Nicole Prent

Nonlinear Optical Properties of Type I Collagen Fibers Studied by Polarization Dependent Second Harmonic Generation Microscopy

Collagen (type I) fibers are readily visualized with second harmonic generation (SHG) microscopy though the molecular origin of the signal has not yet been elucidated. In this study, the molecular origin of SHG from type I collagen is investigated using the time-dependent coupled perturbed Hartree-Fock calculations of the hyperpolarizibilities of glycine, proline, and hydroxyproline. Two effective nonlinear dipoles are found to orient in-the-plane of the amino acids, with one of the dipoles aligning close to the pitch orientation in the triple-helix, which provides the dominant contribution to the SHG polarization properties. The calculated hyperpolarizability tensor element ratios for the collagen triple-helix models: [(Gly3)n]3, [(Gly-Pro2)n]3, and [(Gly-Pro-Hyp)n]3, are used to predict the second-order nonlinear susceptibility ratios, χ(zzz)(2)/χ(iiz)(2) and χ(zii)(2)/χ(iiz)(2) of collagen fibers. From SHG microscopy polarization in, polarization out (PIPO) measurements of type I collagen in human lung tissue, a theoretical method is used to extract the triple-helix orientation angle with respect to the collagen fiber. The study shows the dominant role of amino acid orientation in the triple-helix for determining the polarization properties of SHG and provides a method for determining the triple-helix orientation angle in the collagen fibers.

Influence of semicrystalline order on the second-harmonic generation efficiency in the anisotropic bands of myocytes

The influence of semicrystalline order on the second-harmonic generation (SHG) efficiency in the anisotropic bands of Drosophila melanogaster sarcomeres from larval and adult muscle has been investigated. Differences in the semicrystalline order were obtained by using wild-type and mutant strains containing different amounts of headless myosin. The reduction in semicrystalline order without altering the chemical composition of myofibrils was achieved by observing highly stretched sarcomeres and by inducing a loss of viability in myocytes. In all cases the reduction of semicrystalline order in anisotropic bands of myocytes resulted in a substantial decrease in SHG. Second-harmonic imaging during periodic contractions of myocytes revealed higher intensities when sarcomeres were in the relaxed state compared with the contracted state. This study demonstrates that an ordered semicrystalline arrangement of anisotropic bands plays a determining role in the efficiency of SHG in myocytes.

Nonlinear multicontrast microscopy of hematoxylin-and-eosin-stained histological sections

Imaging hematoxylin-and-eosin-stained cancerous histological sections with multicontrast nonlinear excitation fluorescence, second- and third-harmonic generation (THG) microscopy reveals cellular structures with extremely high image contrast. Absorption and fluorescence spectroscopy together with second hyperpolarizability measurements of the dyes shows that strong THG appears due to neutral hemalum aggregation and is subsequently enhanced by interaction with eosin. Additionally, fluorescence lifetime imaging microscopy reveals eosin fluorescence quenching by hemalums, showing better suitability of only eosin staining for fluorescence microscopy. Multicontrast nonlinear microscopy has the potential to differentiate between cancerous and healthy tissue at a single cell level.

Optical microscopy in photosynthesis.

Emerging as well as the most frequently used optical microscopy techniques are reviewed and image contrast generation methods in a microscope are presented, focusing on the nonlinear contrasts such as harmonic generation and multiphoton excitation fluorescence. Nonlinear microscopy presents numerous advantages over linear microscopy techniques including improved deep tissue imaging, optical sectioning, and imaging of live unstained samples. Nonetheless, with the exception of multiphoton excitation fluorescence, nonlinear microscopy is in its infancy, lacking protocols, users and applications; hence, this review focuses on the potential of nonlinear microscopy for studying photosynthetic organisms. Examples of nonlinear microscopic imaging are presented including isolated light-harvesting antenna complexes from higher plants, starch granules, chloroplasts, unicellular alga Chlamydomonas reinhardtii, and cyanobacteria Leptolyngbya sp. and Anabaena sp. While focusing on nonlinear microscopy techniques, second and third harmonic generation and multiphoton excitation fluorescence microscopy, other emerging nonlinear imaging modalities are described and several linear optical microscopy techniques are reviewed in order to clearly describe their capabilities and to highlight the advantages of nonlinear microscopy.

