Nonlinear optical microscopy can obtain three-dimensionally resolved images within a specimen by exploiting the nonlinear light-matter interaction between the excitation light and sample. However, the image contrast significantly degrades with increasing observation depth because the emitted signal attenuates during light propagation through layers of the tissue before being detected. To obtain high contrast images from deep tissue, we developed saturated excitation (SAX) microscopy using the nonlinearity of near-infrared (NIR) plasmonic scattering from gold nanoshells and gold nanorods. SAX microscopy selectively detects the nonlinear component from the scattering signal generated by nanoparticle probes located at the center of the focal spot. By using this technique, background signals generated at out-of-focal positions are effectively removed. In addition, emitted signals in the NIR from nanoparticle probes efficiently transmit through biological tissue and are ideally suited to image deep parts of the tissue. We experimentally confirmed that scattering intensities from a single gold nanoshell and gold nanorod exhibit nonlinear relations with the excitation intensity of CW laser light at 780 and 1064 nm, respectively. We also demonstrated improvements of image contrast and spatial resolution at the depth of 400 mu m in a phantom of muscle tissue by selectively detecting the nonlinear scattering signal component from gold nanoshells.

Recently, many resolution enhancing techniques are demonstrated, but most of them are severely limited for deep tissue applications. For example, wide-field based localization techniques lack the ability of optical sectioning, and structured light based techniques are susceptible to beam distortion due to scattering/aberration. Saturated excitation (SAX) microscopy, which relies on temporal modulation that is less affected when penetrating into tissues, should be the best candidate for deep-tissue resolution enhancement. Nevertheless, although fluorescence saturation has been successfully adopted in SAX, it is limited by photobleaching, and its practical resolution enhancement is less than two-fold. Recently, we demonstrated plasmonic SAX which provides bleaching-free imaging with three-fold resolution enhancement. Here we show that the three-fold resolution enhancement is sustained throughout the whole working distance of an objective, i.e., 200 μm, which is the deepest super-resolution record to our knowledge, and is expected to extend into deeper tissues. In addition, SAX offers the advantage of background-free imaging by rejecting unwanted scattering background from biological tissues. This study provides an inspirational direction toward deep-tissue super-resolution imaging and has the potential in tumor monitoring and beyond.

Nonlinear optics, when combined with microscopy, is known to provide advantages including novel contrast, deep tissue observation, and minimal invasiveness. In addition, special nonlinearities, such as switch on/off and saturation, can enhance the spatial resolution below the diffraction limit, revolutionizing the field of optical microscopy. These nonlinear imaging techniques are extremely useful for biological studies on various scales from molecules to cells to tissues. Nevertheless, in most cases, nonlinear optical interaction requires strong illumination, typically at least gigawatts per square centimeter intensity. Such strong illumination can cause significant phototoxicity or even photodamage to fragile biological samples. Therefore, it is highly desirable to find mechanisms that allow the reduction of illumination intensity. Surface plasmon, which is the collective oscillation of electrons in metal under light excitation, is capable of significantly enhancing the local field around the metal nanostructures and thus boosting up the efficiency of nonlinear optical interactions of the surrounding materials or of the metal itself. In this mini-review, we discuss the recent progress of plasmonics in nonlinear optical microscopy with a special focus on biological applications. The advancement of nonlinear imaging modalities (including incoherent/ coherent Raman scattering, two/three-photon luminescence, and second/third harmonic generations that have been amalgamated with plasmonics), as well as the novel subdiffraction limit imaging techniques based on nonlinear behaviors of plasmonic scattering, is addressed.

Optical microscopy possesses the capability to image the inside of biological tissues. Two-photon excitation microscopy achieved the spatial resolution in three dimensions by the nonlinear light-material interaction via two-photon excitation of fluorescent probes [1]. However, the contrast of image significantly degrades with increasing the observation depth since the signal light is attenuated during propagation through layers of the tissue before being detected.

A major challenge in tissue imaging is the degradation of resolution with increased depth, due to multiple scattering and refraction. By using long-wavelength lasers, penetration depth can be improved, while resolution further degrades. Recently, superresolution techniques emerged to enhance spatial resolution by switching or saturation of fluorescence. However, fluorescence suffers from photobleaching, and current techniques do not provide deep-tissue imaging capability due to the lack of optical sectioning or the requirement of special beam manipulation. We have recently demonstrated that scattering from a single gold nanoparticle exhibits saturation behavior, which was adopted to significantly enhance resolution by saturated excitation (SAX) microscopy. Compared to fluorophores, scattering from plasmonic nanoparticles is free from bleaching, the cross-section is much larger, and the plasmonic resonance band is broadly tunable with particle shape and size, making it an ideal and robust contrast agent for long-term observation. On the other hand, SAX microscopy does not need any beam engineering and provides intrinsic sectioning with its confocal scheme, suitable for deep-tissue imaging. In this work, we combine the advantages of plasmonics and SAX microscopy to demonstrate resolution enhancement underneath a very deep tissue. One general concern of scattering-based imaging is the background from the strong scattering of the surrounding tissue. Since tissue scattering is linear, and SAX allows the extraction of only nonlinear responses, the background can be fully eliminated, leaving only nanoparticle visible. Therefore, such combination provides a novel tool for not only high-resolution, but also high-contrast, background-free, and long-term imaging deep inside biological tissues.

