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.

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.

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.

Second harmonic generation imaging reveals asymmetry in the rotational helicity of collagen lamellae in chicken corneas.

High tensile strength and optical clarity are unique properties of the cornea. These features are dictated by the three-dimensional architecture of corneal lamellae. Therefore, understanding the microscopic details of the cornea's structural organization may contribute to the development of artificial cornea for the treatment of corneal diseases. In this study, the combination of forward second harmonic generation (SHG) microcopy and fast Fourier-transform based image analysis was used to characterize the depth-dependent superstructure of chicken corneal stroma. Our results show that from the surface, adjacent lamellae of anterior chicken cornea lamella rotate in a counterclockwise direction, and the same rotational helicity is observed in left and right corneas. Furthermore, the overall average rotational pitch of lamellae is 0.92 ± 0.11 degree/µm which persists for 176 ± 14 µm in the anterior stroma. As depth further increased, the rate of lamellar rotation decreases. Upon reaching posterior stroma, lamellar orientation remains constant. Throughout the stroma, collagen lamellae in chicken rotate a total of 169 ± 21 degrees. The lack of lamellar rotation in posterior stroma suggests that packing efficiency cannot be used to explain the helicity of depth-dependent rotation of anterior stroma. In addition, although the right cornea has a higher rotational pitch (0.95 ± 11 vs 0.90 ± 10 degrees/µm) and thinner anterior stroma (173 ± 13 vs 179 ± 14 µm) than the left cornea, the two effects cancel each other out and result in similar total angular rotation of anterior stroma (161 ± 23 and 165 degrees ± 21). Finally, our observation of a total angular rotation of 169 ± 21 degrees shows that within experimental error, chicken cornea lamellae rotate around 180 degrees or half of a complete turn. Additional studies are needed to arrive at an explanation of chicken superstructure in three dimensions.

Dynamic visualization of the recovery of mouse hepatobiliary metabolism to acetaminophen-overdose damage.

Acetaminophen (APAP) overdose is one of the world's leading causes of drug-induced hepatotoxicity. Although traditional methods such as histological imaging and biochemical assays have been successfully applied to evaluate the extent of APAP-induced liver damage, detailed effect of how APAP overdose affect the recovery of hepatobiliary metabolism and is not completely understood. In this work, we used intravital multiphoton microscopy to image and quantify hepatobiliary metabolism of the probe 6-carboxyfluorescein diacetate in APAP-overdose mice. We analyzed hepatobiliary metabolism for up to 7 days following the overdose and found that the excretion of the probe molecule was the most rapid on Day 1 following APAP overdose and slowed down on Days 2 and 3. On Day 7, probe excretion capability has exceeded that of the normal mice, suggesting that newly regenerated hepatocytes have higher metabolic capabilities. Our approach may be further developed applied to studying drug-induced hepatotoxicity in vivo.

In vivo multiphoton kinetic imaging of the toxic effect of carbon tetrachloride on hepatobiliary metabolism.

We used intravital multiphoton microscopy to study the recovery of hepatobiliary metabolism following carbon tetrachloride (CCl4) induced hepatotoxicity in mice. The acquired images were processed by a first order kinetic model to generate rate constant resolved images of the mouse liver. We found that with progression of hepatotoxicity, the spatial gradient of hepatic function disappeared. A CCl4-induced damage mechanism involves the compromise of membrane functions, resulting in accumulation of processed 6-carboxyfluorescein molecules. At day 14 following induction, a restoration of the mouse hepatobiliary function was found. Our approach allows the study of the response of hepatic functions to chemical agents in real time and is useful for studying pharmacokinetics of drug molecules through optical microscopic imaging.

Multiphoton dynamic imaging of the effect of chronic hepatic diseases on hepatobiliary metabolism in vivo.

In this study, intravital multiphoton microscopy was used to quantitatively investigate hepatobiliary metabolism in chronic pathologies of the liver. Specifically, through the use of the probe molecule 6-carboxyfluorescein diacetate, the effects of liver fibrosis, fatty liver, and hepatocellular carcinoma on the metabolic capabilities of mouse liver were investigated. After the acquisition of time-lapse images, a first order kinetic model was used to calculate rate constant resolved images of various pathologies. It was found that the ability of the liver to metabolically process the probe molecules varies among different pathologies, with liver fibrosis and fatty liver disease negatively impacted the uptake, processing, and excretion of molecules. The approach demonstrated in this work allows the study of the response of hepatic functions to different pathologies in real time and is useful for studying processes such as pharmacokinetics through direct optical imaging.

Fibroblasts Slow Conduction Velocity in a Reconstituted Tissue Model of Fibrotic Cardiomyopathy.

Myocardial function deteriorates over the course of fibrotic cardiomyopathy, due to electrophysiological and mechanical effects of myofibroblasts that are not completely understood. Although a range of experimental model systems and associated theoretical treatments exist at the levels of isolated cardiomyocytes and planar co-cultures of myofibroblasts and cardiomyocytes, interactions between these cell types at the tissue level are less clear. We studied these interactions through an engineered heart tissue (EHT) model of fibrotic myocardium and a mathematical model of the effects of cellular composition on EHT impulse conduction velocity. The EHT model allowed for modulation of cardiomyocyte and myofibroblast volume fractions, and observation of cell behavior in a three-dimensional environment that is more similar to native heart tissue than is planar cell culture. The cardiomyocyte and myofibroblast volume fractions determined the retardation of impulse conduction (spread of the action potential) in EHTs as measured by changes of the fluorescence of the Ca2+ probe, Fluo-2. Interpretation through our model showed retardation far in excess of predictions by homogenization theory, with conduction ceasing far below the fibroblast volume fraction associated with steric percolation. Results point to an important multiscale structural role of myofibroblasts in attenuating impulse conduction in fibrotic cardiomyopathy.

