I-Hsiu Chen

Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser.

We demonstrate a novel multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser at 1230 nm. By acquiring the whole nonlinear spectrum in the visible and near-NIR region, this novel technique allows a combination of different imaging modalities, including second-harmonic generation, third-harmonic generation, and multiple-photon fluorescence. Combined with the selected excitation wavelength, which is located in the IR transparency window, this microscopic technique can provide high penetration depth with reduced damage and is ideal for studying living cells.

Wavelength dependent damage in biological multi-photon confocal microscopy: A micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources

Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. Thus induced two-photon absorption and the accompanied multi-photon absorption/ionization not only cause photo-bleaching but also cell damage in the vicinity of the focal point. In this paper, we study the wavelength dependent cell damage induced by high intensity femtosecond near infrared lasers. The study was performed with a Ti:sapphire laser and a Cr:forsterite laser. With a longer output wavelength from a Cr:forsterite laser, multi-photon absorption and auto-fluorescence were found to be significantly suppressed and the destructive plasma formation was found to be greatly reduced. Sustained multi-photon spectra can be observed in most plant specimens with a tightly focused Cr:forsterite laser beam under long term irradiation with more than 100 mW laser average power. In contrast, multi-photon absorption induced destructive plasma formation were frequently observed with a tightly focused Ti:sapphire laser beam within seconds with more than 10 mW laser average power.

Nonlinear bio-photonic crystal effects revealed with multimodal nonlinear microscopy

Highly optically active nonlinear bio‐photonic crystalline and semicrystalline structures in living cells were studied by a novel multimodal nonlinear microscopy. Numerous biological structures, including stacked membranes and aligned protein structures are highly organized on a nanoscale and have been found to exhibit strong optical activities through second‐harmonic generation (SHG) interactions, behaving similarly to man‐made nonlinear photonic crystals. The microscopic technology used in this study is based on a combination of different imaging modes including SHG, third‐harmonic generation, and multiphoton‐induced fluorescence. With no energy release during harmonic generation processes, the nonlinear‐photonic‐crystal‐like SHG activity is useful for investigating the dynamics of structure–function relationships at subcellular levels and is ideal for studying living cells, as minimal or no preparation is required.

Miniaturized video-rate epi-third-harmonic-generation fiber-microscope

With a micro-electro-mechanical system (MEMS) mirror, we successfully developed a miniaturized epi-third-harmonic-generation (epi-THG) fiber-microscope with a video frame rate (31 Hz), which was designed for in vivo optical biopsy of human skin. With a large-mode-area (LMA) photonic crystal fiber (PCF) and a regular microscopic objective, the nonlinear distortion of the ultrafast pulses delivery could be much reduced while still achieving a 0.4 microm lateral resolution for epi-THG signals. In vivo real time virtual biopsy of the Asian skin with a video rate (31 Hz) and a sub-micron resolution was obtained. The result indicates that this miniaturized system was compact enough for the least invasive hand-held clinical use.

Miniaturized multiphoton microscope with a 24Hz frame-rate

With miniaturized tube lenses and a micro-electro-mechanical system (MEMS) mirror, we constructed a miniaturized multiphoton microscope system. Through a two-dimensional asynchronous scanning of the MEMS mirror, 24Hz frame rate can be realized. With a high numerical aperture objective, sub-micron resolution can also be achieved at the same time.

Multiharmonic generation biopsy of skin

Avoiding the on-focus photodamage and phototoxicity problem of two-photon-fluorescence excitation, harmonic generation biopsy based on a /spl sim/1300 nm light source provides a truly noninvasive and highly penetrative optical sectioning of skin.

Three-dimensional electric field visualization utilizing electric-field-induced second-harmonic generation in nematic liquid crystals

An electric-field-induced second-harmonic-generation signal in a nematic liquid crystal is used to map the electric field in an integrated-circuit-like sample. Since the electric-field-induced second-harmonic-generation signal intensity exhibits a strong dependence on the polarization of the incident laser beam, both the amplitude and the orientation of the electric field vectors can be measured. Combined with scanning second-harmonic-generation microscopy, three-dimensional electric field distribution can be easily visualized with high spatial resolution of the order of 1 microm.

Biological photonic crystals revealed by multimodality nonlinear microscopy

A novel multi-modality nonlinear microscopy reveals highly optically-active biophotonic crystal structures in living cells. Numerous biological structures, including stacked membranes and arranged protein structures are highly organized in optical scale and are found to exhibit strong optical activities through second-harmonic-generation (SHG) interactions, behaving similar to man-made photonic crystals. The microscopic technology developed is based on a combination of imaging modalities including not only SHG, but also third-harmonic-generation and multi-photonfluorescence. With no energy deposition during harmonic generation processes, the demonstrated highly-penetrative yet non-invasive microscopy is useful for investigating the dynamics of structure-function relationship at the molecular and subcellular levels and is ideal for studying living cells that require minimal or no preparation.

Miniaturized epi-third harmonic generation microscope with a sub-micron spatial resolution and a video rate

With a 2D scanning MEMS mirror, we demonstrate a miniaturized epi-third-harmonic-generation microscope with a video rate and a 0.7µm transverse resolution. In vivo THG imaging of human skin is demonstrated.

Miniaturized two-photon fluorescence and second harmonic generation microscope with a 24Hz frame-rate

With miniaturized tube lenses and two-dimensional asynchronous scanning of the microelectro-mechanical-system mirror, we demonstrated a 24 Hz frame-rate miniaturized two-photon fluorescence/second-harmonic-generation microscope system. Sub-micron transverse resolution of sectioning images can be achieved.