Bai-g Lin

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.

Multiharmonic-generation biopsy of skin

Because it avoids the in-focus photodamage and phototoxicity problem of two-photon-fluorescence excitation, multiharmonic-generation biopsy based on a 1200-1300-nm light source could provide a truly noninvasive and highly penetrative optical sectioning of skin. We study multiharmonic-generation biopsy of fixed mouse skin. Our preliminary study suggests that this technique could provide submicrometer-resolution deep-tissue noninvasive biopsy images in skin without the use of fluorescence and exogenous markers.

Thickness dependence of optical second harmonic generation in collagen fibrils

Simultaneous backward and forward second harmonic generations from isolated type-I collagen matrix are observed. Optical interference behaviors of these nonlinear optical signals are studied with accurately determined fibril thickness by an atomic force microscope. The nonlinear emission directions are strongly dependent on the coherent interaction within and between collagen fibrils. A linear relationship is obtained to estimate collagen fibril thickness with nanometer precision noninvasively by evaluating the forward/backward second harmonic generation ratio.

Cell manipulation by use of diamond microparticles as handles of optical tweezers

We report the experimental results of our using irregularly shaped diamond microparticles as handles for laser tweezers. Because of their irregular optical shape, control of the rotation of diamond microparticles can easily be achieved in a gradient force optical trap by use of a fixed linearly polarized beam with a fundamental Gaussian mode. By changing the laser focal plane upon a diamond particle near the liquid surface or interfaces, one can fully manipulate both the direction and speed of the rotation. The ability to manipulate a diamond-particle-tagged biological specimen by optical tweezers is discussed. The application of these particles as handles for optical tweezers is demonstrated by optical manipulation of biological cells. Independent movement of linear translation and rotation, with controllable rotation directions and speeds, is successfully achieved.

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.

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.

Coherent interaction of optical second harmonic generation in collagen fibrils

We present the first experimental comparison between optical second harmonic generation images and atomic force microscope images in a matrix of nano-scaled collagen fibrils. Substantial variation of forward and backward propagated second harmonic generation radiation is observed in a single collagen fibril and is nicely correlated with the accurately determined thickness from an atomic force microscope. Contradicting to conventional nonlinear optical theory, our result indicates a linear relationship between fibril thickness and forward / backward second harmonic generation ratio. This is the first demonstration of estimating fibril thickness with nanometer precision by a noninvasive optical method.