3D organizational mapping of collagen fibers elucidates matrix remodeling in a hormone-sensitive 3D breast tissue model.

Hormones play an important role in normal and diseased breast tissue development. However, they can also disrupt cell-matrix interactions and their role in extracellular matrix reorganization during epithelial morphogenesis remains poorly understood, partly due to a lack of sensitive approaches for matrix characterization. Here, we assess the hormonal regulation of matrix reorganization in a three-dimensional (3D) breast tissue culture model using a novel metric, i.e., 3D directional variance, to characterize the 3D organization of collagen fibers visualized via high-resolution, second harmonic generation imaging. This metric enables resolving and quantifying patterns of spatial organization throughout the matrix surrounding epithelial structures treated with 17ß-estradiol (E2) alone, and E2 in combination with either promegestone, a progestogen, or prolactin. Addition of promegestone results in the most disorganized fibers, while the E2 alone treatment leads to the most organized ones. Location-dependent organization mapping indicates that only the prolactin treatment leads to significant heterogeneities in the regional organization of collagen fibers, with higher levels of alignment observed at the end of the elongated epithelial structures. The observed collagen organization patterns for all groups persist for tens of micrometers. In addition, a comparison between 3D directional variance and typical 2D analysis approaches reveals an improved sensitivity of the 3D metric to identify organizational heterogeneities and differences among treatment groups. These results demonstrate that 3D directional variance is sensitive to subtle changes in the extracellular micro-environment and has the potential to elucidate reciprocal cell-matrix interactions in the context of numerous applications involving the study of normal and diseased tissue morphogenesis.

Mapping metabolic changes by noninvasive, multiparametric, high-resolution imaging using endogenous contrast.

Monitoring subcellular functional and structural changes associated with metabolism is essential for understanding healthy tissue development and the progression of numerous diseases, including cancer, diabetes, and cardiovascular and neurodegenerative disorders. Unfortunately, established methods for this purpose either are destructive or require the use of exogenous agents. Recent work has highlighted the potential of endogenous two-photon excited fluorescence (TPEF) as a method to monitor subtle metabolic changes; however, mechanistic understanding of the connections between the detected optical signal and the underlying metabolic pathways has been lacking. We present a quantitative approach to detecting both functional and structural metabolic biomarkers noninvasively, relying on endogenous TPEF from two coenzymes, NADH (reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide). We perform multiparametric analysis of three optical biomarkers within intact, living cells and three-dimensional tissues: cellular redox state, NADH fluorescence lifetime, and mitochondrial clustering. We monitor the biomarkers in cells and tissues subjected to metabolic perturbations that trigger changes in distinct metabolic processes, including glycolysis and glutaminolysis, extrinsic and intrinsic mitochondrial uncoupling, and fatty acid oxidation and synthesis. We demonstrate that these optical biomarkers provide complementary insights into the underlying biological mechanisms. Thus, when used in combination, these biomarkers can serve as a valuable tool for sensitive, label-free identification of changes in specific metabolic pathways and characterization of the heterogeneity of the elicited responses with single-cell resolution.

Variability in responses observed in human white adipose tissue models.

Obesity is a risk factor for a myriad of diseases including diabetes, cardiovascular dysfunction, cirrhosis, and cancer, and there is a need for new systems to study how excess adipose tissue relates to the onset of disease processes. This study provides proof-of-concept patient-specific tissue models of human white adipose tissue to accommodate the variability in human samples. Our 3D tissue engineering approach established lipolytic responses and changes in insulin-stimulated glucose uptake from small volumes of human lipoaspirate, making this methodology useful for patient specific sample source assessments of treatment strategies, drug responses, disease mechanisms, and other responses that vary between patients. Mature unilocular cells were maintained ex vivo in silk porous scaffolds for up to a month of culture and imaged non-invasively with coherent anti-Stokes Raman scattering. Interestingly, differences in responsiveness between tissues were observed in terms of magnitude of lipolysis, ability to suppress lipolysis, differences in glucose uptake, and lipid droplet size. Body mass index was not a factor in determining tissue responsiveness; rather, it is speculated that other unknown variables in the backgrounds of different patients (ethnicity, athleticism, disease history, lifestyle choices, etc.) likely had a more significant effect on the observed differences. This study reinforces the need to account for the variability in backgrounds and genetics within the human population to determine adipose tissue responsiveness. In the future, this tissue system could be used to inform individualized care strategies-enhancing therapeutic precision, improving patient outcomes, and reducing clinical costs.

Label free monitoring of megakaryocytic development and proplatelet formation in vitro.

