Second and third harmonic generation microscopy visualizes key structural components in fresh unprocessed healthy human breast tissue

Real‐time assessment of excised tissue may help to improve surgical results in breast tumor surgeries. Here, as a step towards this purpose, the potential of second and third harmonic generation (SHG, THG) microscopy is explored. SHG and THG are nonlinear optical microscopic techniques that do not require labeling of tissue to generate 3D images with intrinsic depth‐sectioning at sub‐cellular resolution. Until now, this technique had been applied on fixated breast tissue or to visualize the stroma only, whereas most tumors start in the lobules and ducts. Here, SHG/THG images of freshly excised unprocessed healthy human tissue are shown to reveal key breast components—lobules, ducts, fat tissue, connective tissue and blood vessels, in good agreement with hematoxylin and eosin histology. DNA staining of fresh unprocessed mouse breast tissue was performed to aid in the identification of cell nuclei in label‐free THG images. Furthermore, 2‐ and 3‐photon excited auto‐fluorescence images of mouse and human tissue are collected for comparison. The SHG/THG imaging modalities generate high quality images of freshly excised tissue in less than a minute with an information content comparable to that of the gold standard, histopathology. Therefore, SHG/THG microscopy is a promising tool for real‐time assessment of excised tissue during surgery.      

Intraocular multiphoton microscopy with subcellular spatial resolution by infrared femtosecond lasers

We reported on the in situ nonlinear optical sectioning of the corneal and retinal tissues based on the multiphoton microscopy (MPM) with different excitation wavelengths of infrared femtosecond (fs) lasers. The multiphoton nonlinear processing including two-photon fluorescence (2PF) and second harmonic generation (SHG) was induced under condition of high light intensities on an order of MW-GW/cm². The laser beams emitted from the solid-state Ti: sapphire systems were focused in a 0.1 femtoliter focus volume of a high numerous aperture diffraction-limited objective (40 × 1.3 N.A., oil). The corneal layers have been visualized using nonlinear optical tomography. In particular, corneal Bowman’s layer was optically determined in situ. The cellular and collagen components of tissues were selectively displayed with submicron spatial resolution and high efficiency without any assistance of staining or slicing. The preliminary study on retinal optical tomography is here also reported. MPM is a promising and convenient non-invasive technique by which the tissue layers can be visualized and the selective displaying of the tissue microstructures be realized. The optical biopsy based on intrinsic emission of MPM yields details that provide three-dimensional displaying of the tissue component and even have the potential to be used in clinical diagnostics.Dedicated on the occasion of the 66th birthday of Professor Dr. Karl-Juergen Halbhuber     

Three‐dimensional mapping of the orientation of collagen corneal lamellae in healthy and keratoconic human corneas using SHG microscopy

Keratoconus is an eye disorder that causes the cornea to take an abnormal conical shape, thus impairing its refractive functions and causing blindness. The late diagnosis of keratoconus is among the principal reasons for corneal surgical transplantation. This pathology is characterized by a reduced corneal stiffness in the region immediately below Bowman's membrane, probably due to a different lamellar organization, as suggested by previous studies. Here, the lamellar organization in this corneal region is characterized in three dimensions by means of second‐harmonic generation (SHG) microscopy. In particular, a method based on a three‐dimensional correlation analysis allows to probe the orientation of sutural lamellae close to the Bowman's membrane, finding statistical differences between healthy and keratoconic samples. This method is demonstrated also in combination with an epi‐detection scheme, paving the way for a potential clinical ophthalmic application of SHG microscopy for the early diagnosis of keratoconus.

SHG image acquired with sagittal optical sectioning ( A ) of a healthy cornea and ( B ) of a keratoconic cornea. Scale bars: 30 μm.     

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.     

Nonlinear‐optical stain‐free stereoimaging of astrocytes and gliovascular interfaces

