Feasibility of multimodal multiphoton microscopy to facilitate surgical margin assessment in pancreatic cancer.

Pancreatic cancer is a common cancer with poor odds of survival for the patient, with surgical resection offering the only hope of cure. Current surgical practice is time-consuming and, due to time constraints, does not sample the whole cut surface sufficiently to check for remaining cancer. Although microscopy with hematoxylin and eosin (H&E) stain is the gold standard for microscopic evaluation, multiphoton microscopy (MPM) has emerged as an alternative tool for imaging tissue architecture and cellular morphology without labels. We explored the use of multimodal MPM for the label-free identification of normal and cancerous tissue of the pancreas in a mouse model by comparing the images to H&E microscopy. Our early studies indicate that MPM using second-harmonic generation, third-harmonic generation, and multiphoton excitation of endogenous fluorescent proteins can each contribute to the label-free analysis of the pancreatic surgical margin.

Characterization of multiphoton microscopes by the nonlinear knife-edge technique.

Imaging submicron fluorescent microspheres are the standard method for measuring resolution in multiphoton microscopy. However, when using high-energy pulsed lasers, photobleaching and heating of the solution medium may deteriorate the images, resulting in an inaccurate resolution measurement. Moreover, due to the weak higher-order response of fluorescent microspheres, measuring three-photon resolution using three-photon fluorescence (3PEF) and third-harmonic generation (THG) signals is more difficult. In this report, we demonstrate a methodology for complete characterization of multiphoton microscopes based on second- and third-harmonic generation signals from the sharp edge of GaAs wafers. This simple methodology, which we call the nonlinear knife-edge technique, provides fast and consistent lateral and axial resolution measurement with negligible photobleaching effect on semiconductor wafers. In addition, this technique provides information on the field curvature of the imaging system, and perhaps other distortions of the imaging system, adding greater capability compared to existing techniques.

Ultra-strong nonlinear optical processes and trigonal warping in MoS2 layers.

Nonlinear optical processes, such as harmonic generation, are of great interest for various applications, e.g., microscopy, therapy, and frequency conversion. However, high-order harmonic conversion is typically much less efficient than low-order, due to the weak intrinsic response of the higher-order nonlinear processes. Here we report ultra-strong optical nonlinearities in monolayer MoS2 (1L-MoS2): the third harmonic is 30 times stronger than the second, and the fourth is comparable to the second. The third harmonic generation efficiency for 1L-MoS2 is approximately three times higher than that for graphene, which was reported to have a large χ (3). We explain this by calculating the nonlinear response functions of 1L-MoS2 with a continuum-model Hamiltonian and quantum mechanical diagrammatic perturbation theory, highlighting the role of trigonal warping. A similar effect is expected in all other transition-metal dichalcogenides. Our results pave the way for efficient harmonic generation based on layered materials for applications such as microscopy and imaging.Harmonic generation is a nonlinear optical process occurring in a variety of materials; the higher orders generation is generally less efficient than lower orders. Here, the authors report that the third-harmonic is thirty times stronger than the second-harmonic in monolayer MoS2.

Imaging of targeted lipid microbubbles to detect cancer cells using third harmonic generation microscopy.

The use of receptor-targeted lipid microbubbles imaged by ultrasound is an innovative method of detecting and localizing disease. However, since ultrasound requires a medium between the transducer and the object being imaged, it is impractical to apply to an exposed surface in a surgical setting where sterile fields need be maintained and ultrasound gel may cause the bubbles to collapse. Multiphoton microscopy (MPM) is an emerging tool for accurate, label-free imaging of tissues and cells with high resolution and contrast. We have recently determined a novel application of MPM to be used for detecting targeted microbubble adherence to the upregulated plectin-receptor on pancreatic tumor cells. Specifically, the third-harmonic generation response can be used to detect bound microbubbles to various cell types presenting MPM as an alternative and useful imaging method. This is an interesting technique that can potentially be translated as a diagnostic tool for the early detection of cancer and inflammatory disorders.

Optical characterization of directly deposited graphene on a dielectric substrate.

By using scanning multiphoton microscopy we compare the nonlinear optical properties of the directly deposited and transferred to the dielectric substrate graphene. The direct deposition of graphene on oxidized silicon wafer was done by utilizing sacrificial copper catalyst film. We demonstrate that the directly deposited graphene and bi-layered transferred graphene produce comparable third harmonic signals and have almost the same damage thresholds. Therefore, we believe directly deposited graphene is suitable for the use of e.g. nanofabricated optical setups.