Second harmonic (SH) microscopy has proven to be a powerful imaging modality over the past years due to its intrinsic advantages as a multiphoton process with endogenous contrast specificity, which allows pinhole-less optical sectioning, non-invasive observation, deep tissue penetration, and the possibility of easier signal detection at visible wavelengths. Depending on the relative orientation between the polarization of the incoming light and the second-order susceptibility of non-centrosymmetric structures, SH microscopy provides the unique capacity to probe the absolute molecular structure of a broad variety of biological tissues without the necessity for additional labeling. In addition, SH microscopy, when working with polarimetry, provides clear and in-depth insights on the details of molecular orientation and structural symmetry. In this review, the working principles of the polarization resolving techniques and the corresponding implements of SH microscopy are elucidated, with focus on Stokes vector based polarimetry. An overview of the advancements on SH anisotropy measurements are also presented. Specifically, the recent progresses on the following three topics in polarization resolved SH microscopy will be elucidated, which include Stokes vector resolving for imaging molecular structure and orientation, 3-D structural chirality by SH circular dichroism, and correlation with fluorescence lifetime imaging (FLIM) for in vivo wound healing diagnosis. The potentials and challenges for future researches in exploring complex biological tissues are also discussed.

Superresolution microscopies have revolutionized optical imaging field in the last decade by providing a novel capability for nanoscale observation with visible light. Current techniques mostly rely on switching or saturation of fluorescence, but suffer from limited imaging depth due to the requirement of special illumination patterns (STED, SIM), or the lack of optical sectioning capability (localization microscopy). Saturated excitation (SAX) microscopy provides the potential for deep-tissue resolution enhancement due to its laser-scanning nature without additional beam shape engineering. However, for current fluorescence SAX microscopy, it is difficult to achieve resolution better than 100-nm, limited by the difficulty to obtain high order demodulation as well as by photobleaching due to high-intensity illumination. Our recent finding revealed that the bleaching issue in SAX could be resolved by substituting fluorescence with scattering from metallic nanoparticles. From the scattering-based experiment, we realized that the resolution limit of SAX could be significantly improved by proper nonlinear response of emitters. In this paper, we show that with suitable nonlinear power dependence, either scattering or fluorescence, SAX microscopy can provide sub-20-nm spatial resolution at relatively low power. Our work provides not only a new concept to enhance resolution with saturation-based techniques, but also a novel example toward ultrahigh resolution imaging with a laser-scanning scheme.

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Skin is the largest organ of our body serving as the first line defense against patho‐ gens and toxicity. The skin can heal itself if any damage in it occur. Wounds, if not taken care properly, can become chronic and can even cause death. In the field of cosmetics and plastic reconstructive surgery, wounds, are major cause of trauma and costs, which demand proper diagnosis that can help in appropriate treatment. In conventional medicine, wound diagnosis mostly relied on the expertise and experience of physi‐ cians on the basis of non-quantitative observation of clinical signs, or invasive histochemical assessment of biopsies. Methodologies based on light-matter interaction can provide quantitative, noninva‐ sive and real time assessment of a tissue section based on imaging. Depending on the nature of interaction, various contrasts can be achieved by either absorption, scatter‐ ing, or fluorescence, enabling observation of structural or molecular components of tissue sections. Development of multiphoton nonlinear optical detection techniques provide better resolution and tissue penetration depth with optical sectioning ability by using molecular and structural contrasts simultaneously. This chapter discuses and evaluates various optical approaches with special emphasis on multimodal multipho‐ ton imaging of skin tissue components in correlation to physiological processes that affects the wound healing.

The number of publications using FLIM/FRET (Fluorescence lifetime imaging and Forster resonance energy transfer) technique is increasing over the past 10 years (Vogel et al. Sci STKE 18:re2, 2006) [1].

