Distinguishing metastatic triple‐negative breast cancer from nonmetastatic breast cancer using second harmonic generation imaging and resonance Raman spectroscopy

Triple‐negative breast cancer (TNBC) is an aggressive subset of breast cancer that is more common in African‐American and Hispanic women. Early detection followed by intensive treatment is critical to improving poor survival rates. The current standard to diagnose TNBC from histopathology of biopsy samples is invasive and time‐consuming. Imaging methods such as mammography and magnetic resonance (MR) imaging, while covering the entire breast, lack the spatial resolution and specificity to capture the molecular features that identify TNBC. Two nonlinear optical modalities of second harmonic generation (SHG) imaging of collagen, and resonance Raman spectroscopy (RRS) potentially offer novel rapid, label‐free detection of molecular and morphological features that characterize cancerous breast tissue at subcellular resolution. In this study, we first applied MR methods to measure the whole‐tumor characteristics of metastatic TNBC (4T1) and nonmetastatic estrogen receptor positive breast cancer (67NR) models, including tumor lactate concentration and vascularity. Subsequently, we employed for the first time in vivo SHG imaging of collagen and ex vivo RRS of biomolecules to detect different microenvironmental features of these two tumor models. We achieved high sensitivity and accuracy for discrimination between these two cancer types by quantitative morphometric analysis and nonnegative matrix factorization along with support vector machine. Our study proposes a new method to combine SHG and RRS together as a promising novel photonic and optical method for early detection of TNBC.     

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.     

Improved quantification of collagen anisotropy with polarization‐resolved second harmonic generation microscopy

Imaging tissue samples by polarization‐resolved second harmonic generation microscopy provides both qualitative and quantitative insights into collagen organization in a label‐free manner. Polarization‐resolved second harmonic generation microscopy goes beyond simple intensity‐based imaging by adding the laser beam polarization component and applying different quantitative metrics such as the anisotropy factor. It thus provides valuable information on collagen arrangement not available with intensity measurements alone. Current established approaches are limited to calculating the anisotropy factor for only a particular laser beam polarization and no general guidelines on how to select the best laser beam polarization have yet been defined. Here, we introduce a novel methodology for selecting the optimal laser beam polarization for characterizing tissues using the anisotropy in the purpose of identifying cancer signatures. We show that the anisotropy factor exhibits a similar laser beam polarization dependence to the second harmonic intensity and we combine it with the collagen orientation index computed by Fast Fourier Transform analysis of the recorded images to establish a framework for choosing the laser beam polarization that is optimal for an accurate interpretation of polarization‐resolved second harmonic generation microscopy images and anisotropy maps, and hence a better differentiation between healthy and dysplastic areas.

SHG image of skin tissue (a) and a selected area of interest for which we compute the SHG intensity (b) and anisotropy factor (c) dependence on the laser beam polarization and also the FFT spectrum (d) to evaluate the collagen orientation index.     

Multiphoton tomography in differentiation of morphological and molecular subtypes of breast cancer: A quantitative analysis

In this study multiphoton tomography, based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) was used to visualize both the extracellular matrix and tumor cells in different morphological and molecular subtypes of human breast cancer. It was shown, that quantified assessment of the SHG based imaging data has great potential to reveal differences of collagen quantity, organization and uniformity in both low- and highly- aggressive invasive breast cancers. The values of quantity and uniformity of the collagen fibers distribution were significantly higher in low-aggressive breast cancer compared to the highly-aggressive subtypes, while the value representing collagen organization was lower in the former type. Additionally, it was shown, that TPEF detection of elastin fibers and amyloid protein may be used as a biomarker of detection the low-aggressive breast cancer subtype. Thus, TPEF/SHG imaging offers the possibility of becoming a useful tool for the rapid diagnosis of various subtypes of breast cancer during biopsy as well as for the intraoperative determinination of tumor-positive resection margins.     

Collagen ultrastructural symmetry and its malignant alterations in human breast cancer revealed by polarization‐resolved second‐harmonic generation microscopy

Several specific alterations of the extracellular matrix can be considered a distinctive hallmark of cancer. In particular, a different morphology of the collagen scaffold is frequently found within the peritumoural environment. In this study, we report about a significant difference in the ultrastructural organization of collagen at the supra‐molecular level between the perilesional scaffold and the tumour area in human breast carcinoma samples. In particular, we demonstrated that polarization‐resolved second‐harmonic generation (P‐SHG) microscopy is able to link the altered collagen architecture at the ultrastructural level found in perilesional tissue with a different organization of collagen fibrils at the molecular level.     

Polarization-resolved SHG imaging as a fast screening method for collagen alterations during aging: Comparison with light and electron microscopy

Our previous study on rat skin showed that cumulative oxidative pressure induces profound structural and ultrastructural alterations in both rat skin epidermis and dermis during aging. Here, we aimed to investigate the biophotonic properties of collagen as a main dermal component in the function of chronological aging. We used second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) on 5 μm thick skin paraffin sections from 15-day-, 1-month- and 21-month-old rats, respectively, to analyze collagen alterations, in comparison to conventional light and electron microscopy methods. Obtained results show that polarization-resolved SHG (PSHG) images can detect collagen fiber alterations in line with chronological aging and that this method is consistent with light and electron microscopy. Moreover, the β coefficient calculated from PSHG images points out that delicate alterations lead to a more ordered structure of collagen molecules due to oxidative damage. The results of this study also open the possibility of successfully applying this fast and label-free method to previously fixed samples.     

