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.      

Scar tissue classification using nonlinear optical microscopy and discriminant analysis

This paper addresses the scar tissue maturation process that occurs stepwise, and calls for reliable classification. The structure of collagen imaged by nonlinear optical microscopy (NLOM) in post‐burn hypertrophic and mature scar, as well as in normal skin, appeared to distinguish these maturation steps. However, it was a discrimination analysis, demonstrated here, that automated and quantified the scar tissue maturation process. The achieved scar classification accuracy was as high as 96%. The combination of NLOM and discrimination analysis is believed to be instrumental in gaining insight into the scar formation, for express diagnosis of scar and surgery planning. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Optical clearing for improved contrast in second harmonic generation imaging of skeletal muscle

Using second harmonic generation (SHG) imaging microscopy, we have examined the effect of optical clearing with glycerol to achieve greater penetration into specimens of skeletal muscle tissue. We find that treatment with 50% glycerol results in a 2.5-fold increase in achievable SHG imaging depth. Signal processing analyses using fast Fourier transform and continuous wavelet transforms show quantitatively that the periodicity of the sarcomere structure is unaltered by the clearing process and that image quality deep in the tissue is improved with clearing. Comparison of the SHG angular polarization dependence also shows no change in the supramolecular organization of acto-myosin complexes. By contrast, identical treatment of mouse tendon (collagen based) resulted in a strong decrease in SHG response. We suggest that the primary mechanism of optical clearing in muscle with glycerol treatment results from the reduction of cytoplasmic protein concentration and concomitant decrease in the secondary inner filter effect on the SHG signal. The lack of glycerol concentration dependence on the imaging depth indicates that refractive index matching plays only a minor role in the optical clearing of muscle. SHG and optical clearing may provide an ideal mechanism to study physiology in highly scattering skeletal or cardiac muscle tissue with significantly improved depth of penetration and achievable imaging depth.     

Mid‐infrared spectral microimaging of inflammatory skin lesions

Skin is one of the most important organs of the human body because of its characteristics and functions. There are many alterations, either pathological or physiological, that can disturb its functioning. However, at present all methods used to investigate skin diseases, non‐invasive or invasive, are based on clinical examinations by physicians. Thus, diagnosis, prognosis and therapeutic management rely on the expertise of the practitioner, the quality of the method and the accessibility of distinctive morphological characteristics of each lesion. To overcome the high sensitivity of these parameters, techniques based on more objective criteria must be explored. Vibrational spectroscopy has become as a key technique for tissue analysis in the biomedical research field. Based on a non‐destructive light/matter interaction, this tool provides information about specific molecular structure and composition of the analyzed sample, thus relating to its precise physiopathological state and permitting to distinguish lesional from normal tissues. This label‐free optical method can be performed directly on the paraffin‐embedded tissue sections without chemical dewaxing. In this study, the potential of the infrared microspectroscopy, combined with data classification methods was demonstrated, to characterize at the tissular level different types of inflammatory skin lesions, and this independently from conventional histopathology.      

Real‐time optical spectroscopic monitoring of nonirrigated lesion progression within atrial and ventricular tissues

Despite considerable advances in guidance of radiofrequency ablation (RFA) therapy for the treatment of cardiac arrhythmias, success rates have been hampered by a lack of tools for precise intraoperative evaluation of lesion extent. Near‐infrared spectroscopic (NIRS) techniques are sensitive to tissue structural and biomolecular properties, characteristics that are directly altered by radiofrequency (RF) treatment. In this work, a combined NIRS‐RFA catheter is developed for real‐time monitoring of tissue reflectance during RF energy delivery. An algorithm is proposed for processing NIR spectra to approximate nonirrigated lesion depth in both atrial and ventricular tissues. The probe optical geometry was designed to bias measurement influence toward absorption enabling enhanced sensitivity to changes in tissue composition. A set of parameters termed “lesion optical indices” are defined encapsulating spectral differences between ablated and unablated tissue. Utilizing these features, a model for real‐time tissue spectra classification and lesion size estimation is presented. Experimental validation conducted within freshly excised porcine cardiac specimens showed strong concordance between algorithm estimates and post‐hoc tissue assessment.      

Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy

Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.     

