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Second harmonic generation (SHG) microscopy is a commonly used technique to study the organization of collagen within tissues. However, individual collagen fibrils, which have diameters much smaller than the resolution of most optical systems, have not been extensively investigated. Here we probe the structure of individual collagen fibrils using polarization-resolved SHG (PSHG) microscopy and atomic force microscopy. We find that longitudinally polarized light occurring at the edge of a focal volume of a high numerical aperture microscope objective illuminated with linearly polarized light creates a measurable variation in PSHG signal along the axis orthogonal to an individual collagen fibril. By comparing numerical simulations to experimental data, we are able to estimate parameters related to the structure and chirality of the collagen fibril without tilting the sample out of the image plane, or cutting tissue at different angles, enabling chirality measurements on individual nanostructures to be performed in standard PSHG microscopes. The results presented here are expected to lead to a better understanding of PSHG results from both collagen fibrils and collagenous tissues. Further, the technique presented can be applied to other chiral nanoscale structures such as microtubules, nanowires, and nanoribbons.

Circular dichroism second harmonic generation microscopy (CDSHG) is a powerful imaging technique, which allows three-dimensional visualization of collagen fibril orientation in tissues. However, recent publications have obtained contradictory results on whether CDSHG can be used to reveal the relative out-of-plane polarity of collagen fibrils. Here we compare CDSHG images of unstained tendon and tendon which has been stained with hematoxylin and eosin. We find significant differences in the CDSHG between these two conditions, which explain the recent contradictory results within the literature.

Second harmonic generation (SHG) microscopy has emerged as a powerful technique for visualizing collagen organization within tissues. Amongst the many advantages of SHG is its sensitivity to collagen nanoscale organization, and its presumed sensitivity to the relative out of plane polarity of fibrils. Recent results have shown that circular dichroism SHG (CD-SHG), a technique that has been commonly assumed to reveal the relative out of plane polarity of collagen fibrils, is actually insensitive to changes in fibril polarity. However, results from another research group seem to contradict this conclusion. Both previous results have been based on SHG imaging of collagen fibrils within tissues, therefore, to gain a definitive understanding of the sensitivity of SHG to relative out of plane polarity, the results from individual fibrils are desirable. Here we present polarization resolved SHG microscopy (PSHG) data from individual collagen fibrils oriented out of the image plane by buckling on an elastic substrate. We show through correlation with atomic force microscopy measurements that SHG intensity can be used to estimate the out of plane angle of individual fibrils. We then compare the sensitivity of two PSHG techniques, CD-SHG and polarization-in, polarization-out SHG (PIPO-SHG), to the relative out of plane polarity of individual fibrils. We find that for single fibrils CD-SHG is insensitive to relative out of polarity and we also demonstrate the first direct experimental confirmation that PIPO-SHG reveals the relative out of plane polarity of individual collagen fibrils., Competing Interests: The authors declare no conflicts of interest., (© 2023 Optica Publishing Group.)

Alterations in collagen ultrastructure between human gastric adenocarcinoma and normal gastric tissue were investigated using polarization-resolved second harmonic generation (PSHG) microscopy. Cylindrical and trigonal symmetries were assumed to extract quantitative PSHG parameters, ρ , κ and S , from each image pixel. Statistically significant variations in these values were observed for gastric adenocarcinoma, indicating a higher disorder of collagen. Numerical focal volume simulations of crossing fibrils indicate increased S parameter is due to more intersecting collagen fibrils of varying diameters. These parameters were also able to distinguish between different grades of gastric adenocarcinoma indicating that PSHG may be useful for automated cancer diagnosis., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Crystalline maltodextrin particles (CMPs) were investigated using polarization-sensitive second harmonic generation (PSHG) microscopy to determine changes in their crystalline organization due to crystal type (A- and B-type) and hydration for application as starch model systems. Optimization of their synthesis resulted in intense SHG emission, exceeding maize starch granules. PSHG data showed that CMPs have a radial macrostructure with respect to their nucleation regions, fitted ρ values of 2-6, and some similar hydration variations, mimicking starch granules and validating that CMPs may be used as a model system for improved understanding of the SHG properties and applications of starch granules., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