Megakaryopoiesis and platelet production are complex biological processes that require tight regulation of successive lineage commitment steps and are ultimately responsible for maintaining and renewing the pool of circulating platelets in the blood. Despite major advancements in the understanding of megakaryocytic biology, the detailed mechanisms driving megakaryocytic differentiation have yet to be elucidated. Here we show that automated image analysis algorithms applied to two-photon excited fluorescence (TPEF) images can non-invasively monitor structural and metabolic megakaryocyte behavior changes occurring during differentiation and platelet formation in vitro. Our results demonstrate that high-contrast, label-free two photon imaging holds great potential in studying the underlying physiological processes controlling the intricate process of platelet production.

Endogenous Two-Photon Excited Fluorescence Imaging Characterizes Neuron and Astrocyte Metabolic Responses to Manganese Toxicity.

As neurodegenerative conditions are increasingly linked to mitochondrial dysfunction, methods for studying brain cell metabolism at high spatial resolution are needed to elucidate neurodegeneration mechanisms. Two-photon excited fluorescence (TPEF) imaging is a non-destructive, high-resolution technique for studying cell metabolism via endogenous fluorescence of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). We employed TPEF to study the metabolism of primary rat astrocyte and neuronal cultures under normal growth conditions and in response to manganese (Mn) treatment. Histograms of pixel-wise optical redox ratio, defined as FAD/(FAD + NAD(P)H), revealed three distinct redox distributions and significant differences in their relative weights between astrocytes and neurons. When treated with Mn, both cell types exhibited redox ratio shifts consistent with increased oxidative stress. However, the manner in which the redox distributions was affected was distinct for the two cell types. Furthermore, NAD(P)H fluorescence lifetime imaging revealed an increase in bound NAD(P)H fraction upon Mn treatment for neurons, consistent with enhanced apoptosis. Astrocytes showed a decrease in bound fraction, possibly due to a shift towards glycolytic metabolism in response to impaired respiration. These results exhibit TPEF's utility for characterizing detailed metabolic changes of different brain cell types in response to neurotoxins.

Automated quantification of three-dimensional organization of fiber-like structures in biological tissues.

Fiber-like structures are prevalent in biological tissues, yet quantitative approaches to assess their three-dimensional (3D) organization are lacking. We develop 3D directional variance, as a quantitative biomarker of truly 3D fibrillar organization by extending the directional statistics formalism developed for describing circular data distributions (i.e. when 0° and 360° are equivalent) to axial ones (i.e. when 0° and 180° are equivalent). Significant advantages of this analysis include its time efficiency, sensitivity and ability to provide quantitative readouts of organization over different size scales of a given data set. We establish a broad range of applications for this method by characterizing collagen fibers, neuronal axons and fibroblasts in the context of cancer diagnostics, traumatic brain injury and cell-matrix interactions in developing engineered tissues. This method opens possibilities for unraveling in a sensitive, and quantitative manner the organization of essential fiber-like structures in tissues and ultimately its impact on tissue function.

Imaging mitochondrial dynamics in human skin reveals depth-dependent hypoxia and malignant potential for diagnosis.

Active changes in mitochondrial structure and organization facilitate cellular homeostasis. Because aberrant mitochondrial dynamics are implicated in a variety of human diseases, their assessment is potentially useful for diagnosis, therapy, and disease monitoring. Because current techniques for evaluating mitochondrial morphology are invasive or necessitate mitochondria-specific dyes, their clinical translation is limited. We report that mitochondrial dynamics can be monitored in vivo, within intact human skin by relying entirely on endogenous two-photon-excited fluorescence from the reduced metabolic coenzyme nicotinamide adenine dinucleotide (NADH). We established the sensitivity of this approach with in vivo, fast temporal studies of arterial occlusion-reperfusion, which revealed acute changes in the mitochondrial metabolism and dynamics of the lower human epidermal layers. In vitro hypoxic-reperfusion studies validated that the in vivo outcomes were a result of NADH fluorescence changes. To demonstrate the diagnostic potential of this approach, we evaluated healthy and cancerous human skin epithelia. Healthy tissues displayed consistent, depth-dependent morphological and mitochondrial organization patterns that varied with histological stratification and intraepithelial mitochondrial protein expression. In contrast, these consistent patterns were absent in cancerous skin lesions. We exploited these differences to successfully differentiate healthy from cancerous tissues using a predictive classification approach. Collectively, these results demonstrate that our label-free, automated, near real-time assessments of mitochondrial organization-relying solely on endogenous contrast-could be useful for accurate, noninvasive in vivo diagnosis.

Two-photon excited fluorescence of intrinsic fluorophores enables label-free assessment of adipose tissue function.

