the Ag film. By optimizing the Ag film thickness, surface smoothness, and a protective dielectric spacer, efficient TIR TPEF imaging can be achieved through SPCE.

Fabrication of gold nanorods-doped, bovine serum albumin microstructures via multiphoton excited photochemistry.

In this study, three-dimensional (3D) crosslinked bovine serum albumin (BSA) microstructures containing gold nanorods (AuNRs) were fabricated via multiphoton excited photochemistry using Rose Bengal (RB) as the photoactivator. To retain AuNRs in the 3D crosslinked BSA microstructures, the laser wavelength was chosen for two-photon RB absorption for improved two-photon crosslinking efficiency, but not for enhancing the longitudinal plasmon resonance of AuNRs which may result in photothermal damage of AuNRs. Furthermore, with two-photon excitation of RB via AuNRs plasmonics, the laser power can be reduced by about 30%. As a result, 3D BSA microstructures containing AuNRs can be successfully fabricated. The AuNRs-doped BSA microstructures can be applied in biomedical scaffolds with plasmonic properties such as two-photon luminescence imaging and photothermal therapy.

Enhanced two-photon excited fluorescence in three-dimensionally crosslinked bovine serum albumin microstructures.

In this study, the intensity of two-photon excited fluorescence (TPEF) of xanthene dye, Rose Bengal (RB), was significantly enhanced via bovine serum albumin (BSA) microstructures fabricated by the two-photon crosslinking (TPC) technique. The RB was utilized as the photoactivator in the TPC processing and the enhanced TPEF intensity correlates with the concentration of fabricated crosslinked BSA microstructures via the power control and pulse selection of the employed femtosecond laser. As a result, fabrication of three-dimensional BSA microstructures can be simultaneously monitored by the use of TPEF intensity. The crosslinked BSA microstructures synthesized may be used as an ordered biomaterial for fluorescence enhancement.

Studies of intracorneal distribution and cytotoxicity of quantum dots: risk assessment of eye exposure.

The cornea is a potential route of exposure and drug administration for nanoparticles. In this work, we use noninvasive two-photon microscopic imaging to study the distribution and permeability pathway of CdSe/ZnS core/shell quantum dots (QDs) capped with three different functional groups through the cornea. With no additional staining, the two-photon image clearly discloses that fluorescent QDs penetrate and reside within the interlamellar space of second harmonic generating collagenous stroma when the corneal epithelium barrier is injured. An in vitro cytotoxicity test using bovine corneal stromal cells incubated individually with all three kinds of QDs indicates that the cell viability decreases significantly as the QD concentration and incubation period increased. The results also show that the specific QDs influence corneal stromal cell viability up to a significant magnitude of 50% under a relatively low concentration (5-20 nM) and short exposure period (24-48 h). Furthermore, two-photon imaging shows that QDs can be retained within the cornea up to 26 days in an in vivo mouse model. On the basis of our in vivo and in vitro data, we conclude that QDs can penetrate and be retained within cornea long enough to cause consequential cytotoxicity, under the circumstance in which the corneal epithelium barrier is injured. Since corneal abrasion is quite a common situation in daily life, our work raises public attention to the potential risk of eye exposure to nanoparticles.

Comments on "Cell regulation of collagen fibril macrostructure during corneal morphogenesis" by Koudouna et al.

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Hemoglobin autofluorescence as potential long-term glycemic marker in the rat animal model.