Probing Multiscale Collagenous Tissue by Nonlinear Microscopy.

Imaging of thick biological samples has been very challenging because of severe light scattering. The transparent cornea with the unique organization of stromal collagen makes it a good candidate for deep imaging and is responsible for mechanical strength and optical clarity of the eye. However, limitation on traditional histology method provides incomplete spatial information and details on the structural organization of corneal tissue is still not sound. Second harmonic generation (SHG) microscopy is a noninvansive and nonstained technique to characterize the macromolecular organization of collagen in biological tissues. Through the combination of SHG microcopy and optimized Fourier-transform analysis, adult and embryonic chick corneas are investigated. Our results show that the anterior stroma demonstrates a fanlike distribution of rotated fibrous lamellae. In comparison with the anterior structure, the posterior stroma maintains a nonrotating pattern while increasing the depth of corneal tissue. In particular, the rotational pattern in anterior stroma exhibits a potential role of corneal maturation. Moreover, SHG microscopy in combination with the Fourier-transform-based analysis exhibits a useful tool in determination of collagen alignment in biological tissues and discrimination of diseases.

Remodeling by fibroblasts alters the rate-dependent mechanical properties of collagen.

UNLABELLED: The ways that fibroblasts remodel their environment are central to wound healing, development of musculoskeletal tissues, and progression of pathologies such as fibrosis. However, the changes that fibroblasts make to the material around them and the mechanical consequences of these changes have proven difficult to quantify, especially in realistic, viscoelastic three-dimensional culture environments, leaving a critical need for quantitative data. Here, we observed the mechanisms and quantified the mechanical effects of fibroblast remodeling in engineered tissue constructs (ETCs) comprised of reconstituted rat tail (type I) collagen and human fibroblast cells. To study the effects of remodeling on tissue mechanics, stress-relaxation tests were performed on ETCs cultured for 24, 48, and 72h. ETCs were treated with deoxycholate and tested again to assess the ECM response. Viscoelastic relaxation spectra were obtained using the generalized Maxwell model. Cells exhibited viscoelastic damping at two finite time constants over which the ECM showed little damping, approximately 0.2s and 10-30s. Different finite time constants in the range of 1-7000s were attributed to ECM relaxation. Cells remodeled the ECM to produce a relaxation time constant on the order of 7000s, and to merge relaxation finite time constants in the 0.5-2s range into a single time content in the 1s range. Results shed light on hierarchical deformation mechanisms in tissues, and on pathologies related to collagen relaxation such as diastolic dysfunction. STATEMENT OF SIGNIFICANCE: As fibroblasts proliferate within and remodel a tissue, they change the tissue mechanically. Quantifying these changes is critical for understanding wound healing and the development of pathologies such as cardiac fibrosis. Here, we characterize for the first time the spectrum of viscoelastic (rate-dependent) changes arising from the remodeling of reconstituted collagen by fibroblasts. The method also provides estimates of the viscoelastic spectra of fibroblasts within a three-dimensional culture environment. Results are of particular interest because of the ways that fibroblasts alter the mechanical response of collagen at loading frequencies associated with cardiac contraction in humans.

Signal enhancement in multiphoton imaging by the use of coated glass substrates.

In nonlinear optical imaging of biological specimens, more than half of the generated luminescence signal is lost, when signal collection is performed in the epi-illuminated geometry. In this study, we enhanced the collected luminescence signal by the use of alternating multiply-coated layers of tantalum pentoxide (Ta2O5) and silicon dioxide (SiO2) on standard microscope cover glasses that has high transmission in the near-infrared wavelength region and high reflection of the visible, luminescence signal. Our coating is biocompatible, allows visual examination of the specimens and optimize collection of the luminescence signal. We demonstrated this approach on a number of specimens including sulforhodamine solution, fluorescence microspheres, and labeled 3T3 cells. In all cases, the use of coated cover glass enhanced signal, optimally by a factor of about 2. Image analysis of labeled 3T3 cells also shows signal enhancement did not contribute to additional photobleaching. Our results show that properly designed coated cover glass can enhance detected signal in multiphoton microscopy and result in improved image quality.

Physically-induced cytoskeleton remodeling of cells in three-dimensional culture.

Characterizing how cells in three-dimensional (3D) environments or natural tissues respond to biophysical stimuli is a longstanding challenge in biology and tissue engineering. We demonstrate a strategy to monitor morphological and mechanical responses of contractile fibroblasts in a 3D environment. Cells responded to stretch through specific, cell-wide mechanisms involving staged retraction and reinforcement. Retraction responses occurred for all orientations of stress fibers and cellular protrusions relative to the stretch direction, while reinforcement responses, including extension of cellular processes and stress fiber formation, occurred predominantly in the stretch direction. A previously unreported role of F-actin clumps was observed, with clumps possibly acting as F-actin reservoirs for retraction and reinforcement responses during stretch. Responses were consistent with a model of cellular sensitivity to local physical cues. These findings suggest mechanisms for global actin cytoskeleton remodeling in non-muscle cells and provide insight into cellular responses important in pathologies such as fibrosis and hypertension.