Development of Fluorescently Labeled, Functional Type I Collagen Molecules.

While de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are examined. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ≈1.2 labels per collagen molecule preserves these characteristics. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labeled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development at low concentration, the labeled collagen is incorporated into a fine extracellular matrix (ECM) network associated with the cells within 24 h.

Method for measurement of collagen monomer orientation in fluorescence microscopy.

SIGNIFICANCE: Collagen is the most abundant protein in vertebrates and is found in tissues that regularly experience tension, compression, and shear forces. However, the underlying mechanism of collagen fibril formation and remodeling is poorly understood. AIM: We explore how a collagen monomer is visualized using fluorescence microscopy and how its spatial orientation is determined. Defining the orientation of collagen monomers is not a trivial problem, as the monomer has a weak contrast and is relatively small. It is possible to attach fluorescence tags for contrast, but the size is still a problem for detecting orientation using fluorescence microscopy. APPROACH: We present two methods for detecting a monomer and classifying its orientation. A modified Gabor filter set and an automatic classifier trained by convolutional neural network based on a synthetic dataset were used. RESULTS: By evaluating the performance of these two approaches with synthetic and experimental data, our results show that it is possible to determine the location and orientation with an error of ∼37 deg of a single monomer with fluorescence microscopy. CONCLUSIONS: These findings can contribute to our understanding of collagen monomers interaction with collagen fibrils surface during fibril formation and remodeling.

Measuring collagen fibril diameter with differential interference contrast microscopy.

Collagen fibrils, linear arrangements of collagen monomers, 20-500 nm in diameter, comprising hundreds of molecules in their cross-section, are the fundamental structural unit in a variety of load-bearing tissues such as tendons, ligaments, skin, cornea, and bone. These fibrils often assemble into more complex structures, providing mechanical stability, strength, or toughness to the host tissue. Unfortunately, there is little information available on individual fibril dynamics, mechanics, growth, aggregation and remodeling because they are difficult to image using visible light as a probe. The principle quantity of interest is the fibril diameter, which is difficult to extract accurately, dynamically, in situ and non-destructively. An optical method, differential interference contrast (DIC) microscopy has been used to visualize dynamic structures that are as small as microtubules (25 nm diameter) and has been shown to be sensitive to the size of objects smaller than the wavelength of light. In this investigation, we take advantage of DIC microscopy's ability to report dimensions of nanometer scale objects to generate a curve that relates collagen diameter to DIC edge intensity shift (DIC-EIS). We further calibrate the curve using electron microscopy and demonstrate a linear correlation between fibril diameter and the DIC-EIS. Using a non-oil immersion, 40x objective (NA 0.6), collagen fibril diameters between ~100 nm to ~ 300 nm could be obtained with ±11 and ±4 nm accuracy for dehydrated and hydrated fibrils, respectively. This simple, nondestructive, label free method should advance our ability to directly examine fibril dynamics under experimental conditions that are physiologically relevant.

Near infrared spectroscopy for measuring changes in bone hemoglobin content after exercise in individuals with spinal cord injury.

Bone blood perfusion has an essential role in maintaining a healthy bone. However, current methods for measuring bone blood perfusion are expensive and highly invasive. This study presents a custom built near-infrared spectroscopy (NIRS) instrument to measure changes in bone blood perfusion. We demonstrated the efficacy of this device by monitoring oxygenated and deoxygenated hemoglobin changes in the human tibia during and after exercise in able-bodied and in individuals with spinal cord injury (SCI), a population with known impaired peripheral blood perfusion. Nine able-bodied individuals and six volunteers with SCI performed a 10 min rowing exercise (functional electrical stimulation rowing for those with SCI). With exercise, during rowing, able-bodied showed an increase in deoxygenated hemoglobin in the tibia. Post rowing, able-bodied showed an increase in total blood content, characterized by an increase in total hemoglobin content due primarily to an increase in deoxygenated hemoglobin. During rowing and post-rowing, those with SCI showed no change in total blood content in the tibia. The current study demonstrates that NIRS can non-invasively detect changes in hemoglobin concentration in the tibia. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:183-191, 2018.

Super-resolution structured illumination in optically thick specimens without fluorescent tagging.