The origin of second harmonic generation (SHG) signal in otoconia was investigated. SHG signal intensity from otoconia was compared to pure calcite crystals, given calcite is the primary component of otoconia and is known to emit surface SHG. The SHG intensity from calcite was found to be ∼41× weaker than the SHG intensity from otoconia signifying that the SHG signal from otoconia is likely generated from the organic matrix. Furthermore, the SHG intensity from otoconia increased when treated with a chelating agent known to dissolve calcite which confirms that calcite is not the source of SHG. Additionally, polarization-resolved SHG microscopy imaging revealed that the arrangement of the SHG emitters is radial and can form highly ordered domains., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Rope-like structures are ubiquitous in Nature. They are supermolecular assemblies of macromolecules responsible for the structural and mechanical integrity of plant and animal tissues. Collagen fibrils with diameters between 50 and 500 nm and their helical supermolecular structure are good examples of such nanoscale biological ropes. Like man-made laid ropes, fibrils are typically loaded in tension, and due to their large aspect ratio, they are, in principle, prone to buckling and torsional instabilities. One way to study buckling of a rigid rod is to attach it to a stretched elastic substrate that is then returned to its original length. In the case of single collagen fibrils, the observed behavior depends on the degree of hydration. By going from buckling in ambient conditions to immersed in a buffer, fibrils go from the well-known sine wave response to a localized behavior reminiscent of the bird-caging of laid ropes. In addition, in ambient conditions, the sine wave response coexists with the formation of loops along the length of the fibrils, as observed for the torsional instability of a twisted filament when tension is decreased. This work provides direct evidence that single collagen fibrils are highly susceptible to axial compression because of their helical supermolecular structure. As a result, mammals that use collagen fibrils as their main load-bearing element in many tissues have evolved mitigating strategies that protect single fibrils from axial compression damage.

Polarization-sensitive second harmonic generation (SHG) microscopy is an established imaging technique able to provide information related to specific molecular structures including collagen. In this investigation, polarization-sensitive SHG microscopy was used to investigate changes in the collagen ultrastructure between histopathology slides of normal and diseased human thyroid tissues including follicular nodular disease, Grave's disease, follicular variant of papillary thyroid carcinoma, classical papillary thyroid carcinoma, insular or poorly differentiated carcinoma, and anaplastic or undifferentiated carcinoma ex vivo. The second-order nonlinear optical susceptibility tensor component ratios, χ (2) zzz '/χ (2) zxx ' and χ (2) xyz '/χ (2) zxx ', were obtained, where χ (2) zzz '/χ (2) zxx ' is a structural parameter and χ (2) xyz '/χ (2) zxx ' is a measure of the chirality of the collagen fibers. Furthermore, the degree of linear polarization (DOLP) of the SHG signal was measured. A statistically significant increase in χ (2) zzz '/χ (2) zxx ' values for all the diseased tissues except insular carcinoma and a statistically significant decrease in DOLP for all the diseased tissues were observed compared to normal thyroid. This finding indicates a higher ultrastructural disorder in diseased collagen and provides an innovative approach to discriminate between normal and diseased thyroid tissues that is complementary to standard histopathology.

