Chi-Hsiang Lien

Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned excitation

One of the limits of conventional scanning multiphoton microfabrication is its low throughput due to point-by-point processing. In order to surpass this limit, a multiphoton microfabrication system based on spatiotemporal focusing and patterned excitation has been developed to quickly provide three-dimensional (3D) freeform polymer microstructures. 3D freeform polymer microstructures using Rose Bengal as the photoinitiator are created by sequentially stacking two-dimensional fabricating patterns. The size of each fabrication area can be larger than 300 × 170 μm2 (full width at half maximum). Compared to conventional scanning multiphoton excitation and fixed mask pattern generation, this approach offers freeform microstructures and a greater than three-order increase in fabrication speed. Furthermore, the system is capable of optically sectioning the fabricated microstructures for providing 3D inspection.

Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device

In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.

Differentiation of Col I and Col III isoforms in stromal models of ovarian cancer by analysis of second harmonic generation polarization and emission directionality.

A profound remodeling of the extracellular matrix occurs in many epithelial cancers. In ovarian cancer, the minor collagen isoform of Col III becomes upregulated in invasive disease. Here we use second harmonic generation (SHG) imaging microscopy to probe structural differences in fibrillar models of the ovarian stroma comprised of mixtures of Col I and III. The SHG intensity and forward-backward ratios decrease with increasing Col III content, consistent with decreased phasematching due to more randomized structures. We further probe the net collagen α-helix pitch angle within the gel mixtures using what is believed to be a new pixel-based polarization-resolved approach that combines and extends previous analyses. The extracted pitch angles are consistent with those of peptide models and the method has sufficient sensitivity to differentiate Col I from the Col I/Col III mixtures. We further developed the pixel-based approach to extract the SHG signal polarization anisotropy from the same polarization-resolved image matrix. Using this approach, we found that increased Col III results in decreased alignment of the dipole moments within the focal volume. Collectively, the SHG measurements and analysis all indicate that incorporation of Col III results in decreased organization across several levels of collagen organization. Furthermore, the findings suggest that the collagen isoforms comingle within the same fibrils, in good agreement with ultrastructural data. The pixel-based polarization analyses (both excitation and emission) afford determination of structural properties without the previous requirement of having well-aligned fibers, and the approaches should be generally applicable in tissue.

Precise, motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space

Second Harmonic Generation (SHG) microscopy coupled with polarization analysis has great potential for use in tissue characterization, as molecular and supramolecular structural details can be extracted. Such measurements are difficult to perform quickly and accurately. Here we present a new method that uses a liquid crystal modulator (LCM) located in the infinity space of a SHG laser scanning microscope that allows the generation of any desired linear or circular polarization state. As the device contains no moving parts, polarization can be rotated accurately and faster than by manual or motorized control. The performance in terms of polarization purity was validated using Stokes vector polarimetry, and found to have minimal residual polarization ellipticity. SHG polarization imaging characteristics were validated against well-characterized specimens having cylindrical and/or linear symmetries. The LCM has a small footprint and can be implemented easily in any standard microscope and is cost effective relative to other technologies.

Enhanced two-photon excited fluorescence in three-dimensionally crosslinked bovine serum albumin microstructures

In this study, the intensity of two-photon excited fluorescence (TPEF) of xanthene dye, Rose Bengal (RB), was significantly enhanced via bovine serum albumin (BSA) microstructures fabricated by the two-photon crosslinking (TPC) technique. The RB was utilized as the photoactivator in the TPC processing and the enhanced TPEF intensity correlates with the concentration of fabricated crosslinked BSA microstructures via the power control and pulse selection of the employed femtosecond laser. As a result, fabrication of three-dimensional BSA microstructures can be simultaneously monitored by the use of TPEF intensity. The crosslinked BSA microstructures synthesized may be used as an ordered biomaterial for fluorescence enhancement.

