Second Harmonic Microscopy Research Articles

Thermal poling of femtosecond laser-written waveguides in fused silica.

Thermal poling of femtosecond laser written waveguides was investigated using second-harmonic microscopy under three approaches: (1) pre-poling and (2) post-poling in which fused silica substrates were poled before or after waveguide formation, respectively, and (3) double poling in which poling was applied both before and after laser writing. Effective nonlinear waveguide interaction strength was assessed relative to the mode profile and the assessments demonstrated an erasure effect of 81% in pre-poling and an ion migration blocking effect of 26% in post-poling. Double poling was found to recover the nonlinearity over the modal zone, overcoming prior difficulties with combining laser processing and thermal poling, opening up a future avenue for creating active devices through femtosecond laser writing of nonlinear optical circuits in fused silica.

Femoral epiphyseal cartilage matrix changes at predilection sites of equine osteochondrosis: Quantitative MRI, second-harmonic microscopy, and histological findings.

Osteochondrosis is an ischemic chondronecrosis of epiphyseal growth cartilage that results in focal failure of endochondral ossification and osteochondritis dissecans at specific sites in the epiphyses of humans and animals, including horses. The upstream events leading to the focal ischemia remain unknown. The epiphyseal growth cartilage matrix is composed of proteoglycan and collagen macromolecules and encases its vascular tree in canals. The matrix undergoes major dynamic changes in early life that could weaken it biomechanically and predispose it to focal trauma and vascular failure. Subregions in neonatal foal femoral epiphyses (n = 10 osteochondrosis predisposed; n = 6 control) were assessed for proteoglycan and collagen structure/content employing 3T quantitative MRI (3T qMRI: T1ρ and T2 maps). Site-matched validations were made with histology, immunohistochemistry, and second-harmonic microscopy. Growth cartilage T1ρ and T2 relaxation times were significantly increased (p < 0.002) within the proximal third of the trochlea, a site predisposed to osteochondrosis, when compared with other regions. However, this was observed in both control and osteochondrosis predisposed specimens. Microscopic evaluation of this region revealed an expansive area with low proteoglycan content and a hypertrophic-like appearance on second-harmonic microscopy. We speculate that this matrix structure and composition, though physiological, may weaken the epiphyseal growth cartilage biomechanically in focal regions and could enhance the risk of vascular failure with trauma leading to osteochondrosis. However, additional investigations are now required to confirm this. 3T qMRI will be useful for future non-invasive longitudinal studies to track the osteochondrosis disease trajectory in animals and humans. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1743-1752, 2016.

Second-harmonic scanning microscopy of domains in Al wire bonds in IGBT modules.

Scanning second harmonic generation microscopy has been used to investigate crystallographic orientation of the grain structure in Al wire bonds in insulated gate bipolar transistor modules. It was shown that the recorded second harmonic microscopy images revealed the grain structure of the Al sample. Additional information of the individual grain orientation was achieved by using simple interpretations of the recorded rotational anisotropy.

Articular cartilage zonal differentiation via 3D Second-Harmonic Generation imaging microscopy

Purpose: The collagen structure throughout the patella has not been thoroughly investigated by 3D imaging, where the majority of the existing data come from histological cross sections. It is important to have a better understanding of the architecture in normal tissues, where this could then be applied to imaging of diseased states.Methods: To address this shortcoming, we investigated the combined use of collagen-specific Second-Harmonic Generation (SHG) imaging and measurement of bulk optical properties to characterize collagen fiber orientations of the histologically defined zones of bovine articular cartilage. Forward and backward SHG intensities of sections from superficial, middle and deep zones were collected as a function of depth and analyzed by Monte Carlo simulations to extract the SHG creation direction, which is related to the fibrillar assembly.Results: Our results revealed differences in SHG forward–backward response between the three zones, where these are consistent with a previously developed model of SHG emission. Some of the findings are consistent with that from other modalities; however, SHG analysis showed the middle zone had the most organized fibril assembly. While not distinct, we also report bulk optical property values for these different zones within the patella.Conclusions: Collectively, these results provide quantitative measurements of structural changes at both the fiber and fibril assembly of the different cartilage zones and reveals structural information not possible by other microscope modalities. This can provide quantitative insight to the collagen fiber network in normal cartilage, which may ultimately be developed as a biomarker for osteoarthritis.