Wide-field second harmonic generation (SHG) microscopy was developed using a high-power (> 4 W) and high-repetition-rate (MHz range) laser oscillator to achieve fast SHG imaging over a large area (400 µm × 400 µm). The microscope was used for high spatial resolution imaging of contracting muscles in live Drosophila melanogaster larvae. Anisotropic and isotropic bands of striated muscle were distinguished, allowing accurate determination of sarcomere length and SHG intensity from individual sarcomeres. Therefore, wide-field SHG microscopy has applications in basic contractility research and studying arrhythmias, muscular dystrophies and pharmaceutical effects on the muscle contraction dynamics of sarcomeres., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Thin tissue sections of normal and tumorous pancreatic tissues stained with hematoxylin and eosin were investigated using multiphoton excitation fluorescence (MPF), second harmonic generation (SHG), and third harmonic generation (THG) microscopies. The cytoplasm, connective tissue, collagen and extracellular structures are visualized with MPF due to the eosin stain, whereas collagen is imaged with endogenous SHG contrast that does not require staining. Cellular structures, including membranous interfaces and nuclear components, are seen with THG due to the aggregation of hematoxylin dye. Changes in the collagen ultrastructure in pancreatic cancer were investigated by a polarization-sensitive SHG microscopy technique, polarization-in, polarization-out (PIPO) SHG. This involves measuring the orientation of the linear polarization of the SHG signal as a function of the linear polarization orientation of the incident laser radiation. From the PIPO SHG data, the second-order non-linear optical susceptibility ratio, χ (2) zzz '/χ (2) zxx ', was obtained that serves as a structural parameter for characterizing the tissue. Furthermore, by assuming C 6 symmetry, an additional second-order non-linear optical susceptibility ratio, χ (2) xyz '/χ (2) zxx ', was obtained, which is a measure of the chirality of the collagen fibers. Statistically-significant differences in the χ (2) zzz '/χ (2) zxx ' values were found between tumor and normal pancreatic tissues in periductal, lobular, and parenchymal regions, whereas statistically-significant differences in the full width at half maximum (FWHM) of χ (2) xyz '/χ (2) zxx ' occurrence histograms were found between tumor and normal pancreatic tissues in periductal and parenchymal regions. Additionally, the PIPO SHG data were used to determine the degree of linear polarization (DOLP) of the SHG signal, which indicates the relative linear depolarization of the signal. Statistically-significant differences in DOLP values were found between tumor and normal pancreatic tissues in periductal and parenchymal regions. Hence, the differences observed in the χ (2) zzz '/χ (2) zxx ' values, the FWHM of χ (2) xyz '/χ (2) zxx ' values and the DOLP values could potentially be used to aid pathologists in diagnosing pancreatic cancer.

Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning.

Third harmonic generation (THG) microscopy can exploit endogenous harmonophores such as pigment macromolecules for enhanced image contrast, and therefore can be used without exogenous contrast agents. Previous studies have established that carotenoid compounds are ideal harmonophores for THG microscopy; we therefore sought to determine whether THG from endogenous carotenoid-derived compounds, such as retinal in photoreceptor cells, could serve as a new label-free method for developmental studies. Here we study the development of the pupal eye in Drosophila melanogaster and determine the localization of rhodopsin using THG microscopy technique. Additionally, by altering the chromophore or the opsin protein we were able to detect changes in both the retinal distribution morphology and in THG intensity age-dependent profiles. These results demonstrate that THG microscopy can be used to detect altered photoreceptor development and may be useful in clinically relevant conditions associated with photoreceptor degeneration.

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.

Nonlinear optical microscopy has become a powerful tool for high-resolution imaging of cellular and subcellular composition, morphology, and interactions because of its high spatial resolution, deep penetration, and low photo-damage to tissue. Developing specific harmonic probes is essential for exploiting nonlinear microscopic imaging for biomedical applications. We report an organized aggregate of porphyrins (OAP) that formed within lipidic nanoparticles showing fingerprint spectroscopic properties, structure-associated second harmonic generation, and superradiant third harmonic generation. The OAP facilitated harmonic microscopic imaging of living cells with significantly enhanced contrast. The structure-dependent switch between harmonic (OAP-intact) and fluorescence (OAP-disrupted) generation enabled real-time multi-modality imaging of the cellular fate of nanoparticles. Robustly produced under various conditions and easily incorporated into pre-formed lipid nanovesicles, OAP provides a biocompatible nanoplatform for harmonic imaging., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Second harmonic generation (SHG) microscopy is employed to study changes in crystalline organization due to altered gene expression and hydration in barley starch granules. SHG intensity and susceptibility ratio values (R'SHG ) are obtained using reduced Stokes-Mueller polarimetric microscopy. The maximum R'SHG values occur at moderate moisture indicating the narrowest orientation distribution of nonlinear dipoles from the cylindrical axis of glucan helices. The maximum SHG intensity occurs at the highest moisture and amylopectin content. These results support the hypothesis that SHG is caused by ordered hydrogen and hydroxyl bond networks which increase with hydration of starch granules.