Methods of nonlinear optics provide a vast arsenal of tools for label‐free brain imaging, offering a unique combination of chemical specificity, the ability to detect fine morphological features, and an unprecedentedly high, subdiffraction spatial resolution. While these techniques provide a rapidly growing platform for the microscopy of neurons and fine intraneural structures, optical imaging of astroglia still largely relies on filament‐protein‐antibody staining, subject to limitations and difficulties especially severe in live‐brain studies. Once viewed as an ancillary, inert brain scaffold, astroglia are being promoted, as a part of an ongoing paradigm shift in neurosciences, into the role of a key active agent of intercellular communication and information processing, playing a significant role in brain functioning under normal and pathological conditions. Here, we show that methods of nonlinear optics provide a unique resource to address long‐standing challenges in label‐free astroglia imaging. We demonstrate that, with a suitable beam‐focusing geometry and careful driver‐pulse compression, microscopy of second‐harmonic generation (SHG) can enable a high‐resolution label‐free imaging of fibrillar structures of astrocytes, most notably astrocyte processes and their endfeet. SHG microscopy of astrocytes is integrated in our approach with nonlinear‐optical imaging of red blood cells based on third‐harmonic generation (THG) enhanced by a three‐photon resonance with the Soret band of hemoglobin. With astroglia and red blood cells providing two physically distinct imaging contrasts in SHG and THG channels, a parallel detection of the second and third harmonics enables a high‐contrast, high‐resolution, stain‐free stereoimaging of gliovascular interfaces in the central nervous system. Transverse scans of the second and third harmonics are shown to resolve an ultrafine texture of blood‐vessel walls and astrocyte‐process endfeet on gliovascular interfaces with a spatial resolution within 1 μm at focusing depths up to 20 μm inside a brain.     

Nonlinear optical microscopy in decoding arterial diseases

Pathological understanding of arterial diseases is mainly attributable to histological observations based on conventional tissue staining protocols. The emerging development of nonlinear optical microscopy (NLOM), particularly in second-harmonic generation, two-photon excited fluorescence and coherent Raman scattering, provides a new venue to visualize pathological changes in the extracellular matrix caused by atherosclerosis progression. These techniques in general require minimal tissue preparation and offer rapid three-dimensional imaging. The capability of label-free microscopic imaging enables disease impact to be studied directly on the bulk artery tissue, thus minimally perturbing the sample. In this review, we look at recent progress in applications related to arterial disease imaging using various forms of NLOM.     

A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms

Background information. CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest). Results. We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron‐microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high‐pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 μm distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of non‐correlative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron‐microscopic protocols, making them more accessible to a wider range of researchers. Conclusions. We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time‐demanding procedure of specimen preparation for any kind of TEM.     

Multimodal and multiplex spectral imaging of rat cornea ex vivo using a white‐light laser source

We applied our multimodal nonlinear spectral imaging microscope to the measurement of rat cornea. We successfully obtained multiple nonlinear signals of coherent anti‐Stokes Raman scattering (CARS), third‐order sum frequency generation (TSFG), and second harmonic generation (SHG). Depending on the nonlinear optical processes, the cornea tissue was visualized with different image contrast mechanism simultaneously. Due to white‐light laser excitation, multiplex CARS and TSFG spectra were obtained. Combined multimodal and spectral analysis clearly elucidated the layered structure of rat cornea with molecular structural information. This study indicates that our multimodal nonlinear spectral microscope is a promising bioimaging method for tissue study.

Multimodal nonlinear spectral images of rat cornea at corneal epithelium and corneal stroma in the in‐plane (XY) direction. With use of the combinational analysis of different nonlinear optical processes, detailed molecular structural information is available without staining or labelling.     

二次谐波显微成像技术

二次谐波非线性显微成像技术是近年发展起来的一种新型光学成像方法,已广泛应用于生物医学的各个领域。介绍了光学二次谐波产生的原理、成像装置及其技术发展,描述了二次谐波的成像特点和它与双光子荧光成像的异同,并对其在生物医学上的应用及发展前景做出展望。     

Advances in nonlinear optical microscopy techniques for in vivo and in vitro neuroimaging

Understanding the mechanism of the brain via optical microscopy is one of the challenges in neuroimaging, considering the complex structures. Advanced neuroimaging techniques provide a more comprehensive insight into patho-mechanisms of brain disorders, which is useful in the early diagnosis of the pathological and physiological changes associated with various neurodegenerative diseases. Recent advances in optical microscopy techniques have evolved powerful tools to overcome scattering of light and provide improved in vivo neuroimaging with sub-cellular resolution, endogenous contrast specificity, pinhole less optical sectioning capability, high penetration depth, and so on. The following article reviews the developments in various optical imaging techniques including two-photon and three-photon fluorescence, second-harmonic generation, third-harmonic generation, coherent anti-Stokes Raman scattering, and stimulated Raman scattering in neuroimaging. We have outlined the potentials and drawbacks of these techniques and their possible applications in the investigation of neurodegenerative diseases.     

Collagen chirality and three‐dimensional orientation studied with polarimetric second‐harmonic generation microscopy

Polarization‐dependent second‐harmonic generation (P‐SHG) microscopy is used to characterize molecular nonlinear optical properties of collagen and determine a three‐dimensional (3D) orientation map of collagen fibers within a pig tendon. C₆ symmetry is used to determine the nonlinear susceptibility tensor components ratios in the molecular frame of reference and , where the latter is a newly extracted parameter from the P‐SHG images and is related to the chiral structure of collagen. The is observed for collagen fibers tilted out of the image plane, and can have positive or negative values, revealing the relative polarity of collagen fibers within the tissue. The P‐SHG imaging was performed using a linear polarization‐in polarization‐out (PIPO) method on thin sections of pig tendon cut at different angles. The nonlinear chiral properties of collagen can be used to construct the 3D organization of collagen in the tissue and determine the orientation‐independent molecular susceptibility ratios of collagen fibers in the molecular frame of reference.      

