Mihaela Balu

Two-photon absorption cross-sections of common photoinitiators

Recent interests in and applications of two-photon absorption (2PA) induced photopolymerization have afforded advanced opportunities to perform three-dimensionally resolved polymerization, resulting in intricate microfabrication and imaging. Many of the reported 2PA-induced polymerizations make use of commercially available photoinitiators, and a key parameter to consider is the two-photon absorption cross-section (δ) of the initiator. To date, there has been no comprehensive investigation of two-photon absorptivity of commercial photoinitiators, though a few studies presenting novel photoinitiators for two-photon polymerization have appeared. Herein, we report the 2PA properties of common, commercially available photoinitiators typically utilized in conventional radiation curing science and technologies, and often used in 2PA-based polymerizations. Z-scan and white-light continuum (WLC) pump–probe techniques were utilized to obtain two-photon absorption cross-sections ( δ). The results for most compounds were found to yield good agreement between the two methods. Most of the photoinitiators studied possess low δ, except Irgacure OXE01, indicating a need for the development of new photoinitiators with improved properties optimized for 2PA applications. A compound prepared in our laboratories exhibits high 2PA and was useful as a two-photon free-radical photoinitiator.

Fiber delivered probe for efficient CARS imaging of tissues

We demonstrate a fiber-based probe for maximum collection of the coherent anti-Stokes Raman scattering (CARS) signal in biological tissues. We discuss the design challenges including capturing the backscattered forward generated CARS signal in the sample and the effects of fiber nonlinearities on the propagating pulses. Three different single mode fibers (fused silica fiber, photonic crystal fiber and double-clad photonic crystal fiber) were tested for the probe design. We investigated self-phase modulation, stimulated Raman scattering (SRS) and four-wave-mixing (FWM) generation in the fiber: nonlinear processes expected to occur in a two-beam excitation based probe. While SPM and SRS induced spectral broadening was negligible, a strong non phase-matched FWM contribution was found to be present in all the tested fibers for excitation conditions relevant to CARS microscopy of tissues. To spectrally suppress this strong contribution, the pro design incorporates separate fibers for excitation light delivery and for signal detection, in combination with dichroic optics. CARS images of the samples were recorded by collecting the back-scattered forward generated CARS signal in the sample through a multi-mode fiber. Different biological tissues were imaged ex vivo in order to assess the performance of our fiber-delivered probe for CARS imaging, a tool which we consider an important advance towards label-free, in vivo probing of superficial tissues.

Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media

We present a comparative study of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging in turbid media at 800- and 1300-nm excitation. The depth-dependent decay of TPEF and SHG signals in turbid tissue phantoms is used to estimate the impact of light scattering on excitation intensity at each wavelength. A 50 to 80% increase in scattering length is observed using 1300-nm excitation, while peak TPEF emission intensity is obtained 10 to 20 microm beneath the surface for both sources. The increased penetration depth at 1300 nm is confirmed by TPEF and SHG microscopy of tissue phantoms composed of gelatin/microspheres and 3-D organotypic collagen-fibroblast cultures, respectively. Our results establish the feasibility of 1.3-microm excitation in nonlinear optical microscopy.

Dispersion of nonlinear refraction and two-photon absorption using a white-light continuum Z-scan

We use a white-light continuum (WLC) Z-scan technique to measure the degenerate two-photon absorption spectrum and associated dispersion of the nonlinear refraction in ZnSe. The spectral components of the WLC source are separated by using a narrow band variable filter to minimize nondegenerate nonlinearities. We observe a change in sign of the ultrafast nonlinear refractive index around 0.7 of the bandgap energy as predicted by theory.

