Shih-Peng Tai

Two-photon fluorescence microscope with a hollow-core photonic crystal fiber

Self-phase-modulation and group velocity dispersion of near IR femtosecond pulses in fibers restrict their use in two-photon fluorescence microscopy (TPFM). Here we demonstrate a hollow-core photonic crystal fiber based two-photon fluorescence microscope with low nonlinearity and dispersion effects. We use this fiber-based TPFM system to take two-photon fluorescence (chlorophyll) images of mesophyll tissue in the leaf of Rhaphidophora aurea. With less than 2mW average power exposure on the leaf at 755nm, the near zero-dispersion wavelength, chloroplasts distribution inside the mesophyll cells can be identified with a sub-micron spatial resolution. The acquired image quality is comparable to that acquired by the conventional fiber-free TPFM system, due to the negligible temporal pulse broadening effect.

In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy

Elastin is an essential and widespread structural protein in charge of the integrity on tissues and organs. In this study, we demonstrate that elastin is a major origin of the third-harmonic-generation (THG) contrast under Cr:forsterite laser excitation operating at 1230nm, with selective visualization inside many tissues such as lung tissues and arteries. In vivo imaging of the nude mouse elastic cartilage beneath the hypodermis by epi- THG microscopy keeps the high resolution and contrast in all three dimensions. Combined with second-harmonic-generation microscopy, THG microscopy exhibits the ability to show the extraordinary proliferation of elastic fibers for the ophthalmic disease of pterygium and the capability of distinguishable visualization from collagen.

Cell tracking and detection of molecular expression in live cells using lipid-enclosed CdSe quantum dots as contrast agents for epi-third harmonic generation microscopy.

We demonstrated that lipid-enclosed CdSe quantum dots (LEQDs) can function as versatile contrast agents in epi-detection third harmonic generation (THG) microscopy for biological applications in vivo. With epi-THG intensities 20 times stronger than corresponding fluorescence intensities from the same LEQDs under the same conditions of energy absorption, such high brightness LEQDs were proved for the abilities of cell tracking and detection of specific molecular expression in live cancer cells. Using nude mice as an animal model, the distribution of LEQD-loaded tumor cells deep in subcutaneous tissues were imaged with high THG contrast. This is the first demonstration that THG contrast can be manipulated in vivo with nanoparticles. By linking LEQDs with anti-Her2 antibodies, the expression of Her2/neu receptors in live breast cancer cells could also be easily detected through THG. Compared with fluorescence modalities, the THG modality also provides the advantage of no photobleaching and photoblinkin g effects. Combined with a high penetration 1230 nm laser, these novel features make LEQDs excellent THG contrast agents for in vivo deep-tissue imaging in the future.

Measuring plasmon-resonance enhanced third-harmonic χ(3) of Ag nanoparticles

By coinciding the plasmon frequency with the third-harmonic frequency of the excitation light, the authors determined the plasmon-resonance enhanced optical third-harmonic-generation (THG) susceptibility of a polyvinylpyrrolidone-coated Ag nanoparticle with a 5–7nm diameter. With dispersed Ag nanoparticles on a quartz surface and through measuring the frequency dependent THG intensities, interface THG showed evident enhancement when the third harmonic of excitation matched the Ag-nanoparticle’s plasmon-resonant frequency. According to the effective medium theory and by analyzing the interface THG under focused Gaussian beams, the ensemble-averaged χ(3)(3ω:ω,ω,ω) of a Ag nanoparticle can be estimated to be on the order of 2×10−11esu.

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.

Selective imaging in second-harmonic-generation microscopy by polarization manipulation

Second-harmonic-generation (SHG) has proved itself as an important contrast mechanism in microscopic applications. Its noninvasiveness, optical sectioning capability, and high-penetrability provide attractive features in observation of thick biological tissues. Fibrous proteins, such as myosin and collagen, are dominant SHG harmonophores in vertebrates. Due to their biophotonic crystal nature, SHGs from these proteins are known to exhibit specific polarization dependencies, reflecting local molecule arrangements. Here the authors demonstrate a scheme to distinguish SHG from myosin-based muscle fibers and intertwined collagenous perimysium through polarization selection, without complicated staining or sample/image processing required.

Optical signal degradation study in fixed human skin using confocal microscopy and higher-harmonic optical microscopy

Confocal and nonlinear optical microscopies have been applied for dermatological studies because of their capability to provide sub-surface three-dimensional images with sub-microm spatial resolutions. Optical signal degradation as the imaging plane being moved toward deeper regions in skin specimens is the key factor that limits the observation depth for the laser scanning based linear or nonlinear imaging modalities. In this article, we studied the signal degradation in fixed human skin specimens using reflection confocal microscopy and higher-harmonic optical microscopy based on a Cr:forsterite femtosecond laser centered at 1230-nm. By analyzing the optical properties through these linear and nonlinear imaging modalities, we found that the optical signal degradation in the studied human skin specimen is dominated by the distortion of the point spread function.

Compact fiber-delivered Cr:forsterite laser for nonlinear light microscopy.

We demonstrate a compact and self-starting fiber-delivered femtosecond Cr:forsterite laser for nonlinear light microscopy. A semiconductor saturable absorber mirror provides the self-starting mechanism and maintains long-term stability in the laser cavity. Four double-chirped mirrors are employed to reduce the size of the cavity and to compensate for group velocity dispersion. Delivered by a large-mode-area photonic crystal fiber, the generated laser pulses can be compressed down to be with a nearly transform-limited pulse width with 2.2-nJ fiber-output pulse energy. Based on this fiber-delivered Cr:forsterite laser source, a compact and reliable two-photon fluorescence microscopy system can thus be realized.

Biomolecular imaging based on far-red fluorescent protein with a high two-photon excitation action cross section

Received October 14, 2005; revised January 7, 2006; accepted January 9, 2006; posted January 12, 2006 (Doc. ID 65391) The two-photon excitation action cross section of Hc-Red fluorescent proteins (Hc-RFPs) is measured and found to be of the same order as that of enhanced green fluorescent proteins. With a 618 nm emission wavelength in the far-red region and with an excitation wavelength around 1200 nm, Hc-RPF-based two-photon fluorescence microscopy (2PFM) can offer deep penetration capability inside live samples and is ideal for in vivo gene expression study and biomolecular imaging in live objects. In vivo 2PFM of the developing heart deep inside a transgenic zebrafish embryo tagged by Hc-RFP is also successfully demonstrated.

Selective imaging in second-harmonic-generation microscopy with anisotropic radiation

As a novel modality of optical microscopy, second-harmonic generation (SHG) provides attractive features including intrinsic optical sectioning, noninvasiveness, high specificity, and high penetrability. For a biomedical application, the epicollection of backward propagating SHG is necessary. But due to phase-matching constraint, SHG from thick tissues is preferentially forward propagation. Myosin and collagen are two of the most abundant fibrous proteins in vertebrates, and both exhibit a strong second-harmonic response. We find that the radiation patterns of myosin-based muscle fibers and collagen fibrils are distinct due to coherence effects. Based on these asymmetric radiation patterns, we demonstrate selective imaging between intertwining muscle fibers and type I collagen fibrils with forward and backward SHG modalities, respectively. Thick muscle fibers dominate the forward signal, while collagen fibril distribution is preferentially resolved in the backward channel without strong interference from muscle. Moreover, we find that well-formed collagen fibrils are highlighted by forward SHG, while loosely arranged collagen matrix is outlined by backward signal.