Lukas Kontenis

Molecular Organization of Crystalline β‑Carotene in Carrots Determined with Polarization-Dependent Second and Third Harmonic Generation Microscopy

Polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and third harmonic generation (THG) microscopy was used to study the crystalline organization of β-carotene molecules within individual aggregates contained in the chromoplasts of orange carrots in vivo. Multimodal PIPO SHG and PIPO THG studies of the aggregates revealed one dominant SHG and THG dipole signifying that β-carotene molecules are oriented along a single axis. Three-dimensional visualization of the orientation of β-carotene molecules with respect to the aggregate axis was also performed with both microscopy modalities and revealed organization of the aggregates as ribbon-like structures consisting of twists and folds. Therefore, PIPO SHG and PIPO THG microscopy provides information on the crystalline organization and the orientation of ordered biological structures in vivo where multimodal polarization dependent SHG and THG investigations are particularly advantageous as both noncentrosymmetric and centrosymmetric crystalline organizations can be probed.

Changes of collagen ultrastructure in breast cancer tissue determined by second-harmonic generation double Stokes-Mueller polarimetric microscopy

Second-harmonic generation (SHG) double Stokes-Mueller polarimetric microscopy is applied to study the alteration of collagen ultrastructure in a tissue microarray containing three pathological human breast cancer types with differently overexpressed estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Kleinman symmetry is experimentally validated in breast tissue for 1028 nm laser wavelength and it has been shown that measurements with only linearly polarized incoming and outgoing states can determine molecular nonlinear susceptibility tensor component ratio, average in-plane orientation of collagen fibers and degree of linear polarization of SHG. Increase in the susceptibility ratio for ER, PgR, HER2 positive cases, reveals ultrastructural changes in the collagen fibers while the susceptibility ratio increase and decrease in degree of linear polarization for ER and PgR positive cases indicate alteration of the ultrastructure and increased disorder of the collagen fibers within each focal volume. The study demonstrates a potential use of polarimetric SHG microscopy for collagen characterization and cancer diagnostics.

Second harmonic generation double stokes Mueller polarimetric microscopy of myofilaments

The experimental implementation of double Stokes Mueller polarimetric microscopy is presented. This technique enables a model-independent and complete polarimetric characterization of second harmonic generating samples using 36 Stokes parameter measurements at different combinations of incoming and outgoing polarizations. The degree of second harmonic polarization and the molecular nonlinear susceptibility ratio are extracted for individual focal volumes of a fruit fly larva wall muscle.

Interferometric backward third harmonic generation microscopy for axial imaging with accuracy beyond the diffraction limit.

A new nonlinear microscopy technique based on interference of backward-reflected third harmonic generation (I-THG) from multiple interfaces is presented. The technique is used to measure height variations or changes of a layer thickness with an accuracy of up to 5 nm. Height variations of a patterned glass surface and thickness variations of fibroblasts are visualized with the interferometric epi-THG microscope with an accuracy at least two orders of magnitude better than diffraction limit. The microscopy technique can be broadly applied for measuring distance variations between membranes or multilayer structures inside biological tissue and for surface height variation imaging.

Third-harmonic generation Stokes-Mueller polarimetric microscopy

An experimental implementation of the nonlinear Stokes-Mueller polarimetric (NSMP) microscopy in third-harmonic generation modality is presented. The technique is able to extract all eight 2D-accessible χ(3) components for any sample from 64 polarization measurements, and can be applied to noninvasive ultrastructural characterization. The polarization signature of an isotropic glass coverslip is presented, and carotenoid crystallites in the root of orange carrot (Daucus carota) are investigated, showing complex χ(3) components with a significant chiral contribution.

Experimental implementation of THG Stokes-Mueller polarimetric microscopy

A polarimetric third-harmonic generation microscopy based on nonlinear Stokes-Mueller polarimetry theory is presented. All eight observable χ(3) tensor components of a β-carotene crystallite are extracted and a chiral component is identified.

Experimental demonstration of third-harmonic generation nonlinear Stokes-Mueller polarimetric microscopy

First experimental results of third-harmonic generation (THG) nonlinear Stokes-Mueller polarimetric (NSMP) microscopy are presented. Per-pixel polarimetric analysis allows all eight normalized observable χ(3) tensor elements to be extracted.

Nonlinear stokes mueller polarimetry for microscopic imaging of ordered biological structures

A microscopic nonlinear Stokes Mueller (SM) polarimetric imaging of ordered biological structures is presented. The nonlinear SM polarization microscopy provides the information about ultrastructural organization of molecules in biological tissue.

Nonlinear Stokes Mueller Polarimetric Microscopy

The two- and three-photon Stokes Mueller (SM) formalism is presented for the second and third harmonic generation polarimetry, respectively. The nonlinear SM polarization microscopy provides the information about ultrastructural organization of molecules in biological tissue.

Second-Harmonic Generation Double Stokes Mueller Polarimetric Microscopy

A novel double Stokes Mueller polarimetry (DSMP) technique is employed for nonlinear optical microscopy of biological samples. Complete per-voxel sample polarimetric characterization, extraction of the susceptibility tensor values and reconstruction of biological structure is presented.