Changes in collagen ultrastructure between malignant and normal human thyroid tissue were investigated ex vivo using polarization second harmonic generation (SHG) microscopy. The second-order nonlinear optical susceptibility tensor component ratio and the degree of linear polarization (DOLP) of the SHG signal were measured. The ratio values are related to the collagen ultrastructure, while DOLP indicates the relative amount of coherent signal and incoherent scattering of SHG. Increase in ratio values and decrease in DOLP were observed for tumor tissue compared to normal thyroid, indicating higher ultrastructural disorder in tumor collagen.

The origin of second harmonic generation (SHG) in starch granules was investigated using ab initio quantum mechanical modeling and experimentally examined using polarization-in, polarization-out (PIPO) second harmonic generation microscopy. Ab initio calculations revealed that the largest contribution to the SHG signal from A- and B-type allomorphs of starch originates from the anisotropic organization of hydroxide and hydrogen bonds mediated by aligned water found in the polymers. The hypothesis was experimentally tested by imaging maize starch granules under various hydration and heat treatment conditions that alter the hydrogen bond network. The highest SHG intensity was found in fully hydrated starch granules, and heat treatment diminished the SHG intensity. The PIPO SHG imaging showed that dried starch granules have a much higher nonlinear optical susceptibility component ratio than fully hydrated granules. In contrast, deuterated starch granules showed a smaller susceptibility component ratio demonstrating that SHG is highly sensitive to the organization of the hydroxyl and hydrogen bond network. The polarization SHG imaging results of potato starch granules, representing starch allomorph B, were compared to those of maize starch granules representing allomorph A. The results showed that the amount of aligned water was higher in the maize granules. Nonlinear microscopy of starch granules provides evidence that varying hydration conditions leads to significant changes in the nonlinear susceptibility ratio as well as the SHG intensity, supporting the hypothesis from ab initio calculations that the dominant contribution to SHG is due to the ordered hydroxide and hydrogen bond network.

We demonstrate a noninvasive optical microscopy technique based on polarization-dependent second harmonic generation for determining the crystal lattice structure and microscopic heterogeneities within individual nanostructures. Differentiation between periodically twinned and wurtzite ZnSe nanowires (NWs) was demonstrated, and measurement of the cubic lattice rotation orientation around the NW axis was determined within 1° accuracy. Zinc blende NWs were differentiated from wurtzite. The technique can be used for quality inspection and optimization of growth conditions for nanostructures.

Nonlinear optical microscopy was used to image the localization of astaxanthin accumulation in the green alga, Haematococcus pluvialis. Polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and third harmonic generation (THG) microscopy was applied to study the crystalline organization of astaxanthin molecules in light-stressed H. pluvialis in vivo. Since astaxanthin readily forms H- and J-aggregates in aqueous solutions, PIPO THG studies of astaxanthin aggregates contained in red aplanospores were compared to PIPO THG of in vitro self-assembled H- and J-aggregates of astaxanthin. The PIPO THG data clearly showed an isotropic organization of astaxanthin in red aplanospores of H. pluvialis. This is in contrast to the highly anisotropic organization of astaxanthin in synthetic H- and J-aggregates, which showed to be uniaxial. Since carotenoids in vitro preferentially form H- and J-aggregates, but in vivo form a randomly organized structure, this implies that astaxanthin undergoes a different way of packing in biological organisms, which is either due to the unique physical environment of the alga or is controlled enzymatically.

Polarization second harmonic microscopy was used for collagen imaging in human non-small cell lung carcinoma and normal lung tissues ex vivo and revealed significant differences in the nonlinear susceptibility component ratio, demonstrating potential use in cancer diagnosis.

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.

Photosynthetic structures when imaged with nonlinear optical microscopy give rise to high third harmonic generation (THG) signal intensity due to the presence of chlorophylls and xanthophylls which have large second hyperpolarizabilitiy (γ) values. The γ value of trimers of the light-harvesting chlorophyll a/b pigment-protein complex of photosystem II (LHCII) isolated from pea (Pisum sativum) plants was investigated by the THG ratio technique at 1028 nm wavelength and found to have the value (-1600 ± 400) × 10(-41) m(2) V(-2). The large negative γ value of trimeric LHCII is due to the presence of chlorophyll a and chlorophyll b which have large negative γ values, while positive γ values of xanthophylls reduce the magnitude of the THG signal. Variation was observed between the measured γ value of LHCII and the approximated γ value of LHCII obtained by adding individual γ values of chlorophylls and xanthophylls. This difference can be attributed to the differing inter-pigment interactions of oriented chlorophylls and xanthophylls in the pigment-protein complex compared to randomly oriented non-interacting pigments in solution, as well as a differing dielectric environment of the pigments within LHCII versus the surrounding organic solvent.