A direction‐selective local‐thresholding method,DSLT, in combination with a dye‐based method for automated three‐dimensional segmentation of cells and airspaces in developing leaves

Quantifying the anatomical data acquired from three‐dimensional (3D) images has become increasingly important in recent years. Visualization and image segmentation are essential for acquiring accurate and detailed anatomical data from images; however, plant tissues such as leaves are difficult to image by confocal or multi‐photon laser scanning microscopy because their airspaces generate optical aberrations. To overcome this problem, we established a staining method based on Nile Red in silicone‐oil solution. Our staining method enables color differentiation between lipid bilayer membranes and airspaces, while minimizing any damage to leaf development. By repeated applications of our staining method we performed time‐lapse imaging of a leaf over 5 days. To counteract the drastic decline in signal‐to‐noise ratio at greater tissue depths, we also developed a local thresholding method (direction‐selective local thresholding, DSLT) and an automated iterative segmentation algorithm. The segmentation algorithm uses the DSLT to extract the anatomical structures. Using the proposed methods, we accurately segmented 3D images of intact leaves to single‐cell resolution, and measured the airspace volumes in intact leaves.     

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)     

High‐intensity‐focused ultrasound and phase‐sensitive optical coherence tomography for high resolution surface acoustic wave elastography

Elastography has the ability of quantitatively evaluating the mechanical properties of soft tissue; thus it is helpful for diagnosis and treatment monitoring of many diseases, for example, skin diseases. Surface acoustic waves (SAWs) have been proven to be a non‐invasive, non‐destructive method for accurate characterization of tissue elastic properties. Current SAW elastography using high‐energy laser pulse or mechanical shaker still have some problems. In order to improve SAW elastography in medical application, a new technique was proposed in this paper, which combines high‐intensity‐focused ultrasound as a SAWs impulse inducer and phase‐sensitive optical coherence tomography as a SAWs detector. A 2% agar‐agar phantom and ex‐vivo porcine skin were tested. The data were processed by a new algorithm based on the Fourier analysis. The results show that the proposed method has the capability of quantifying the elastic properties of soft tissue‐mimicking materials. The lateral resolution of the elastogram has been significantly improved and the different layers in heterogeneous material could also been distinguished. Our improved technique of SAW elastography has a large potential to be widely applied in clinical use for skin disease diagnosis and treatment monitoring.      

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.      

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.     

Polarization second harmonic generation microscopy provides quantitative enhanced molecular specificity for tissue diagnostics

Due to specific structural organization at the molecular level, several biomolecules (e.g., collagen, myosin etc.) which are strong generators of second harmonic generation (SHG) signals, exhibit unique responses depending on the polarization of the excitation light. By using the polarization second harmonic generation (p‐SHG) technique, the values of the second order susceptibility components can be used to differentiate the types of molecule, which cannot be done by the use of a standard SHG intensity image. In this report we discuss how to implement p‐SHG on a commercial multiphoton microscope and overcome potential artifacts in susceptibility (χ) image. Furthermore we explore the potential of p‐SHG microscopy by applying the technique to different types of tissue in order to determine corresponding reference values of the ratio of second‐order χ tensor elements. These values may be used as a bio‐marker to detect any structural alterations in pathological tissue for diagnostic purposes.

The SHG intensity image (red) in ( a ) shows the distribution of collagen fibers in ovary tissue but cannot determine the type of collagen fiber. However, the histogram distribution ( b ) for the values of the χ tensor element ratio can be used to quantitatively identify the types of collagen fibers.     

Grid computing in image analysis

Diagnostic surgical pathology or tissue–based diagnosis still remains the most reliable and specific diagnostic medical procedure. The development of whole slide scanners permits the creation of virtual slides and to work on so-called virtual microscopes. In addition to interactive work on virtual slides approaches have been reported that introduce automated virtual microscopy, which is composed of several tools focusing on quite different tasks. These include evaluation of image quality and image standardization, analysis of potential useful thresholds for object detection and identification (segmentation), dynamic segmentation procedures, adjustable magnification to optimize feature extraction, and texture analysis including image transformation and evaluation of elementary primitives. Grid technology seems to possess all features to efficiently target and control the specific tasks of image information and detection in order to obtain a detailed and accurate diagnosis. Grid technology is based upon so-called nodes that are linked together and share certain communication rules in using open standards. Their number and functionality can vary according to the needs of a specific user at a given point in time. When implementing automated virtual microscopy with Grid technology, all of the five different Grid functions have to be taken into account, namely 1) computation services, 2) data services, 3) application services, 4) information services, and 5) knowledge services. Although all mandatory tools of automated virtual microscopy can be implemented in a closed or standardized open system, Grid technology offers a new dimension to acquire, detect, classify, and distribute medical image information, and to assure quality in tissue–based diagnosis.     

Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres

Several biologically important protein structures give rise to strong second-harmonic generation (SHG) in their native context. In addition to high-contrast optical sections of cells and tissues, SHG imaging can provide detailed structural information based on the physical constraints of the optical effect. In this study we characterize, by biochemical and optical analysis, the critical structures underlying SHG from the complex muscle sarcomere. SHG emission arises from domains of the sarcomere containing thick filaments, even within nascent sarcomeres of differentiating myocytes. SHG from isolated myofibrils is abolished by extraction of myosin, but is unaffected by removal or addition of actin filaments. Furthermore, the polarization dependence of sarcomeric SHG is not affected by either the proportion of myosin head domains or the orientation of myosin heads. By fitting SHG polarization anisotropy readings to theoretical response curves, we find an orientation for the elemental harmonophore that corresponds well to the pitch of the myosin rod alpha-helix along the thick filament axis. Taken together, these data indicate that myosin rod domains are the key structures giving SHG from striated muscle. This study should guide the interpretation of SHG contrast in images of cardiac and skeletal muscle tissue for a variety of biomedical applications.     

Nonlinear laser imaging of skin lesions

We investigated different kinds of human ex‐vivo skin samples by combined two‐photon intrinsic fluorescence (TPE), second‐harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), and multispectral two‐photon emission detection (MTPE). Morphological and spectroscopic differences were found between healthy and pathological skin samples, including tumors. In particular, we examined tissue samples from normal and pathological scar tissue (keloid), and skin tumors, including basal cell carcinoma (BCC), and malignant melanoma (MM). By using combined TPE‐SHG microscopy we investigated morphological features of different skin regions. Further comparative analysis of healthy skin and neoplastic samples was performed using FLIM, and MTPE. Finally, we demonstrated the use of methyl‐aminolevulinate as a contrast agent to increase the contrast in BCC border detection. The results obtained represent further support for in‐vivo noninvasive imaging of diseased skin. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Cover Picture: J. Biophotonics 10/2012

Pixel‐resolution mapping of collagen fiber spatial orientation in bovine leg tendon (upper row), chicken skin (middle row) and chicken leg tendon (bottom row) was achieved using polarization‐resolved SHG microscopy. Shown in the left column are SHG intensity images acquired by circularly polarized femtosecond laser. In addition, maps of fiber azimuthal angles are shown in the middle column. Finally, SHG image at different depths for bovine tendon (right column, upper panel) and fiber elevation angle maps for chicken skin and chicken leg tendon are shown in right column. Individual image size: 120 × 120 mm². (Picture: V. A. Hovhannisyan et al., pp. 768–776 in this issue)     

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.     

Label‐free quantitative chemical imaging and classification analysis of adipogenesis using mouse embryonic stem cells

Stem cells have received much attention recently for their potential utility in regenerative medicine. The identification of their differentiated progeny often requires complex staining procedures, and is challenging for intermediary stages which are a priori unknown. In this work, the ability of label‐free quantitative coherent anti‐Stokes Raman scattering (CARS) micro‐spectroscopy to identify populations of intermediate cell states during the differentiation of murine embryonic stem cells into adipocytes is assessed. Cells were imaged at different days of differentiation by hyperspectral CARS, and images were analysed with an unsupervised factorization algorithm providing Raman‐like spectra and spatially resolved maps of chemical components. Chemical decomposition combined with a statistical analysis of their spatial distributions provided a set of parameters that were used for classification analysis. The first 2 principal components of these parameters indicated 3 main groups, attributed to undifferentiated cells, cells differentiated into committed white pre‐adipocytes, and differentiating cells exhibiting a distinct protein globular structure with adjacent lipid droplets. An unsupervised classification methodology was developed, separating undifferentiated cell from cells in other stages, using a novel method to estimate the optimal number of clusters. The proposed unsupervised classification pipeline of hyperspectral CARS data offers a promising new tool for automated cell sorting in lineage analysis.