Applications of second-harmonic generation imaging microscopy in ovarian and breast cancer.

In this perspective, we discuss how the nonlinear optical technique of second-harmonic generation (SHG) microscopy has been used to greatly enhance our understanding of the tumor microenvironment (TME) of breast and ovarian cancer. Striking changes in collagen architecture are associated with these epithelial cancers, and SHG can image these changes with great sensitivity and specificity with submicrometer resolution. This information has not historically been exploited by pathologists but has the potential to enhance diagnostic and prognostic capabilities. We summarize the utility of image processing tools that analyze fiber morphology in SHG images of breast and ovarian cancer in human tissues and animal models. We also describe methods that exploit the SHG physical underpinnings that are effective in delineating normal and malignant tissues. First we describe the use of polarization-resolved SHG that yields metrics related to macromolecular and supramolecular structures. The coherence and corresponding phase-matching process of SHG results in emission directionality (forward to backward), which is related to sub-resolution fibrillar assembly. These analyses are more general and more broadly applicable than purely morphology-based analyses; however, they are more computationally intensive. Intravital imaging techniques are also emerging that incorporate all of these quantitative analyses. Now, all these techniques can be coupled with rapidly advancing miniaturization of imaging systems to afford their use in clinical situations including enhancing pathology analysis and also in assisting in real-time surgical determination of tumor margins.

Cellulose amorphization by swelling in ionic liquid/water mixtures: a combined macroscopic and second-harmonic microscopy study.

Amorphization of cellulose by swelling in ionic liquid (IL)/water mixtures at room temperature is a suitable alternative to the dissolution-precipitation pretreatment known to facilitate enzymatic digestion. When soaking microcrystalline cellulose in the IL 1-ethyl-3-methylimidazolium acetate containing 20 wt % water, the crystallinity of the cellulose sample is strongly reduced. As less than 4 % of the cellulose dissolves in this mixture, this swelling method makes a precipitation step and subsequent energy-intensive IL purification redundant. Second-harmonic generation (SHG) microscopy is used as a structure-sensitive technique for in situ monitoring of the changes in cellulose crystallinity. Combined optical and SHG observations confirm that in the pure IL complete dissolution takes place, while swelling without dissolution in the optimal IL/water mixture yields a solid cellulose with a significantly reduced crystallinity in a single step.

Capturing the optical phase response of nanoantennas by coherent second-harmonic microscopy.

The ultrafast coherent control of light localization in resonant plasmonic nanostructures is intricately related to the phase response of the involved plasmon resonances. In this work, we exploit the second harmonic signal generated by single optical nanoantennas subject to broadband phase-controlled femtosecond pulses to study and tailor the coherent resonance response. Our results reveal that both the spectral phase and the amplitude components associated with the plasmon resonance of arbitrary individual nanoantennas can be accurately determined.

Optimization-based wavefront sensorless adaptive optics for multiphoton microscopy.

Optical aberrations have detrimental effects in multiphoton microscopy. These effects can be curtailed by implementing model-based wavefront sensorless adaptive optics, which only requires the addition of a wavefront shaping device, such as a deformable mirror (DM) to an existing microscope. The aberration correction is achieved by maximizing a suitable image quality metric. We implement a model-based aberration correction algorithm in a second-harmonic microscope. The tip, tilt, and defocus aberrations are removed from the basis functions used for the control of the DM, as these aberrations induce distortions in the acquired images. We compute the parameters of a quadratic polynomial that is used to model the image quality metric directly from experimental input-output measurements. Finally, we apply the aberration correction by maximizing the image quality metric using the least-squares estimate of the unknown aberration.