This research extends the work of Hoffman et al. to provide both sectioning and super-resolution using random patterns within thick specimens. Two methods of processing structured illumination in reflectance have been developed without the need for a priori knowledge of either the optical system or the modulation patterns. We explore the use of two deconvolution algorithms that assume either Gaussian or sparse priors. This paper will show that while both methods accomplish their intended objective, the sparse priors method provides superior resolution and contrast against all tested targets, providing anywhere from ∼1.6× to ∼2× resolution enhancement. The methods developed here can reasonably be implemented to work without a priori knowledge about the patterns or point spread function. Further, all experiments are run using an incoherent light source, unknown random modulation patterns, and without the use of fluorescent tagging. These additional modifications are challenging, but the generalization of these methods makes them prime candidates for clinical application, providing super-resolved noninvasive sectioning in vivo.

Enhanced tagging of light utilizing acoustic radiation force with speckle pattern analysis.

In optical imaging, the depth and resolution are limited due to scattering. Unlike light, scattering of ultrasound (US) waves in tissue is negligible. Hybrid imaging methods such as US-modulated optical tomography (UOT) use the advantages of both modalities. UOT tags light by inducing phase change caused by modulating the local index of refraction of the medium. The challenge in UOT is detecting the small signal. The displacement induced by the acoustic radiation force (ARF) is another US effect that can be utilized to tag the light. It induces greater phase change, resulting in a stronger signal. Moreover, the absorbed acoustic energy generates heat, resulting in change in the index of refraction and a strong phase change. The speckle pattern is governed by the phase of the interfering scattered waves; hence, speckle pattern analysis can obtain information about displacement and temperature changes. We have presented a model to simulate the insonation processes. Simulation results based on fixed-particle Monte Carlo and experimental results show that the signal acquired by utilizing ARF is stronger compared to UOT. The introduced mean irradiance change (MIC) signal reveals both thermal and mechanical effects of the focused US beam in different timescales. Simulation results suggest that variation in the MIC signal can be used to generate a displacement image of the medium.

Single-image structured illumination using Hilbert transform demodulation.

Structured illumination microscopy (SIM) achieves sectioning at depth by removing undesired light from out-of-focus planes within a specimen. However, it generally requires at least three modulated images with discrete phase shifts of 0, 120, and 240 deg to produce sectioning. Using a Hilbert transform demodulation, it is possible to produce both sectioning and depth information relative to a reference plane (i.e., a coverslip) using only a single image. The specimen is modulated at a known frequency, and the unmodulated portion of the image is estimated. These two components are used to provide a high-quality sectioned image containing both axial and lateral information of an object. The sectioning resolution with a single image is on par with that of a control three-image SIM. We are also able to show that when used with three images of discrete phase, this method produces better contrast within a turbid media than the traditional SIM technique. Because the traditional SIM requires alignment of three different phases, small differences in optical path length can introduce strong artifacts. Using the single-image technique removes this dependency, greatly improving sectioning in turbid media. Multiple targets with various depths and opaqueness are considered, including human skin in vivo, demonstrating a quick and useful way to provide noninvasive sectioning in real time.

Diffusion model for ultrasound-modulated light.

Researchers use ultrasound (US) to modulate diffusive light in a highly scattering medium like tissue. This paper analyzes the US-optical interaction in the scattering medium and derives an expression for the US-modulated optical radiance. The diffusion approximation to the radiative transport equation is employed to develop a Green's function for US-modulated light. The predicted modulated fluence and flux are verified using finite-difference time-domain simulations. The Green's function is then utilized to illustrate the modulated reflectance as the US-optical interaction increases in depth. The intent of this paper is to focus on high US frequencies necessary for high-resolution imaging because they are of interest for applications such as phase conjugation.

Analysis and modeling of an ultrasound-modulated guide star to increase the depth of focusing in a turbid medium.

The effects of strong scattering in tissue limit the depth to which light may be focused. However, it has been shown that scattering may be reduced utilizing adaptive optics with a focused ultrasound (US) beam guidestar. The optical signal traveling through the US beam waist is frequency shifted and may be isolated with demodulation. This paper utilizes a multiphysics simulation to model the optical and US interactions in both synthetic tissue and random scattering media. The results illustrate that optical energy may be focused within a turbid medium utilizing a US guidestar. The results also suggest that optical energy travels preferentially along optical channels within a turbid medium.

Stepwise multiphoton activation fluorescence reveals a new method of melanin detection.