Current methods for evaluating adipose tissue function are destructive or have low spatial resolution. These limit our ability to assess dynamic changes and heterogeneous responses that occur in healthy or diseased subjects, or during treatment. Here, we demonstrate that intrinsic two-photon excited fluorescence enables functional imaging of adipocyte metabolism with subcellular resolution. Steady-state and time-resolved fluorescence from intracellular metabolic co-factors and lipid droplets can distinguish the functional states of excised white, brown, and cold-induced beige fat. Similar optical changes are identified when white and brown fat are assessed in vivo. Therefore, these studies establish the potential of non-invasive, high resolution, endogenous contrast, two-photon imaging to identify distinct adipose tissue types, monitor their functional state, and characterize heterogeneity of induced responses.

Endogenous two-photon fluorescence imaging elucidates metabolic changes related to enhanced glycolysis and glutamine consumption in precancerous epithelial tissues.

Alterations in the balance between different metabolic pathways used to meet cellular bioenergetic and biosynthetic demands are considered hallmarks of cancer. Optical imaging relying on endogenous fluorescence has been used as a noninvasive approach to assess tissue metabolic changes during cancer development. However, quantitative correlations of optical assessments with variations in the concentration of relevant metabolites or in the specific metabolic pathways that are involved have been lacking. In this study, we use high-resolution, depth-resolved imaging, relying entirely on endogenous two-photon excited fluorescence in combination with invasive biochemical assays and mass spectrometry to demonstrate the sensitivity and quantitative nature of optical redox ratio tissue assessments. We identify significant differences in the optical redox ratio of live, engineered normal and precancerous squamous epithelial tissues. We establish that while decreases in the optical redox ratio are associated with enhanced levels of glycolysis relative to oxidative phosphorylation, increases in glutamine consumption to support energy production are associated with increased optical redox ratio values. Such mechanistic insights in the origins of optical metabolic assessments are critical for exploiting fully the potential of such noninvasive approaches to monitor and understand important metabolic changes that occur in live tissues at the onset of cancer or in response to treatment.

Quantitative characterization of mineralized silk film remodeling during long-term osteoblast-osteoclast co-culture.

The goal of this study was to explore quantitative assessments of mineralized silk protein biomaterial films by co-cultures of human mesenchymal stem cell-derived osteoblasts and human acute monocytic leukemia cell line-derived osteoclasts during long-term culture (8-32 weeks). The remodeled films were quantitatively assessed using three different techniques during this extended cultivation to provide more comprehensive insight into the impact of co-cultures on surface remodeling. Scanning electron microscopy (SEM) with three dimensional surface reconstructions was used to quantitatively determine various surface morphological features and measures of roughness indicative of remodeling by the cells. Additionally, reconstructed surfaces were converted to depth images for Fourier analysis to quantify the potential fractal organization of biomineralization. The long-term remodeled films were also imaged using confocal reflectance microscopy and micro-computed tomography (micro-CT) to further quantify morphological changes. Films remodeled in co-culture demonstrated increased roughness parameters, fractal organization, and volume compared to films remodeled by osteoblasts alone. The combination of these techniques to quantify remodeling of mineralized protein films shows promise for quantifying processes related to mineralized surfaces.

Harmonic mode locking in a sliding-frequency fiber laser.

We demonstrate a sliding-frequency mode-locked (SFM) erbium fiber laser generating 20 ps pulses with center wavelengths rapidly sweeping across a spectral range of 50 nm. Excess optical nonlinearity in the laser cavity leads to multipulsing, with a tendency to tight pulse bunching (<3 ns) at the fundamental cavity frequency of 25 MHz. The addition of a parallel optical delay line, with a path difference equal to a rational fraction of the cavity length, distributes the pulses uniformly across the entire cavity and achieves a harmonic SFM up to 1 GHz. The result establishes cavity nonlinearity as a critical design parameter for picosecond wavelength-swept lasers.

Two-photon microscopy by wavelength-swept pulses delivered through single-mode fiber.

Nonlinear microscopy through flexible fiber-optic catheters has potential in clinical diagnostic applications. Here, we demonstrate a new approach based on wavelength-swept narrowband pulses that permits simple fiber-optic delivery without need of the dispersion management and allows nonmechanical beam scanning. Using 0.86 ps pulses rapidly tuned from 789 nm to 822 nm at a sweep rate of 200 Hz, we demonstrate two-photon fluorescence and second-harmonic generation imaging through a 5-m-long standard single-mode fiber.

Optical microassembly platform for constructing reconfigurable microenvironments for biomedical studies.

Cellular development is highly influenced by the surrounding microenvironment. We propose user-reconfigurable microenvironments and bio-compatible scaffolds as an approach for understanding cellular development processes. We demonstrate a model platform for constructing versatile microenvironments by fabricating morphologically complex microstructures by two-photon polymerization (2PP) and then assembling these archetypal building blocks into various configurations using multiple, real-time configurable counterpropagating-beam (CB) traps. The demonstrated capacity for handling feature-rich microcomponents may be further developed into a generalized microassembly platform.