Cellular micropattering has been increasingly adopted in quantitative biological experiments. A Q-switched pulsed neodymium-doped yttrium ortho-vanadate (Nd∶YVO4) laser directed in-situ microfabrication technique for cell patterning is presented. A platform is designed uniquely to achieve laser ablation. The platform is comprised of thin gold coating over a glass surface that functions as a thermal transducer and is over-layered by a cell repellant polymer layer. Micropatterns are engraved on the platform, subsequently exposing specific cell adhesive micro-domains by ablating the gold-polymer coating photothermally. Experimental results indicate that the proposed approach is applicable under culture conditions, viable toward cells, and has a higher engraving speed. Possible uses in arraying isolated single cells on the platform are also shown. Additionally, based on those micro-patterns, dynamic cellular morphological changes and migrational speed in response to geometrical barriers are studied to demonstrate the potential applications of the proposed approach. Our results further demonstrate that cells in narrower geometry had elongated shapes and higher migrational speed than those in wider geometry. Importantly, the proposed approach will provide a valuable reference for efforts to study single cell dynamics and cellular migration related processes for areas such as cell division, wound healing, and cancer invasion.

Institute of Biophotonics, National Yang-M Taipei, Taiwan. E-mail: kao@ym.edu.tw; T Centre for Interdisciplinary Science, Natio Photonics Centre, 1001 Ta Hsueh Rd., H nctu.edu.tw Department of Applied Chemistry and Instit Tung University, Tin-Ka Ping Photonics Cen Taiwan † Electronic supplementary informa 10.1039/c4ra06492e ‡ These authors contributed equally to th Cite this: RSC Adv., 2014, 4, 44100

We report a novel method for studying cellular migration in vitro. Cytophilic microdomains were formed on a cytophobic substrate by laser ablation. HeLa cells were grown on those domains until confluence, and then channels were formed to guide cellular migration. Two-photon excitation fluorescence-lifetime imaging of NADH revealed metabolic variation among migrating and nonmigrating cells.

Multiphoton microscopy was employed to study normal skin wound healing in live rats noninvasively. Wound healing is a process involving series of biochemical events. This study evaluates the regeneration of collagen and change in cellular metabolic activity during wound healing in rats, with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), respectively. In eukaryotic cells ATP is the molecule that holds the energy for cellular functioning. Whereas NADH is an electron donor in the metabolic pathways, required to generate ATP. Fluorescence lifetime of NADH free to protein bound ratio was evaluated to determine the relative metabolic activity. The FLIM data were acquired by a TCSPC system using SPCM software and analyzed by SPCImage software. Additionally, polarization resolved SHG signals were also collected to observe the changes in optical birefringence and hence the anisotropy of regenerated collagens from rat wound biopsy samples. Mat lab programming was used to process the data to construct the anisotropy images. Results indicated that, cells involved in healing had higher metabolic activity during the first week of healing, which decreases gradually and become equivalent to normal skin upon healing completes. A net degradation of collagen during the inflammatory phase and net regeneration starting from day 5 were observed in terms of SHG signal intensity change. Polarization resolved SHG imaging of the wound biopsy sample indicates higher value of anisotropy in proliferative phase, from day 4th to 8th, of wound formation; however the anisotropy decreases upon healing.

We reports a fluorescence lifetime imaging (FLIM) based mapping of NADH cellular metabolic activity in response to geometrical constrains. A novel Q-switched pulsed neodymium-doped yttrium ortho-vanadate (Nd:YVO4) laser directed in situ micro-fabrication technique was implemented to make the different geometrical micro-domains. The uniquely designed platform was comprised of thin gold coating over a glass surface that functions as a thermal transducer and was overlayered by a cell repellant polymer layer. Micro-patterns were engraved on the platform, subsequently exposing specific cell adhesive micro-domains by ablating the gold-polymer coating photo-thermally [1]. Rectangular micro-patterns of three different sizes of widths 200 µm, 100 µm, 30 µm were made. The morphology of those cells in response to different geometry was observed. Experimental results indicated that cells were aligned along the length of the geometry when the width was comparable to the size of a cell around 30 µm for HeLa cells. In contrast, for micro domain with higher width length, cells were in normal morphology. FLIM technique has been used to study the NADH metabolic activity of the cells in response to these three geometries. Ti:sapphire laser was used to achieve two photon autofluorescence excitation of NADH from an intracellular region. Upon binding to mitochondrial membrane proteins, NADH molecules get associated with the energy generation pathway [2]. Therefore, evaluating the ratio between free and enzyme-bound forms of the fluorophore (NADH) by FLIM can provide further insight into the relative metabolic state of cells [3]. We have used the phasor approach to study the FLIM data and compare the average lifetime (τm) and free and enzyme bound ratio of NADH for the cells in different domains. The results indicated that cells those are in narrower domain (30 µm) have lower metabolic activity than those are in wider geometry (100 µm, 200 µm).

A pulsed Nd:YVO4 laser ablative substrate is fabricated by coating a layer of carbon and gold separately on glass slides over layered by cytophobic MPC polymer. Micro patterns were engraved exposing the cell adhesive domains in situ, where singlet cells were arrayed.