Distinguishing between benign and malignant melanocytic nevi by in vivo multiphoton microscopy

Monitoring of atypical nevi is an important step in early detection of melanoma, a clinical imperative in preventing the disease progression. Current standard diagnosis is based on biopsy and histopathologic examination, a method that is invasive and highly dependent upon physician experience. In this work, we used a clinical multiphoton microscope to image in vivo and noninvasively melanocytic nevi at three different stages: common nevi without dysplastic changes, dysplastic nevi with structural and architectural atypia, and melanoma. We analyzed multiphoton microscopy (MPM) images corresponding to 15 lesions (five in each group) both qualitatively and quantitatively. For the qualitative analysis, we identified the morphologic features characteristic of each group. MPM images corresponding to dysplastic nevi and melanoma were compared with standard histopathology to determine correlations between tissue constituents and morphology and to evaluate whether standard histopathology criteria can be identified in the MPM images. Prominent qualitative correlations included the morphology of epidermal keratinocytes, the appearance of nests of nevus cells surrounded by collagen fibers, and the structure of the epidermal-dermal junction. For the quantitative analysis, we defined a numerical multiphoton melanoma index (MMI) based on three-dimensional in vivo image analysis that scores signals derived from two-photon excited fluorescence, second harmonic generation, and melanocyte morphology features on a continuous 9-point scale. Indices corresponding to common nevi (0-1), dysplastic nevi (1-4), and melanoma (5-8) were significantly different (P < 0.05), suggesting the potential of the method to distinguish between melanocytic nevi in vivo.

Broadband Z-scan characterization using a high-spectral-irradiance, high-quality supercontinuum

We generate a high-spectral-irradiance, high-quality continuum by weakly focusing femtosecond pulses in Kr gas. We use this continuum as a source for rapid Z-scan measurements of the degenerate nonlinear absorption spectrum and the associated dispersion of the nonlinear refraction in optical materials throughout the visible. We measure the degenerate two-photon absorption spectra and the dispersion of the nonlinear refractive index, n2, of two well-characterized semiconductors (ZnSe and ZnS) as reference samples for our method, along with dilute solutions of organic materials. The latter materials demonstrate application of the technique to samples with lower nonlinearities.

Three- and four-photon absorption of a multiphoton absorbing fluorescent probe

The utility of multiphoton excitation processes has been the subject of increased attention due to their potential applications in biophotonics, biology, and medicine through three-dimensional fluorescence imaging and photodynamic therapy. Evidence of this are the multiple applications of two-photon absorption (2PA) in fluorescence spectroscopy and 3D imaging over the last several years because of its large effective Stoke¿s Shift and high spatial resolution.[l,2] However, because the irradiation penetration depth of 2PA is limited in medical and biological applications due to the unwanted absorption and scattering when two red photons are used, the scientific communib recently started to explore higher order absorption processes at longer wavelengths such as three- (3PA) and four-photon absorption (IPA) that minimize the scattered light losses, and reduce the unwanted linear absorption in the living organism transparency window.

Production of iron-oxide nanoparticles by laser-induced pyrolysis of gaseous precursors

Laser-assisted pyrolysis in a continuous flow reactor has been applied to synthesise iron-oxide nanoparticles. The scope of the present contribution was to investigate the possibility of increasing the reaction yield in order to obtain powder amounts suitable for practical applications. To this aim, a gas mixture containing Fe(CO)5 and N2O has been submitted to CO2 c.w. laser pyrolysis. As a reaction sensitiser gas, SF6 has been preferred to C2H4 to avoid ethylene fragmentation in N2O presence and the formation of iron carbides. Due to unexpected SF6 dissociation, the synthesis process led to the preferential formation of iron fluoride compounds. Powder samples, submitted to calcining treatment (400°C, 3 h), showed an almost complete transformation to α- and γ-iron oxides retaining the nanostructure feature of the powder.

Two-photon excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo

Abstract. Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λex=1000  nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6±0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI=0.5±0.05 and 0.17±0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo.

Evaluation of Stimulated Raman Scattering Microscopy for Identifying Squamous Cell Carcinoma in Human Skin

There is a need to develop non‐invasive diagnostic tools to achieve early and accurate detection of skin cancer in a non‐surgical manner. In this study, we evaluate the capability of stimulated Raman scattering (SRS) microscopy, a potentially non‐invasive optical imaging technique, for identifying the pathological features of s squamous cell carcinoma (SCC) tissue.