Laser projection using generalized phase contrast.

We demonstrate experimental laser projection of a gray-level photographic image with 74% light efficiency using the generalized phase contrast (GPC) method. In contrast with a previously proposed technique [Alonzo, New J. Phys. 9, 132 (2007)], a new approach to image construction via GPC is introduced. An arbitrary phase shift filter eliminates the need for high-frequency modulation and conjugate phase encoding. This lowers device performance requirements and allows practical implementation with currently available dynamic spatial light modulators.

Computerized "drag-and-drop" alignment of GPC-based optical micromanipulation system.

In the past, aligning the counterpropagating beams in our 3D real-time generalized phase contrast (GPC) trapping system has been a task requiring moderate skills and prior experience with optical instrumentation. A ray transfer matrix analysis and computer-controlled actuation of mirrors, objective, and sample stage has made this process user friendly. The alignment procedure can now be done in a very short time with just a few drag-and-drop tasks in the user-interface. The future inclusion of an image recognition algorithm will allow the alignment process to be executed completely without any user interaction. An automated sample loading tray with a loading precision of a few microns has also been added to simplify the switching of samples under study. These enhancements have significantly reduced the level of skill and experience required to operate the system, thus making the GPC-based micromanipulation system more accessible to people with little or no technical expertise in optics.

2D optical manipulation and assembly of shape-complementary planar microstructures.

Optical trapping and manipulation offer great flexibility as a non-contact microassembly tool. Its application to the assembly of microscale building blocks may open new doors for micromachine technology. In this work, we demonstrate all-optical assembly of microscopic puzzle pieces in a fluidic environment using programmable arrays of trapping beams. Identical shape-complimentary pieces are optically fabricated with submicron resolution using two-photon polymerization (2PP) technique. These are efficiently assembled into space-filling tessellations by a multiple-beam optical micromanipulation system. The flexibility of the system allows us to demonstrate both user-interactive and computer-automated modes of serial and parallel assembly of microscale objects with high spatial and angular positioning precision.

Generalized phase contrast matched to Gaussian illumination.

We show that the generalized phase contrast method (GPC) can be used as a versatile tool for shaping an incident Gaussian illumination into arbitrary lateral beam profiles. For illustration, we use GPC in an energy-efficient phase-only implementation of various apertures that do not block light but instead effectively redirect the available photons from a bell-shaped light distribution. GPC-based generation of lateral beam profiles can thus be achieved using a simplified optical implementation as it eliminates the need for a potentially lossy initial beam shaping. The required binary phase input is simple to fabricate for static applications and can be easily reconfigured up to device frame refresh rates for dynamic applications.

Autonomous and 3D real-time multi-beam manipulation in a microfluidic environment.

The Generalized Phase Contrast (GPC) method of optical 3D manipulation has previously been used for controlled spatial manipulation of live biological specimen in real-time. These biological experiments were carried out over a time-span of several hours while an operator intermittently optimized the optical system. Here we present GPC-based optical micromanipulation in a microfluidic system where trapping experiments are computer-automated and thereby capable of running with only limited supervision. The system is able to dynamically detect living yeast cells using a computer-interfaced CCD camera, and respond to this by instantly creating traps at positions of the spotted cells streaming at flow velocities that would be difficult for a human operator to handle. With the added ability to control flow rates, experiments were also carried out to confirm the theoretically predicted axially dependent lateral stiffness of GPC-based optical traps.

GPC-based optical micromanipulation in 3D real-time using a single spatial light modulator.

Using a novel dual-beam readout with the generalized phase contrast (GPC) method, a multiple-beam 3D real-time micromanipulation system requiring only one spatial light modulator (SLM) has been realized. A theoretical framework for the new GPC scheme with two parallel illumination beams is presented and corroborated with an experimental demonstration. Three-dimensional arrays of polystyrene microbeads were assembled in the newly described system. The use of air immersion objective lenses with GPC-based optical trapping allowed the simultaneous viewing of the assemblies in two orthogonal bright-field imaging perspectives.

Helico-conical optical beams: a product of helical and conical phase fronts.

Helico-conical optical beams, different from higher-order Bessel beams, are generated with a parallel-aligned nematic liquid crystal spatial light modulator (SLM) by multiplying helical and conical phase functions leading to a nonseparable radial and azimuthal phase dependence. The intensity distributions of the focused beams are explored in two- and threedimensions. In contrast to the ring shape formed by a focused optical vortex, a helico-conical beam produces a spiral intensity distribution at the focal plane. Simple scaling relationships are found between observed spiral geometry and initial phase distributions. Observations near the focal plane further reveal a cork-screw intensity distribution around the propagation axis. These light distributions, and variations upon them, may find use for optical trapping and manipulation of mesoscopic particles.