Diabetes is a serious disease whose patients often require long-term care. Blood glucose and intermediate glycation product of glycated hemoglobin (HbA1c) are, at best, surrogate biomarkers of disease progression. There is indication that advanced glycation end products (AGEs) better reflect diabetic risks. In this study, we explored the use of red blood cells (RBCs) and lysed hemoglobin (Hb) autofluorescence (AF) as potential biomarkers of diabetic complication. AF spectra measured under 370 nm excitation reveals that both RBC and Hb fluorescence in the 420 to 600 nm region. At early time points following diabetic induction in rats, AF increase in lysed Hb is more dramatic compared to that of RBCs. Moreover, we found significance variance of Hb autofluorescence despite relatively constant HbA1c levels. Furthermore, we found that although a correlation exists between AGE autofluorescence and HbA1c levels, the lack of complete correspondence suggests that the rate of AGE production differs significantly among different rats. Our results suggest that with additional development, both RBC and Hb autofluorescence from lysed RBCs may be used act long-term glycemic markers for diabetic complications in patients.

Multiphoton imaging of the effect of monosaccharide diffusion and formation of fluorescent advanced end products in porcine aorta.

Prolonged exposure of tissues to elevated blood sugar levels lead to the formation of advanced glycation end products (AGEs), thus contributing to diabetic complications. Since the vascular system is in immediate contact with blood, diabetic effects on aorta is a major health concern. However, the relative effect of the diffusion of sugar molecular through the vascular wall and the rate of AGE formation is not known. In this study, we aim to address this issue by incubating excised porcine aorta in D-glucose, D-galactose, and D-fructose solutions for different periods. The tissue specimens were then excised for multiphoton imaging of autofluorescence intensity profiles across the aorta wall. We found that for Days 4 to 48 incubation, autofluorescence is constant along the radial direction of the aorta sections, suggesting that monosaccharide diffusion is rapid in comparison to the rate of formation of fluorescent AGEs (fAGEs). Moreover, we found that in porcine aorta, the rate of fAGE formation of D-fructose and D-glucose are factors 2.08 and 1.14 that of D-galactose. Our results suggest that for prolonged exposure of the cardiovascular system to elevated monosaccharides 4 days or longer, damage to the aorta is uniform throughout the tissues.

Single-Shot Quantitative Polarization Imaging of Complex Birefringent Structure Dynamics.

Polarization light microscopes are powerful tools for probing molecular order and orientation in birefringent materials. While a number of polarization microscopy techniques are available to access steady-state properties of birefringent samples, quantitative measurements of the molecular orientation dynamics on the millisecond time scale have remained a challenge. We propose polarized shearing interference microscopy (PSIM), a single-shot quantitative polarization imaging method, for extracting the retardance and orientation angle of the laser beam transmitting through optically anisotropic specimens with complex structures. The measurement accuracy and imaging performance of PSIM are validated by imaging a birefringent resolution target and a bovine tendon specimen. We demonstrate that PSIM can quantify the dynamics of a flowing lyotropic chromonic liquid crystal in a microfluidic channel at an imaging speed of 506 frames per second (only limited by the camera frame rate), with a field-of-view of up to 350 × 350 µm2 and a diffraction-limit spatial resolution of ~2 µm. We envision that PSIM will find a broad range of applications in quantitative material characterization under dynamical conditions.

Second harmonic generation imaging - a new method for unraveling molecular information of starch.

We present a new method, second harmonic generation (SHG) imaging for the study of starch structure. SHG imaging can provide the structural organization and molecular orientation information of bio-tissues without centrosymmetry. In recent years, SHG has proven its capability in the study of crystallized bio-molecules such as collagen and myosin. Starch, the most important food source and a promising future energy candidate, has, for a decade, been shown to exhibit strong SHG response. By comparing SHG intensity from different starch species, we first identified that the SHG-active molecule is amylopectin, which accounts for the crystallinity in starch granules. With the aid of SHG polarization anisotropy, we extracted the complete χ((2)) tensor of amylopectin, which reflects the underlying molecular details. Through χ((2)) tensor analysis, three-dimensional orientation and packing symmetry of amylopectin are determined. The helical angle of the double-helix in amylopectin is also deduced from the tensor, and the value corresponds well to previous X-ray studies, further verifying amylopectin as SHG source. It is noteworthy that the nm-sized structure of amylopectin inside a starch granule can be determined by this far-field optical method with 1-µm excitation wavelength. Since SHG is a relatively new tool for plant research, a detailed understanding of SHG in starch structure will be useful for future high-resolution imaging and quantitative analyses for food/energy applications.