The stepwise multiphoton activated fluorescence (SMPAF) of melanin, activated by a continuous-wave mode near infrared (NIR) laser, reveals a broad spectrum extending from the visible spectra to the NIR and has potential application for a low-cost, reliable method of detecting melanin. SMPAF images of melanin in mouse hair and skin are compared with conventional multiphoton fluorescence microscopy and confocal reflectance microscopy (CRM). By combining CRM with SMPAF, we can locate melanin reliably. However, we have the added benefit of eliminating background interference from other components inside mouse hair and skin. The melanin SMPAF signal from the mouse hair is a mixture of a two-photon process and a third-order process. The melanin SMPAF emission spectrum is activated by a 1505.9-nm laser light, and the resulting spectrum has a peak at 960 nm. The discovery of the emission peak may lead to a more energy-efficient method of background-free melanin detection with less photo-bleaching.

Structured illumination microscopy using random intensity incoherent reflectance.

Depth information is resolved from thick specimens using a modification of structured illumination. By projecting a random projection pattern with varied spatial frequencies that is rotated while capturing images, sectioning can be performed using an incoherent light source in reflectance only. This provides a low-cost solution to obtaining information similar to that produced in confocal microscopy and other methods of structured illumination, without the requirement of complex or elaborate equipment, coherent light sources, or fluorescence. The broad line width of the light emitting diode minimizes artifacts associated with speckle from the laser while also increasing the safety of the instrument. Single diffusers and cascaded diffusers are compared to provide the most efficient method for sectioning at depth. By using reflectance only, in vivo images are produced on a human subject, generating high-contrast images and providing depth information about subsurface objects.

Deep-tissue focal fluorescence imaging with digitally time-reversed ultrasound-encoded light.

Fluorescence imaging is one of the most important research tools in biomedical sciences. However, scattering of light severely impedes imaging of thick biological samples beyond the ballistic regime. Here we directly show focusing and high-resolution fluorescence imaging deep inside biological tissues by digitally time-reversing ultrasound-tagged light with high optical gain (~5×10(5)). We confirm the presence of a time-reversed optical focus along with a diffuse background-a corollary of partial phase conjugation-and develop an approach for dynamic background cancellation. To illustrate the potential of our method, we image complex fluorescent objects and tumour microtissues at an unprecedented depth of 2.5 mm in biological tissues at a lateral resolution of 36 µm×52 µm and an axial resolution of 657 µm. Our results set the stage for a range of deep-tissue imaging applications in biomedical research and medical diagnostics.

Optimization of pupil design for point-scanning and line-scanning confocal microscopy.

Both point-scanning and line-scanning confocal microscopes provide resolution and optical sectioning to observe nuclear and cellular detail in human tissues, and are being translated for clinical applications. While traditional point-scanning is truly confocal and offers the best possible optical sectioning and resolution, line-scanning is partially confocal but may offer a relatively simpler and lower-cost alternative for more widespread dissemination into clinical settings. The loss of sectioning and loss of contrast due to scattering in tissue is more rapid and more severe with a line-scan than with a point-scan. However, the sectioning and contrast may be recovered with the use of a divided-pupil. Thus, as part of our efforts to translate confocal microscopy for detection of skin cancer, and to determine the best possible approach for clinical applications, we are now developing a quantitative understanding of imaging performance for a set of scanning and pupil conditions. We report a Fourier-analysis-based computational model of confocal microscopy for six configurations. The six configurations are point-scanning and line-scanning, with full-pupil, half-pupil and divided-pupils. The performance, in terms of on-axis irradiance (signal), resolution and sectioning capabilities, is quantified and compared among these six configurations.

Enhanced melanin fluorescence by stepwise three-photon excitation.

The fluorescence of eumelanin (from Sepia officinalis and black human hair) was activated and enhanced by almost three orders of magnitude by exposure to near-infrared radiation. No activation or enhanced emission was observed when the samples were heated up to 100°C. The near-infrared irradiation caused obvious changes to the eumelanin and could be seen by fluorescence and bright field imaging. The area of enhanced emission appeared to originate from a region with changes in the morphology of the eumelanin's granule and increased with exposure time. At least two different components with enhanced fluorescence were activated and could be distinguished by their excitation properties. One component could be excited efficiently with wavelengths in the visible region and exhibited linear absorption dependence with respect to the laser power level. The second component could be excited efficiently using near-infrared wavelengths by a nonlinear process and exhibited a third-order dependence on the excitation. The third-order dependence is explained by a step-wise excited-state absorption process since the same third-order dependence was present when either continuous wave or femtosecond pulsed laser, with similar average-power levels, was used.

Mechanical and optical dynamic model of lung.