High power NIR fiber-optic femtosecond Cherenkov radiation and its application on nonlinear light microscopy

We reported a record high power (>250 mW) and compact near-infrared fiber-optic femtosecond Cherenkov radiation source and its new application on nonlinear light microscopy for the first time (to our best knowledge). The high power femtosecond Cherenkov radiation was generated by 1.03 μm femtosecond pulses from a portable diode-pumped laser and a photonic crystal fiber as a compact, flexible, and highly efficient wavelength convertor. Sectioned nonlinear light microscopy images from mouse brain blood vessel network and rat tail tendon were then performed by the demonstrated light source. Due to the advantages of its high average output power (>250 mW), high pulse energy (>4 nJ), excellent wavelength conversion efficiency (>40%), compactness, simplicity in configuration, and turn-key operation, the demonstrated femtosecond Cherenkov radiation source could thus be widely applicable as an alternative excitation source to mode-locked Ti:Sapphire lasers for future clinical nonlinear microscopy or other applications requiring synchronized multi-wavelength light sources.

Dynamic particle tracking via temporal focusing multiphoton microscopy with astigmatism imaging

A three-dimensional (3D) single fluorescent particle tracking strategy based on temporal focusing multiphoton excitation microscopy (TFMPEM) combined with astigmatism imaging is proposed for delivering nanoscale-level axial information that reveals 3D trajectories of single fluorospheres in the axially-resolved multiphoton excitation volume without z-axis scanning. Whereas other scanning spatial focusing multiphoton excitation schemes induce optical trapping interference, temporal focusing multiphoton excitation produces widefield illumination with minimum optical trapping force on the fluorospheres. Currently, the lateral and axial positioning resolutions of the dynamic particle tracking approach are about 14 nm and 21 nm in standard deviation, respectively. Furthermore, the motion behavior and diffusion coefficients of fluorospheres in glycerol solutions with different concentrations are dynamically measured at a frame rate up to 100 Hz. This TFMPEM with astigmatism imaging holds great promise for exploring dynamic molecular behavior deep inside biotissues via its superior penetration, reduced trapping effect, fast frame rate, and nanoscale-level positioning.

Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned generation

One of the limits of a conventional multiphoton microfabrication is its low throughput due to the sequential nature of scanning process. In this study, a multiphoton microfabrication system based on spatiotemporal focusing and patterned excitation has been developed to provide freeform polymer microstructures fast. The system integrates a 10 kHz repetition rate ultrafast amplifier featuring strong instantanrror device generating designed patterns at the focal plane. As the result, three-dimensional freeform polymer microstructures using Rose Bengal as the photoinitiator are created by sequentially stacking up two-dimensional (2D) structures layer-by-layer. The size of each 2D fabrication area can be larger than 100 × 100 μm2. Compared with scanning process or fixed mask pattern generation, this approach provides two- or three-fold fabrication speed and freeform microstructures. Furthermore, the system is capable of optical sectioning the fabricated microstructures for providing 3D inspection.

Surface plasmon-enhanced and quenched two-photon excited fluorescence

This study investigated theoretically and experimentally that two-photon excited fluorescence is enhanced and quenched via surface plasmons (SPs) excited by total internal reflection with a silver film. The fluorescence intensity is fundamentally affected by the local electromagnetic field enhancement and the quantum yield change according to the surrounding structure and materials. By utilizing the Fresnel equation and classical dipole radiation modeling, local electric field enhancement, fluorescence quantum yield, and fluorescence emission coupling yield via SPs were theoretically analyzed at different dielectric spacer thicknesses between the fluorescence dye and the metal film. The fluorescence lifetime was also decreased substantially via the quenching effect. A two-photon excited total internal reflection fluorescence (TIRF) microscopy with a time-correlated single photon counting device has been developed to measure the fluorescence lifetimes, photostabilities, and enhancements. The experimental results demonstrate that the fluorescence lifetimes and the trend of the enhancements are consistent with the theoretical analysis. The maximum fluorescence enhancement factor in the surface plasmon-total internal reflection fluorescence (SP-TIRF) configuration can be increased up to 30 fold with a suitable thickness SiO2 spacer. Also, to compromise for the fluorescence enhancement and the fluorophore photostability, we find that the SP-TIRF configuration with a 10 nm SiO2 spacer can provide an enhanced and less photobleached fluorescent signal via the assistance of enhanced local electromagnetic field and quenched fluorescence lifetime, respectively.

Polarization Resolved SHG Imaging in Ovarian Cancer

Pixel-based SHG polarization techniques quantitatively probe macromolecular structure of collagen I and III in fibrillar models of ovarian cancer. The methods are sensitive to the triple helix structure of the isoforms and resulting fibril assembly.