Differential polarization nonlinear optical microscopy has the potential to become an indispensable tool for structural investigations of ordered biological assemblies and microcrystalline aggregates. Their microscopic organization can be probed through fast and sensitive measurements of nonlinear optical signal anisotropy, which can be achieved with microscopic spatial resolution by using time-multiplexed pulsed laser beams with perpendicular polarization orientations and photon-counting detection electronics for signal demultiplexing. In addition, deformable membrane mirrors can be used to correct for optical aberrations in the microscope and simultaneously optimize beam overlap using a genetic algorithm. The beam overlap can be achieved with better accuracy than diffraction limited point-spread function, which allows to perform polarization-resolved measurements on the pixel-by-pixel basis. We describe a newly developed differential polarization microscope and present applications of the differential microscopy technique for structural studies of collagen and cellulose. Both, second harmonic generation, and fluorescence-detected nonlinear absorption anisotropy are used in these investigations. It is shown that the orientation and structural properties of the fibers in biological tissue can be deduced and that the orientation of fluorescent molecules (Congo Red), which label the fibers, can be determined. Differential polarization microscopy sidesteps common issues such as photobleaching and sample movement. Due to tens of megahertz alternating polarization of excitation pulses fast data acquisition can be conveniently applied to measure changes in the nonlinear signal anisotropy in dynamically changing in vivo structures.

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 chlorophylls'γ values is negative while carotenoids have positive γ values., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The use of carotenoids as biologically friendly labels for third harmonic generation (THG) microscopy is demonstrated. Carotenoid containing liposomes are used to label cell structures via liposome cell fusion. The THG microscopy labels, called harmonophores, were characterized by measuring the third-order nonlinear susceptibility (χ((3))) of carotenoids: violaxanthin, neoxanthin, lutein, β-carotene, zeaxanthin, canthaxanthin and astaxanthin. The THG ratio method was used, which is based on measuring the THG intensity from two interfaces using a nonlinear optical microscope. The second hyperpolarizability values of carotenoids were extracted from χ((3)) measurements taking into account the refractive index at fundamental and third harmonic wavelengths. The length dependence of the second hyperpolarizability of conjugated polyenes from 9 to 13 double bonds with varying oxygen functional groups was investigated. It appears that the presence of epoxides can have a higher influence than an additional conjugated double bond. Furthermore, labelling of both Drosophila Schneider 2 cells and Drosophila melanogaster larvae myocytes with β-carotene was achieved. This study demonstrates that THG enhancement by carotenoids can be used for nontoxic in vivo labelling of subcellular structures for third 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.

With polarization dependent second harmonic generation (SHG) microscopy becoming a more popular method for investigating the structure of biological materials, there is a need to develop tools with which to understand and interpret the observed SHG properties. Quantum mechanical calculations of the hyperpolarizability tensor have become a popular method for understanding the SHG properties of biomolecules. Visualization of the full hyperpolarizability tensor, termed the unit sphere representation, has been developed to provide insight and intuition on the relationship between SHG properties and molecules. A single vector representation is also presented, which approximates the SHG properties of molecules for certain cases, where the anisotropy is negligible., (Copyright © 2010 Wiley Periodicals, Inc.)

We present a new laser system and nonlinear microscope, designed for differential nonlinear microscopy. The microscope features time-correlated single photon counting of multiphoton fluorescence generated by an alternating pulse-train of orthogonally polarized pulses. The generated nonlinear signal is separated using home-built electronics. Results are presented on fluorescence-detected nonlinear absorption linear anisotropy (FDNALA) of chloroplasts in Asparagus Sprengerii Regel and of Congo Red-stained cellulose.

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.

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., (© Springer Science+Business Media B.V. 2009)

We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences.

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.