Harmonic optical tomography of nonlinear structures.

Second-harmonic generation microscopy is a valuable label-free modality for imaging non-centrosymmetric structures and has important biomedical applications from live-cell imaging to cancer diagnosis. Conventional second-harmonic generation microscopy measures intensity signals that originate from tightly focused laser beams, preventing researchers from solving the scattering inverse problem for second-order nonlinear materials. Here, we present harmonic optical tomography (HOT) as a novel modality for imaging microscopic, nonlinear and inhomogeneous objects. The HOT principle of operation relies on inter-ferometrically measuring the complex harmonic field and using a scattering inverse model to reconstruct the three-dimensional distribution of harmonophores. HOT enables strong axial sectioning via the momentum conservation of spatially and temporally broadband fields. We illustrate the HOT operation with experiments and reconstructions on a beta-barium borate crystal and various biological specimens. Although our results involve second-order nonlinear materials, we show that this approach applies to any coherent nonlinear process.

Demonstration of flat-top beam illumination in widefield multiphoton microscopy.

Multiphoton microscopy provides a suitable technique for imaging biological tissues with submicrometer resolution. Usually a Gaussian beam (GB) is used for illumination, leading to a reduced power efficiency in the multiphoton response and vignetting for a square-shaped imaging area. A flat-top beam (FTB) provides a uniform spatial intensity distribution that equalizes the probability of a multiphoton effect across the imaging area. We employ a customized widefield multiphoton microscope to compare the performance of a square-shaped FTB illumination with that based on using a GB, for both two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging. The variation in signal-to-noise ratio across TPF images of fluorescent dyes spans ∼5.6 dB for the GB and ∼1.2 dB for the FTB illumination, respectively. For the GB modality, TPF images of mouse colon and Convallaria root, and SHG images of chicken tendon and human breast biopsy tissue showcase ∼20 % area that are not imaged due to either insufficient or lack of illumination. For quantitative analysis that depends on the illuminated area, this effect can potentially lead to inaccuracies. This work emphasizes the applicability of FTB illumination to multiphoton applications.

Chemical and physical changes of microplastics during sterilization by chlorination.

Wastewater treatment plants are known to release microplastics that have been detected in aquatic and terrestrial organisms constituting part of the human diet. Chlorination of wastewater-borne microplastics was hypothesized to induce chemical and physical changes detectable by Raman spectroscopy and differential scanning calorimetry (DSC). In the laboratory, virgin plastics (∼0.05 × 2 × 2 mm) were exposed to differing sterilization conditions representative of dosages used in the disinfection of drinking water, wastewater, and heavily contaminated surfaces. Polypropylene (PP) was most resistant to chlorination, followed by high density polyethylene (HDPE) and polystyrene (PS). Polystyrene showed degradation, indicated by changes in Raman peak widths, at concentration-time regimes (CT values) as low as 75 mg min/L, whereas HDPE and PP remained unaltered even at chlorine doses characteristic of wastewater disinfection (150 mg min/L). However, HDPE and PS were not completely resistant to oxidative attack by chlorination. Under extremely harsh conditions, shifts in Raman peaks and the formation of new bonds were observed. These results show that plastics commonly used in consumer products can be chemically altered, some even under conditions prevailing during wastewater treatment. Changes in polymer properties, observed for HDPE and PP under extreme exposure conditions only, are predicted to alter the risk microplastics pose to aquatic and terrestrial biota, since an increase in carbon-chlorine (C-Cl) bonds is known to increase toxicity, rendering the polymers more hydrophobic and thus more prone to adsorb, accumulate, and transport harmful persistent pollutants to biota in both aquatic and terrestrial environments.

Second-harmonic patterned polarization-analyzed reflection confocal microscopy of stromal collagen in benign and malignant breast tissues.

We present the results of polarimetric analysis of collagen on varying pathologies of breast tissues using second-harmonic patterned polarization-analyzed reflection confocal (SPPARC) microscopy. Experiments are conducted on a breast tissue microarray having benign tissues (BT), malignant invasive lobular carcinoma (ILC), and benign stroma adjacent to the malignant tissues (called the benign adjacent tissue, or BAT). Stroma in BAT and ILC exhibit the largest parameter differences. We observe that stromal collagen readings in ILC show lower depolarization, lower diattenuation and higher linear degree-of-polarization values than stromal collagen in BAT. This suggests that the optical properties of collagen change most in the vicinity of tumors. A similar trend is also exhibited in the non-collagenous extrafibrillar matrix plus cells (EFMC) region. The three highlighted parameters show greatest sensitivity to changes in the polarization response of collagen between pathologies.