A multiscale, multiphysics model generates synthetic images of alveolar compression under spherical indentation at the visceral pleura of an inflated lung. A mechanical model connects the millimeter scale of an indenter tip to the behavior of alveoli, walls, and membrane at the micrometer scale. A finite-difference model of optical coherence tomography (OCT) generates the resulting images. Results show good agreement with the experiments performed using a unique indenter-OCT system. The images depict the physical result with the addition of refractive artifacts and speckle. Compression of the alveoli alters the refractive effects, which introduce systematic errors in the computation of alveolar volume. The complete computational model is useful to evaluate new proposed imaging instrumentation and to develop algorithms for obtaining quantitative data on deformation. Among the potential applications, a better understanding of recruitment of alveoli during inflation of a lung, obtained through a combination of models and imaging could lead to improvements in noninvasive treatment of atelectasis.

Small-angle light scattering to detect strain-directed collagen degradation in native tissue.

It has been demonstrated that there is a mechanochemical relationship between collagen and collagenolytic enzymes such that increased tensile mechanical strain reduces the enzymatic cutting rate. This mechanochemical relationship has the potential to permit directed remodelling of tissue-engineered constructs in vitro and to shed light on the generation of load-adapted collagen-based connective tissue. In this investigation, we demonstrate that small-angle light scattering (SALS) has the sensitivity to dynamically detect the preferential enzymatic degradation of a subset of unloaded collagen fibrils within differentially loaded native tissue. Detection of the difference in the relative degradation rate of unloaded fibrils versus loaded fibrils was manifested through changes in the spatial distribution of the SALS signal. Specifically, we found a linear increase in the eccentricity of the SALS data that was consistent with preferential retention of the collagen fibrils aligned with the applied tensile strain. We conclude that SALS is simple, inexpensive and may provide a useful optical screening method permitting real-time monitoring of strain-controlled tissue and construct remodelling.

Refractive effects on optical measurement of alveolar volume: a 2-D ray-tracing approach.

Lung imaging and assessment of alveoli geometry in the lung tissue is of great importance. Optical coherence tomography (OCT) is a real-time imaging technique used for this purpose, based on near-infrared interferometry, that can image several layers of distal alveoli in the lung tissue. The OCT measurements use low coherence interferometry, where light reflections from surfaces in the tissue are used to construct 2D images of the tissue. OCT images provide better depth compared to other optical microscopy techniques such as confocal reflectance and two-photon microscopy. Therefore, it is important to detect and verify optical distortions that happens with OCT, including refractive effect at the tissue-air alveoli wall interface which is not taken into account in the OCT imaging model. In this paper, the refractive effect at the tissue-air interface of the alveoli wall is modeled using exact ray tracing and direct implementation of Snell's law, and differences between alveoli area computed from OCT imaging and those measured by exact ray tracing of the OCT signal are analyzed.

Mechanical strain stabilizes reconstituted collagen fibrils against enzymatic degradation by mammalian collagenase matrix metalloproteinase 8 (MMP-8).

BACKGROUND: Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain. METHODOLOGY/PRINCIPAL FINDINGS: The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded. CONCLUSIONS/SIGNIFICANCE: In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen.

Mechanical strain enhances survivability of collagen micronetworks in the presence of collagenase: implications for load-bearing matrix growth and stability.

There has been great interest in understanding the methods by which collagen-based load-bearing tissue is constructed, grown and maintained in vertebrate animals. To date, the responsibility for this process has largely been placed with mesenchymal fibroblastic cells that are thought to fully control the morphology of load-bearing extracellular matrix (ECM). However, given clear limitations in the ability of fibroblastic cells to precisely place or remove single collagen molecules to sculpt tissue, we have hypothesized that the material itself must play a critical role in the determination of the form of structural ECM. We here demonstrate directly, using live, dynamic, differential interference contrast imaging, that mechanically strained networks of collagen fibrils, exposed to collagenase (Clostridium histolyticum), degrade preferentially. Specifically, unstrained fibrils are removed 'quickly', while strained fibrils persist significantly longer. The demonstration supports the idea that collagen networks are mechanosensitive in that they are stabilized by mechanical strain. Thus, collagen molecules (together with their complement enzymes) may comprise the basis of a smart, load-adaptive, structural material system. This concept has the potential to drastically simplify the assumed role of the fibroblast, which would need only to provide ECM molecules and mechanical force to sculpt collagenous tissue.