Polarization‐dependent second‐harmonic generation for collagen‐based differentiation of breast cancer samples

Nonlinear optical imaging techniques have been widely used to reveal biological structures for accurate diagnosis at the cellular as well as the tissue level. In the present study, polarization‐dependent second‐harmonic generation (PSHG) was used to determine collagen orientation in breast cancer biopsy tissues (grades 0, I, II and III). The obtained data were processed using fast Fourier transform (FFT) analysis, while second‐harmonic generation (SHG) anisotropy and the “ratio parameter” values were also calculated. Such measurements were shown to be able to distinguish collagen structure modifications in different cancer grades tested. The analysis presented herein suggests that PSHG imaging could provide a quantitative evaluation of the tumor state and the distinction of malignant from benign breast tissues. The obtained results also allowed the development of a biophysical model, which can explain the aforementioned differentiations and is in agreement with the simulations relating the SHG anisotropy values with the mechanical tension applied to the collagen during cancer progression. The current approach could be a step forward for the development of new, nondestructive, label free optical diagnostic tools for cancer reducing the need of recalls and unnecessary biopsies, while potentially improving cancer detection rates.     

Dual‐ and single‐shot susceptibility ratio measurements with circular polarizations in second‐harmonic generation microscopy

Polarization‐resolved second‐harmonic generation (P‐SHG) microscopy is a technique capable of characterizing nonlinear optical properties of noncentrosymmetric biomaterials by extracting the nonlinear susceptibility tensor components ratio , with z‐axis parallel and x‐axis perpendicular to the C₆ symmetry axis of molecular fiber, such as a myofibril or a collagen fiber. In this paper, we present two P‐SHG techniques based on incoming and outgoing circular polarization states for a fast extraction of : A dual‐shot configuration where the SHG circular anisotropy generated using incident right‐ and left‐handed circularly‐polarized light is measured; and a single‐shot configuration for which the SHG circular anisotropy is measured using only one incident circular polarization state. These techniques are used to extract the of myosin fibrils in the body wall muscles of Drosophila melanogaster larva. The results are in good agreement with values obtained from the double Stokes‐Mueller polarimetry. The dual‐ and single‐shot circular anisotropy measurements can be used for fast imaging that is independent of the in‐plane orientation of the sample. They can be used for imaging of contracting muscles, or for high throughput imaging of large sample areas.     

Imaging directed photothermolysis through two‐photon absorption demonstrated on mouse skin – a potential novel tool for highly targeted skin treatment

One‐photon absorption based traditional laser treatment may not necessarily be selective at the microscopic level, thus could result in un‐intended tissue damage. Our objective is to test whether two‐photon absorption (TPA) could provide highly targeted tissue alteration of specific region of interest without damaging surrounding tissues. TPA based laser treatments (785 nm, 140 fs pulse width, 90 MHz) were performed on ex vivo mouse skin using different average power levels and irradiation times. Reflectance confocal microscopy (RCM) and combined second‐harmonic‐generation (SHG) and two‐photon fluorescence (TPF) imaging channels were used to image before, during, and after each laser treatment. The skin was fixed, sectioned and H & E stained after each experiment for histological assessment of tissue alterations and for comparison with the non‐invasive imaging assessments. Localized destruction of dermal fibers was observed without discernible epidermal damage on both RCM and SHG + TPF images for all the experiments. RCM and SHG + TPF images correlated well with conventional histological examination. This work demonstrated that TPA‐based light treatment provides highly localized intradermal tissue alteration. With further studies on optimizing laser treatment parameters, this two‐photon absorption photothermolysis method could potentially be applied in clinical dermatology. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Quantification of collagen fiber structure using second harmonic generation imaging and two‐dimensional discrete Fourier transform analysis: Application to the human optic nerve head

Second harmonic generation (SHG) microscopy is widely used to image collagen fiber microarchitecture due to its high spatial resolution, optical sectioning capabilities and relatively nondestructive sample preparation. Quantification of SHG images requires sensitive methods to capture fiber alignment. This article presents a two‐dimensional discrete Fourier transform (DFT)–based method for collagen fiber structure analysis from SHG images. The method includes integrated periodicity plus smooth image decomposition for correction of DFT edge discontinuity artefact, avoiding the loss of peripheral image data encountered with more commonly used windowing methods. Outputted parameters are as follows: the collagen fiber orientation distribution, aligned collagen content and the degree of collagen fiber dispersion along the principal orientation. We demonstrate its application to determine collagen microstructure in the human optic nerve head, showing its capability to accurately capture characteristic structural features including radial fiber alignment in the innermost layers of the bounding sclera and a circumferential collagen ring in the mid‐stromal tissue. Higher spatial resolution rendering of individual lamina cribrosa beams within the nerve head is also demonstrated. Validation of the method is provided in the form of correlative results from wide‐angle X‐ray scattering and application of the presented method to other fibrous tissues.      