Label-free multi-photon imaging of dysplasia in Barrett's esophagus.

Barrett's esophagus (BE) is a metaplastic disorder where dysplastic and early cancerous changes are invisible to the naked eye and where the practice of blind biopsy is hampered by large sampling errors. Multi-photon microscopy (MPM) has emerged as an alternative solution for fast and label-free diagnostic capability for identifying the histological features with sub-micron accuracy. We developed a compact, inexpensive MPM system by using a handheld mode-locked fiber laser operating at 1560nm to study mucosal biopsies of BE. The combination of back-scattered THG, back-reflected forward THG and SHG signals generate images of cell nuclei and collagen, leading to label-free diagnosis in Barrett's.

Silicon nanoridge array waveguides for nonlinear and sensing applications.

We fabricate and characterize waveguides composed of closely spaced and longitudinally oriented silicon ridges etched into silicon-on-insulator wafers. Through both guided mode and bulk measurements, we demonstrate that the patterning of silicon waveguides on such a deeply subwavelength scale is desirable for nonlinear and sensing applications alike. The proposed waveguide geometry simultaneously exhibits comparable propagation losses to similar schemes proposed in literature, an enhanced effective third-order nonlinear susceptibility, and high sensitivity to perturbations in its environment.

Investigation of second- and third-harmonic generation in few-layer gallium selenide by multiphoton microscopy.

Gallium selenide (GaSe) is a layered semiconductor and a well-known nonlinear optical crystal. The discovery of graphene has created a new vast research field focusing on two-dimensional materials. We report on the nonlinear optical properties of few-layer GaSe using multiphoton microscopy. Both second- and third-harmonic generation from few-layer GaSe flakes were observed. Unexpectedly, even the peak at the wavelength of 390 nm, corresponding to the fourth-harmonic generation or the sum frequency generation from third-harmonic generation and pump light, was detected during the spectral measurements in thin GaSe flakes.

Multiphoton microscopy as a detection tool for photobleaching of EO materials.

Multi-photon microscopy operating at 1550 nm is employed as a rapid characterization tool for studying the photostability of three well-known electro-optical materials. Different nonlinear optical responses such as multi-photon excitation fluoresence, second-, and third-harmonic generation can be used as detection probes to reveal the degradation mechanisms. This technique is rapid, accurate, and can be used to study the photostability of a broad range of materials.

High-quality crystallinity controlled ALD TiO2 for waveguiding applications.

We demonstrate a novel atomic layer deposition (ALD) process to make high-quality nanocrystalline titanium dioxide (TiO(2)) with intermediate Al(2)O(3) layers to limit the crystal size. The process is based on titanium chloride (TiCl(4))+water and trimethyl aluminum (TMA)+ozone processes at 250°C deposition temperature. The waveguide losses measured using a prism coupling method for 633 and 1551 nm wavelengths are as low as 0.2±0.1 dB/mm with the smallest crystal size, with losses increasing with crystal size. In comparison, plain TiO(2) deposited at 250°C without the intermediate Al(2)O(3) layers shows high scattering losses and is not viable as waveguide material. The third-order optical nonlinearity decreases with smaller crystal size as verified by third-harmonic generation microscopy but still remains high for all samples. Crystallinity controlled ALD-grown TiO(2) is an excellent candidate for various optical applications, where good thermal stability and high third-order optical nonlinearity are needed.

Rapid large-area multiphoton microscopy for characterization of graphene.

Single- and few-layer graphene was studied with simultaneous third-harmonic and multiphoton-absorption-excited fluorescence microscopy using a compact 1.55 µm mode-locked fiber laser source. Strong third-harmonic generation (THG) and multiphoton-absorption-excited fluorescence (MAEF) signals were observed with high contrast over the signal from the substrate. High contrast was also achieved between single- and bilayer graphene. The measurement is straightforward and very fast compared to typical Raman mapping, which is the conventional method for characterization of graphene. Multiphoton microscopy is also proved to be an extremely efficient method for detecting certain structural features in few-layer graphene. The accuracy and speed of multiphoton microscopy make it a very promising characterization technique for fundamental research as well as large-scale fabrication of graphene. To our knowledge, this is the first time simultaneous THG and MAEF microscopy has been utilized in the characterization of graphene. This is also the first THG microscopy study on graphene using the excitation wavelength of 1.55 µm, which is significant in telecommunications and signal processing.