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.

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.

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.

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.

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.

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.

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.

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.