Intermyofilament dynamics of myocytes revealed by second harmonic generation microscopy

Drosophila melanogaster larva myocytes are imaged with second harmonic generation (SHG) microscopy undergoing forced stretching and rhythmic contractions to determine the nature of the SHG signal. During stretching, double peaked SHG profiles of the anisotropic (A-) bands evolve into single peaks with a higher SHG intensity. The dip in the intensity profile at the center of the A-band is attributed to destructive interference from out-of-phase second harmonic radiating myosin molecules that, in the central region of myofilaments, are arranged antiparallel. An intensity increase at the center of the A-band appears during forced stretching due to a small, less than 100 nm, intermyofilament separation of the antiparallel myosin molecules leading to constructive interference of the SHG radiation. In addition, the same phenomenon occurs during periodic contractions of the myocyte, where an SHG intensity increase with the lengthening of sarcomeres is observed. The SHG intensity dependence on sarcomere length can be used for imaging myocyte contractions with low resolution microscopy, and can be applied for the development of diagnostic tools where monitoring of muscle contraction dynamics is required.

Measuring the molecular second hyperpolarizability in absorptive solutions by the third harmonic generation ratio technique.

Measurement of the second hyperpolarizability (γ) values of compounds can provide insight into the molecular structural requirements for enhancement of third harmonic generation (THG) signal. A convenient method for measuring the γ of compounds in solutions was developed by implementing the THG ratio method which is based on measuring the THG intensity from two interfaces using a nonlinear optical microscope while accounting for the refractive index of solutions at the fundamental and third harmonic wavelengths. We demonstrated that the difference in refractive index at both wavelengths strongly influenced the calculation of γ values when compounds have absorption near the third harmonic or fundamental wavelength. To this end, a refractometer with the wavelength tuning range from UV to near IR was constructed, and the measured refractive indices were used to extract the γ values. The γ values of carotenoids and chlorophylls found in photosynthetic pigment-protein complexes were explored. Large differences in the refractive index at third harmonic and fundamental wavelengths for chlorophylls result in γ values that are more than two orders of magnitude larger than γ values for carotenoids as well as the sign of chlorophyllsγ values is negative while carotenoids have positive γ values.

Applications of nonlinear microscopy for studying the structure and dynamics in biological systems

Laser scanning nonlinear optical microscopy is used to study structure and dynamics of cellular and sub-cellular structures in vivo. Under tight focusing conditions with a high numerical aperture objective, nonlinear optical signals such as third harmonic generation (THG), second harmonic generation (SHG), and multiphoton excitation fluorescence (MPF) are simultaneously produced. MPF is extensively used in biological imaging. Unfortunately, fluorescence is accompanied by heat dissipation in the sample and photobleaching effects. On the other hand, parametric processes such as SHG and THG are free of photobleaching since they involve only virtual electronic states where there is no transfer of energy into the medium. There are many naturally occurring structures that exhibit harmonic generation effects, and hence, do not require dyes that can potentially disrupt the normal functionality of the system. SHG is efficiently generated in non-centrosymmetric media, such as chiral structures and interfaces. The THG signal is generated due to a break in symmetry at interfaces and can be enhanced by the presence of multilamellar structures, as in the mitochondria or chloroplasts. Many interesting biological processes, such as signal transduction in neurons or ATP synthesis in mitochondria, involve the movement of ions across membranes. THG and SHG are sensitive to changing electric potential gradients, and hence are ideally suited for dynamical investigations of these biological processes. The present work will expose the structural factors and conditions that influence THG and SHG generation efficiencies in biological samples. Examples of visualizing chloroplasts and mitochondria will illustrate the advantages of harmonic generation microscopy for studying structural and functional properties of the in vivo systems.