Multiple release kinetics of targeted drug from gold nanorod embedded polyelectrolyte conjugates induced by near-infrared laser irradiation.

The conjugates of gold nanorods and the model drug, fluorescein isothiocyanate (FITC), embedded inside polyelectrolytes (GNRs/FITC@PLE) were synthesized to study the release kinetics of FITC under femtosecond near-infrared (NIR) laser irradiation. The optical and structural properties of GNRs/FITC@PLE conjugates before and after laser treatments were examined using UV-vis spectroscopy, confocal microscopy, and transmission electron microscopy (TEM). The release of FITC from the conjugates was induced by the heat generated from gold nanorods under laser irradiation. The concentration of released FITC was measured as the time of continuous and periodic laser irradiation was varied. Within 5 min of the laser exposure, the release rates of FITC exhibited zero-order and first-order kinetics under continuous and periodic irradiation, respectively. Furthermore, a drug release system was designed based on the conjugates of gold nanorods and the anticancer drug, paclitaxel (PTX), embedded inside polyelectrolytes (GNRs/PTX@PLE). The conjugates were applied for in vitro studies with breast cancer cells. The release of PTX from the conjugates was triggered by NIR laser irradiation, and the inhibition rates of the cells showed strong dependencies on the irradiation modes and time. The results suggested that the multiple releases of PTX from the conjugates can be controlled by laser irradiation within a long period of time. Our system holds great potential for future therapeutic applications on breast cancers.

Photophysical mechanisms of collagen modification by 80 MHz femtosecond laser.

Photophysical mechanisms of collagen photomodification (CFP) by the use of a 80 MHz, 780 nm femtosecond titanium-sapphire laser were investigated. Our observation that the decrease in collagen second harmonic generation and increase in two-photon autofluorescence intensity occurred primarily at sites where photoproducts were present suggested that the photoproducts may act to facilitate the CFP process. Laser power study of CFP indicated that the efficiency of the process depended on the sixth power of the laser intensity. Furthermore, it was demonstrated that CFP can be used for bending and cutting of collagen fibers and creating 3D patterns within collagen matrix with high precision (~2 µm).

Multiphoton fabrication of freeform polymer microstructures with gold nanorods.

In this study, three-dimensional (3D) polyacrylamide microstructures containing gold nanorods (AuNRs) were fabricated by two-photon polymerization (TPP) using Rose Bengal (RB) as the photoinitiator. To retain AuNRs in the 3D polymer microstructures, the laser wavelength was chosen for two-photon RB absorption for improved TPP efficiency, but not for enhancing the longitudinal plasmon resonance of AuNRs which may result in photothermal damage of AuNRs. After TPP processing, the laser wavelength was tuned for the longitudinal plasmon resonance and the laser power was increased to beyond the damage threshold of the AuNRs for reshaping the AuNRs into gold nanospheres. As a result, AuNRs in designated positions of the fabricated 3D microstructures can be achieved. Two-photon luminescence from the doped AuNRs can also act as contrast agent for the visualization of 3D polymer microstructures.

Quantification of collagen structural changes during chick corneal development.

As the most abundant structural mammalian protein, collagen has been implicated in the pathogenesis of numerous diseases such as osteogenesis imperfecta, and cancer. In the case of cornea, abnormal cornea development can lead to conditions such as agenesis, megalocornea, microcornea, and cornea plana. Therefore, understanding the mechanisms of collagen assembly during development may contribute to the prevention or treatment of corneal diseases. In this study, we applied fast Fourier transform second harmonic generation microscopy to quantify parameters of corneal structures during chick development. Our results show that both the rotational pitch and overall rotational angle of corneal stroma modulate between E9 and E19. In addition, we found that corneal structures between left and right corneas are highly correlated during development.