Nonlinear optical microscopy with sub-30 fs pulses from an Yb-fiber laser, approximately three times shorter than typical fiber laser pulses, leads to an order of magnitude brighter third harmonic generation imaging. Multiphoton fluorescence, second and third harmonic generation modalities are compared on stained microspheres and unstained biological tissues.

Third-harmonic-generation (THG) has been emerged as an important noninvasive intravital imaging modality of in vivo biological research [1][2][3][4] in recent years with the advantages including intrinsic optical sectioning capability due to the highorder nonlinearity nature and no energy release due to the virtual-state-transition characteristic, [5][6][7][8][9] thus allowing much improved cell viability [3,4] in contrast to current absorptionbased fluorescence technologies. Although THG nonlinearity exists in all bio-materials, the Gouy phase shift effect substantially limits THG to be observed in the vicinity of interfaces where the first order or third order susceptibility discontinues.[10] Therefore, THG is generally regarded as a morphological imaging tool due to its interface-sensitive nature, with limited capability for molecular imaging. It is thus highly desirable to develop exogenous THG contrast agents to trace the functions of a specific molecule, taking advantage of the noninvasive nature of the THG process. Recently, noble metal nanoparticles have been proved to be able to enhance various nonlinear optical signals through surface plasmon resonance. [11][12][13][14][15][16] It should be ideal to adopt nanoparticles as molecular contrast agents of THG microscopy. However, these previous experiments proposed to enhance nonlinear emissions by matching excitation energy with the plasmon resonance energy of metal nanoparticles, which could induce strong laser absorption in nanoparticles while the induced temperature increase might alter the behaviors of the targeted bio-molecules or even induce thermal damages in the studied biological specimens.In this letter, we demonstrate molecular THG microscopy by using silver nanoparticles as exogenous THG contrast agents. This demonstration was performed in cultured mouse bladder carcinoma cells (MBT2) and the matched cell line with knocked-down Her2/neu expression by RNAi. Through matching surface plasmon wavelength to THG wavelength, strong contrast can be provided by the silver nanoparticles under a THG microscope, while the laser wavelength is located in the biological penetration window and laser absorption in nanoparticles is also strongly reduced due to the huge spectral difference between the laser excitation wavelength and the plasmon resonance wavelength. By successfully conjugating anti-her2 antibodies with the citrated silver nanoparticles, Her2/neu in the cancerous cell membranes is successfully imaged with THG microscopy.With the help of surface plasmon-resonance, nanometersized noble metals can serve as a nanoscopic optical resonant cavity. Metal nanoparticles with the plasmon resonance at the third harmonic of optical excitation, in the macroscopic point of view, is analogous to an optical third-harmonic oscillator. [17,18] We chose silver nanoparticles for its blue-violet plasmon resonance wavelengths when soaked in water. For nonlinear biological in vivo imaging, near-infrared (NIR) femtosecond lasers are preferred as the THG excitation sources ...

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.

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.

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.