Retinal synaptic regeneration via microfluidic guiding channels.

In vitro culture of dissociated retinal neurons is an important model for investigating retinal synaptic regeneration (RSR) and exploring potentials in artificial retina. Here, retinal precursor cells were cultured in a microfluidic chip with multiple arrays of microchannels in order to reconstruct the retinal neuronal synapse. The cultured retinal cells were physically connected through microchannels. Activation of electric signal transduction by the cells through the microchannels was demonstrated by administration of glycinergic factors. In addition, an image-based analytical method was used to quantify the synaptic connections and to assess the kinetics of synaptic regeneration. The rate of RSR decreased significantly below 100 µM of inhibitor glycine and then approached to a relatively constant level at higher concentrations. Furthermore, RSR was enhanced by chemical stimulation with potassium chloride. Collectively, the microfluidic synaptic regeneration chip provides a novel tool for high-throughput investigation of RSR at the cellular level and may be useful in quality control of retinal precursor cell transplantation.

Image-inspired 3D multiphoton excited fabrication of extracellular matrix structures by modulated raster scanning.

Multiphoton excited photochemistry is a powerful 3D fabrication tool that produces sub-micron feature sizes. Here we exploit the freeform nature of the process to create models of the extracellular matrix (ECM) of several tissues, where the design blueprint is derived directly from high resolution optical microscopy images (e.g. fluorescence and Second Harmonic Generation). To achieve this goal, we implemented a new form of instrument control, termed modulated raster scanning, where rapid laser shuttering (10 MHz) is used to directly map the greyscale image data to the resulting protein concentration in the fabricated scaffold. Fidelity in terms of area coverage and relative concentration relative to the image data is ~95%. We compare the results to an STL approach, and find the new scheme provides significantly improved performance. We suggest the method will enable a variety of cell-matrix studies in cancer biology and also provide insight into generating scaffolds for tissue engineering.

Mesenchymal stem cell interactions with 3D ECM modules fabricated via multiphoton excited photochemistry.

To understand complex micro/nanoscale ECM stem cell interactions, reproducible in vitro models are needed that can strictly recapitulate the relative content and spatial arrangement of native tissue. Additionally, whole ECM proteins are required to most accurately reflect native binding dynamics. To address this need, we use multiphoton excited photochemistry to create 3D whole protein constructs or "modules" to study how the ECM governs stem cell migration. The constructs were created from mixtures of BSA/laminin (LN) and BSA alone, whose comparison afforded studying how the migration dynamics are governed from the combination of morphological and ECM cues. We found that mesenchymal stem cells interacted for significantly longer durations with the BSA/LN constructs than pure BSA, pointing to the importance of binding cues of the LN. Critical to this work was the development of an automated system with feedback based on fluorescence imaging to provide quality control when synthesizing multiple identical constructs.

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.

The discrimination of type I and type II collagen and the label-free imaging of engineered cartilage tissue.

Using excitation polarization-resolved second harmonic generation (SHG) microscopy, we measured SHG intensity as a function of the excitation polarization angle for type I and type II collagens. We determined the second order susceptibility (χ((2))) tensor ratios of type I and II collagens at each pixel, and displayed the results as images. We found that the χ((2)) tensor ratios can be used to distinguish the two types of collagen. In particular, we obtained χ(zzz)/χ(zxx) = 1.40 ± 0.04 and χ(xzx)/χ(zxx) = 0.53 ± 0.10 for type I collagen from rat tail tendon, and χ(zzz)/χ(zxx) = 1.14 ± 0.09 and χ(xzx)/χ(zxx) = 0.29 ± 0.11 for type II collagen from rat trachea cartilage. We also applied this methodology on the label-free imaging of engineered cartilage tissue which produces type I and II collagen simultaneously. By displaying the χ((2)) tensor ratios in the image format, the variation in the χ((2)) tensor ratios can be used as a contrast mechanism for distinguishing type I and II collagens.

Discrimination of collagen in normal and pathological skin dermis through second-order susceptibility microscopy.

Polarization-resolved, second harmonic generation (P-SHG) microscopy at single pixel resolution is utilized for medical diagnosis of pathological skin dermis. In analyzing the large area, pixel by pixel, second-order susceptibility of normal and pathological skin dermis, we found that P-SHG can be used to distinguish normal and dermal pathological conditions of keloid, morphea, and dermal elastolysis. Specifically, we found that the second order susceptibility tensor ratio of d(33)/d(31) for normal skins is 1.27+/-0.20, while the corresponding values for keloid, morphea, and dermal elastolysis are respectively 1.67+/-0.29, 1.79+/-0.30, and 1.75+/-0.31. We also found that the histograms of the d(33)/d(31) ratio for the pathological skins contain two peak values and are 1.5 times wider than that of the normal case, suggesting that the pathological dermal collagen fibers tend to be more structurally heterogeneous. Our work demonstrates that pixel-resolved, second-order susceptibility microscopy is effective for detecting heterogeneity in spatial distribution of collagen fibers and maybe used for future clinical diagnosis and in vivo studies of collagen pathological conditions.

Characterization of optical-aberration-induced lateral and axial image inhomogeneity in multiphoton microscopy.

The effects of off-axis optical aberration in multiphoton microscopy and the resulting lateral and axial image inhomogeneity are investigated. The lateral inhomogeneity of the scanning field is demonstrated by second harmonic generation (SHG) imaging of fasciae and two-photon fluorescence (TPF) microscopy of thin fluorescent samples. Furthermore, refractive index mismatch-caused intensity attenuation of the TPF signal at central and peripheral regions of the scanning frame is measured using homogeneous 10-microM sulforhodamine B samples with refractive indexes of 1.33 and around 1.465. In addition to characterizing image field convexity, we also found that image resolution degrades away from the optical axis. These effects need to be accounted for in both qualitative and quantitative multiphoton imaging applications.

Image heterogeneity correction in large-area, three-dimensional multiphoton microscopy.

Large-area multiphoton laser scanning microscopy (LMLSM) can be applied in biology and medicine for high sensitivity and resolution tissue imaging. However, factors such as refractive index mismatch induced spherical aberration, emission/excitation absorption and scattering can result in axial intensity attenuation and lateral image heterogeneity, affecting both qualitative and quantitative image analysis. In this work, we describe an image correction algorithm to improve three-dimensional images in LMLSM. The method consists of multiplying the measured nonlinear signal by a three-dimensional correction factor, determined by the use of twophoton images of the appropriate specimens and specimen absorption and scattering properties at the excitation and emission wavelengths. The proposed methodology is demonstrated in correcting multiphoton images of objects imbedded in uniform fluorescent background, lung tissue, and Drosophila larva.