Self‐assembling amyloid‐like peptides as exogenous second harmonic probes for bioimaging applications

Amyloid‐like peptides are an ideal model for the mechanistic study of amyloidosis, which may lead to many human diseases, such as Alzheimer disease. This study reports a strong second harmonic generation (SHG) effect of amyloid‐like peptides, having a signal equivalent to or even higher than those of endogenous collagen fibers. Several amyloid‐like peptides (both synthetic and natural) were examined under SHG microscopy and shown they are SHG‐active. These peptides can also be observed inside cells (in vitro). This interesting property can make these amyloid‐like peptides second harmonic probes for bioimaging applications. Furthermore, SHG microscopy can provide a simple and label‐free approach to detect amyloidosis. Lattice corneal dystrophy was chosen as a model disease of amyloidosis. Morphological difference between normal and diseased human corneal biopsy samples can be easily recognized, proving that SHG can be a useful tool for disease diagnosis.     

Automatic and label‐free identification of blood vessels in gliomas using the combination of multiphoton microscopy and image analysis

Currently, the targeted treatment of tumor based on the tumor microenvironment is newly developed. Blood vessels are the key parts in the tumor microenvironment, which is taken as a new visible target for tumor therapy. Multiphoton microscopy (MPM), based on the second harmonic generation and two‐photon excited fluorescence, is available to make the label‐free analysis on the blood vessels in human gliomas. MPM can reveal the vascular morphological characteristics in gliomas, including vascular malformation, intense vascular proliferation, perivascular collagen deposition, perivascular lymphocytes aggregation and microvascular proliferation. In addition, the image analysis algorithms were developed to automatically calculate the perivascular collagen content, vascular cavity area, lumen area, wall area and vessel number. Thus, the vascular morphology, the perivascular collagen deposition and intense vascular proliferation degree can be further quantitatively characterized. Compared with the pathological analysis, the combination of MPM and image analysis has potential advantages in making a quantitative and qualitative analyzing on vascular morphology in glioma microenvironment. As micro‐endoscope and two‐photon fiberscope are technologically improved, this combined method will be a useful imaging way to make the real‐time research on the targeting tumor microenvironment in gliomas.     

Comparison of higher‐order multiphoton signal generation and collection at the 1700‐nm window based on transmittance measurement of objective lenses

One benefit of excitation at the 1700‐nm window is the more accessible modalities of multiphoton signal generation. It is demonstrated here that the transmittance performance of the objective lens is of vital importance for efficient higher‐order multiphoton signal generation and collection excited at the 1700‐nm window. Two commonly used objective lenses for multiphoton microscopy (MPM) are characterized and compared, one with regular coating and the other with customized coating for high transmittance at the 1700‐nm window. Our results show that, fourth harmonic generation imaging of mouse tail tendon and 5‐photon fluorescence of carbon quantum dots using the regular objective lens shows an order of magnitude signal higher than those using the customized objective lens. Besides, the regular objective lens also enables a 3‐photon fluorescence imaging depth of >1600 μm in mouse brain in vivo. Our results will provide guidelines for objective lens selection for MPM at the 1700‐nm window.     

Signal generation and Raman‐resonant imaging by non‐degenerate four‐wave mixing under tight focusing conditions

The authors demonstrate Raman‐resonant imaging based on the simultaneous generation of several nonlinear frequency mixing processes resulting from a 3‐color coherent anti‐Stokes Raman scattering (CARS) experiment. The interaction of three coincident short‐pulsed laser beams simultaneously generates both 2‐color (degenerate) CARS and 3‐color (non‐degenerate) CARS signals, which are collected and characterized spectroscopically – allowing for resonant, doubly‐resonant, and non‐resonant contrast mechanisms. Images obtained from both 2‐color and 3‐color CARS signals are compared and found to provide complementary information. The 3‐color CARS microscopy scheme provides a versatile multiplexed modality for biological imaging, which may extend the capabilities of label‐free non‐linear microscopy, e.g. by probing multiple Raman resonances. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)