Second harmonic generation polarization properties of myofilaments

Abstract. Second harmonic generation (SHG) polarization microscopy was used to investigate the organization of myosin nanomotors in myofilaments of muscle cells. The distribution of the second-order nonlinear susceptibility component ratio χzzz(2)/χzxx(2) along anisotropic bands of sarcomeres revealed differences between the headless and head-containing regions of myofilaments. The polarization-in polarization-out SHG measurements of headless myosin mutants of indirect flight muscle in Drosophila melanogaster confirmed a lower susceptibility component ratio compared to the head-containing myocytes with wild-type myosins. The increase in the ratio is assigned to the change in the deflection angle of the myosin S2 domain and possible contribution of myosin heads. The nonlinear susceptibility component ratio is a sensitive indicator of the myosin structure, and therefore, it can be used for conformational studies of myosin nanomotors. The measured ratio values can also be used as the reference for ab initio calculations of nonlinear optical properties of different parts of myosins.

Time and structural crosscorrelation image analysis of microscopic volumes, simultaneously recorded with second harmonic generation, third harmonic generation, and multiphoton excitation fluorescence microscopy

Our newly developed multimodal microscope enables simultaneous collection of second harmonic generation (SHG), third harmonic generation (THG) and multiphoton excitation fluorescence (MPF) signals. The signals can be generated within different or the same intercellular structures. In comparing two signals, traditional methods of image crosscorrelation analysis using Pearsons coefficient provide a general parameter as to whether the images are similar, however it does not give detailed information about correlation of different structures inside the images. We present here a new technique that employs a pixel by pixel analysis over an entire area or volume that is used to correlate the structures appearing in the images. The result of the analysis reveals structures within the sample that are generated by both nonlinear signals as well as highlighting the structures that are generated by only one of the nonlinear signals. The algorithm provides a means to colocalize different structures revealed by the different nonlinear contrast mechanisms. Structural correlation maps are useful in identifying the origin of structures in one nonlinear contrast mechanism when the origin of structures in another is known. Image analysis has also been exploited for sequences of images taken in time. The intensity fluctuations in time for each pixel reveal regions of intense physiological activity in biological samples. Correlation of time dependent fluctuations from different pixels in the image time series allows construction of the structural map that undergoes similar time behavior or appears out of phase. These structural correlation analysis techniques are demonstrated based on polystyrene beads and cardiomyocytes.

Dynamic investigation of Drosophila myocytes with second harmonic generation microscopy

The functional dynamics and structure of both larval and adult Drosophila melanogaster muscle were investigated with a nonlinear multimodal microscope. Imaging was carried out using a home built microscope capable of recording the multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation signals simultaneously at a scanning rate of up to ~12 frames/sec. The sample was excited by a home built femtosecond Ti:Sapphire laser at 840 nm, or by a Yb-ion doped potassium gadolinium tungstate (Yb:KGW) crystal based oscillator at 1042 nm. There was no observable damage detected in the myocyte after prolonged scanning with either of the lasers. Microscopic second harmonic generation (SHG) appears particularly strong in the myocytes. This allows the fast contraction dynamics of the myocytes to be followed. The larger sarcomere size observed in the larvae myocytes is especially well suited for studying the contraction dynamics. Microscopic imaging of muscle contractions showed different relaxation and contraction rates. The SHG intensities were significantly higher in the relaxed state of the myocyte compared to the contracted state. The imaging also revealed disappearance of SHG signal in highly stretched sarcomeres, indicating that SHG diminishes in the disordered structures. The study illustrates that SHG microscopy, combined with other nonlinear contrast mechanisms, can help to elucidate physiological mechanisms of contraction. This study also provides further insight into the mechanisms of harmonic generation in biological tissue and shows that crystalline arrangement of macromolecules has a determining factor for the high efficiency second harmonic generation from the bulk structures.