Effect of the domain shape on noncollinear second-harmonic emission in disordered quadratic media

We study the role of the individual ferroelectric domain shape on the second-harmonic emission in strontium barium niobate featuring a random quadratic nonlinearity. The noncollinearly emitted second-harmonic signal is scanned in the far-field at different incident angles for different domain size distributions. This offers the possibility to retrieve the Fourier spectrum, corresponding to the spatial domain distribution and domain shape. Based on images of the domain structures retrieved by Čerenkov-type second-harmonic microscopy, domain patterns are simulated, the second-harmonic intensities are calculated, and finally compared with the measurements.

Numerical simulation of polarization-resolved second-harmonic microscopy in birefringent media

Polarization-resolved second-harmonic microscopy has recently emerged as a valuable technique for in situ imaging of collagen structure in tissues. Nevertheless, collagen-rich tissues such as tendon, ligament, skin dermis, bone, cornea, or artery exhibit a heterogeneous and anisotropic architecture that results in complex optical properties. While experimental evidence of polarization distortions has been reported in various tissues, the physics of second-harmonic imaging within such tissues is not fully understood yet. In this work, we performed numerical simulations of polarization-resolved second-harmonic generation in a strongly focused regime within a birefringent tissue. We show that vectorial components due to strong focusing have a rather small effect on the measurement of the second-harmonic tensorial response, while birefringence and optical dispersion may affect these measurements dramatically. We show indeed that a difference in the focal field distribution for ordinary and extraordinary waves results in different phase-matching conditions, which strongly affects the relative efficacy of second-harmonic generation for different polarizations. These results are of great interest for extracting reliable quantitative parameters from second-harmonic images.

Fluorescence and second-harmonic generation correlative microscopy to probe space charge separation and silver cluster stabilization during direct laser writing in a tailored silver-containing glass

We report on fluorescence and second-harmonic generation correlative microscopy of femtosecond direct laser-induced structures in a tailored silver-containing phosphate glass. We compare the spatial distributions of the related permanent electric field and silver clusters. The latter appear to be co-localized where the associated electric potential ensures favorable reduction-oxidation conditions for their formation and stabilization. Space charge separation is shown to occur prior the cluster formation. The associated electric field is a key parameter for silver clustering, thanks to electric field assisted silver ion motion. Future photonic structures combining 3D laser-structured fluorescence and nonlinear optical properties in such tailored glass will require an optimal control of the induced electric field distribution.

Precision increase with two orthogonal analyzers in polarization-resolved second-harmonic generation microscopy

We analyze the increase in precision of parameters estimation for polarization-resolved second-harmonic generation imaging microscopy when two intensities are measured with two orthogonal analyzers. The analysis is performed for measuring anisotropy parameters and molecule orientation for samples with cylindrical symmetry in the presence of photon noise with Poisson statistics. The improvement in comparison to global intensity measurement (i.e., without analyzer) is discussed.

Thermal Transitions of Fibrillar Collagen Unveiled by Second-Harmonic Generation Microscopy of Corneal Stroma

The thermal transitions of fibrillar collagen are investigated with second-harmonic generation polarization anisotropy microscopy. Second-harmonic generation images and polarization anisotropy profiles of corneal stroma heated in the 35–80°C range are analyzed by means of a theoretical model that is suitable to probe principal intramolecular and interfibrillar parameters of immediate physiological interest. Our results depict the tissue modification with temperature as the interplay of three destructuration stages at different hierarchical levels of collagen assembly including its tertiary structure and interfibrillar alignment, thus supporting and extending previous findings. This method holds the promise of a quantitative inspection of fundamental biophysical and biochemical processes and may find future applications in real-time and postsurgical functional imaging of collagen-rich tissues subjected to thermal treatments.

Observing the localization of light in space and time by ultrafast second-harmonic microscopy

Multiple coherent scattering and the constructive interference of certain scattering paths form the common scheme of several remarkable localization phenomena of classical and quantum waves in randomly disordered media1. Prominent examples are electron transport in disordered conductors2,3, the localization of excitons in semiconductor nanostructures4,5, surface plasmon polaritons at rough metallic films6,7 or light in disordered dielectrics8,9,10,11 and amplifying media1,12,13,14. However, direct observation of the fundamental spatiotemporal dynamics of the localization process remains challenging15. This holds true, in particular, for the localization of light occurring on exceedingly short femtosecond timescales and nanometre length scales. Here, we combine second harmonic microscopy with few-cycle time resolution to probe the spatiotemporal localization of light waves in a random dielectric medium. We find lifetimes of the photon modes of several femtoseconds and a broad distribution of the local optical density of states, revealing central hallmarks of the localization of light. By combining high-resolution nonlinear optical microscopy with few-cycle time resolution, scientists show that they are able to probe the spatiotemporal localization of light waves in random dielectric nanostructures. The findings will aid the study of light localization dynamics in a variety of passive and active random media.

Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging

A series of chromophores with enhanced second- and third-order nonlinear optical properties were engineered for use in combined second-harmonic and two-photon fluorescence microscopy. Electron-accepting moieties imparted nonlinear optical properties to the chromophores. The electron-rich carbazole core served as a template towards one- or two-dimensional chromophores. More efficient acceptor groups (pyridinium, benzazolium, benzothiazolium) on the carbazole donor core resulted in improved second- and third-order nonlinear optical properties. A selection of these chromophores was tested in a cellular environment with a multimodal multiphoton microscope. The structural differences of the chromophores resulted in high selectivity for mitochondria or the nucleus in two-photon fluorescence and ranging from no signal to high selectivity for mitochondria in the SHG channel.

Second-harmonic confocal microscopy of layered microstructures based on porous silicon

Layered structures based on porous silicon have been studied by confocal microscopy of the second optical harmonic. A linear increase in the second-harmonic intensity with increasing porosity of the layers has been observed. This behavior may result from spatial fluctuations in the dipole quadratic response of the pore walls.

Beam position stabilization for a confocal multiphoton microscope

The influence of beam-pointing on scanning confocal microscopy is investigated. The beam displacement is measured using a quadrant photodiode, and the apparent movement of a sub-micron-sized particle observed by second-harmonic microscopy is linked to the beam displacement. A simple beam-pointing stabilization is implemented, and improvement of beam stability by three orders of magnitude on long time scales is achieved.

Ensemble and Individual Characterization of the Nonlinear Optical Properties of ZnO and BaTiO3 Nanocrystals

Hyper Rayleigh scattering (HRS) and second harmonic (SH) microscopy were used to study the second order nonlinear optical response of ZnO and BaTiO3 nanocrystals, which have been recently proposed as new markers for bioimaging. HRS, combined with dynamic light scattering, was first used to retrieve hyperpolarizabilities and nonlinear coefficients of both type of nanocrystals. The results indicate that the main contribution to the SH signal is related to the bulk noncentrosymmetric structure of the particles. Successively, we carried out the inspection of individual nanocrystals by SH microscopy, interpreting their response using the nonlinear coefficients deduced from HRS.

Second-harmonic microscopy for fluence-dependent investigation of laser-induced surface reactions

Spatially resolved optical second-harmonic generation (SHG), that is, SHG microscopy, is shown to be a versatile tool for the investigation of femtosecond laser-induced surface processes such as adsorbate diffusion and desorption and their dependence on absorbed laser fluence. As an example, time-resolved measurements on the diffusion of O from the steps onto the terraces of a vicinal Pt(111) surface at low substrate temperature were performed. Using the sensitivity of SHG on step coverage and analyzing the signal change across the laser beam profile via SHG microscopy during laser-induced diffusion, the strong nonlinear fluence dependence of the diffusion rate could be determined. Using SHG microscopy in combination with a two-pulse correlation scheme, time-resolved information on the surface reaction was obtained as a function of absorbed laser fluence.

Progress in creating second-order optical nonlinearity in silicate glasses and waveguides through thermal poling

This paper describes progress in characterizing the distribution and localization of the second-order nonlinearity induced in thermally poled silicate glasses and optical waveguides, in particular, optical fibers. It starts by describing the basics of the poling technique, especially the most commonly used “thermal poling” technique. Then results of systematic investigation of the distribution of the second-order nonlinearity in poled glass and special fibers using second-harmonic microscopy are presented. Interesting issues such as the effectiveness of the poling technique for waveguides formed by ultrafast laser pulses are also discussed.