Evaluation of breast carcinoma regression after preoperative chemotherapy by label‐free multiphoton imaging and image analysis

Neoadjuvant chemotherapy is increasingly being used in breast carcinoma as it significantly improves the prognosis and consistently leads to an increased rate of breast preservation. How to accurately assess tumor response after treatment is a crucial factor for developing reasonable therapeutic strategy. In this study, we were in an attempt to monitor tumor response by multimodal multiphoton imaging including two‐photon excitation fluorescence and second‐harmonic generation imaging. We found that multiphoton imaging can identify different degrees of tumor response such as a slight, significant, or complete response and can detect morphological alteration associated with extracellular matrix during the progression of breast carcinoma following preoperative chemotherapy. Two quantitative optical biomarkers including tumor cellularity and collagen content were extracted based on automatic image analysis to help monitor changes in tumor and its microenvironment. Furthermore, tumor regression grade diagnosis was tried to evaluate by multiphoton microscopy. These results may offer a basic framework for using multiphoton microscopic imaging techniques as a helpful diagnostic tool for assessing breast carcinoma response after presurgical treatment.     

Multiphoton tomography visualizes collagen fibers in the tumor microenvironment that maintain cancer‐cell anchorage and shape

Second harmonic generation (SHG) multiphoton imaging can visualize fibrillar collagen in tissues. SHG has previously shown that fibrillar collagen is altered in various types of cancer. In the present study, in vivo high resolution SHG multi‐photon tomography in living mice was used to study the relationship between cancer cells and intratumor collagen fibrils. Using green fluorescent protein (GFP) to visualize cancer cells and SHG to image collagen, we demonstrated that collagen fibrils provide a scaffold for cancer cells to align themselves and acquire optimal shape. These results suggest a new paradigm for a stromal element of tumors: their role in maintaining anchorage and shape of cancer cells that may enable them to proliferate. J. Cell. Biochem. 114: 99–102, 2012. © 2012 Wiley Periodicals, Inc.     

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.      

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.     

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.     

Comparison of optically‐derived biomarkers in healthy and brain tumor tissue under one‐ and two‐photon excitation

The surgical outcome of brain tumor resection and needle biopsy is significantly correlated to the patient's prognosis. Brain tumor surgery is limited to resecting the solid portion of the tumor as current intraoperative imaging modalities are incapable of delineating infiltrative regions. For accurate delineation, in situ tissue interrogation at the submicron scale is warranted. Additionally, multimodal detection is required to remediate the genetically and molecularly heterogeneous nature of brain tumors, notably, that of gliomas, meningioma and brain metastasis. Multimodal detection, such as spectrally‐ and temporally‐resolved fluorescence under one‐ and two‐photon excitation, enables characterizing tissue based on several endogenous optical contrasts. In order to assign the optically‐derived parameters to different tissue types, construction of an optical database obtained from biopsied tissue is warranted. This report showcases the different quantitative and semi‐quantitative optical markers that may comprise the tissue discrimination database. These include: the optical index ratio, the optical redox ratio, the relative collagen density, spectrally‐resolved fluorescence lifetime parameters, two‐photon fluorescence imaging and second harmonic generation imaging.     

Strategies for High-Resolution Imaging of Epithelial Ovarian Cancer by Laparoscopic Nonlinear Microscopy

Ovarian cancer remains the most frequently lethal of the gynecologic cancers owing to the late detection of this disease. Here, by using human specimens and three mouse models of ovarian cancer, we tested the feasibility of nonlinear imaging approaches, the multiphoton microscopy (MPM) and second harmonic generation (SHG) to serve as complementary tools for ovarian cancer diagnosis. We demonstrate that MPM/SHG of intrinsic tissue emissions allows visualization of unfixed, unsectioned, and unstained tissues at a resolution comparable to that of routinely processed histologic sections. In addition to permitting discrimination between normal and neoplastic tissues according to pathological criteria, the method facilitates morphometric assessment of specimens and detection of very early cellular changes in the ovarian surface epithelium. A red shift in cellular intrinsic fluorescence and collagen structural alterations have been identified as additional cancer-associated changes that are indiscernible by conventional pathologic techniques. Importantly, the feasibility of in vivo laparoscopic MPM/SHG is demonstrated by using a “stick” objective lens. Intravital detection of neoplastic lesions has been further facilitated by low-magnification identification of an indicator for cathepsin activity followed by MPM laparoscopic imaging. Taken together, these results demonstrate that MPM may be translatable to clinical settings as an endoscopic approach suitable for high-resolution optical biopsies as well as a pathology tool for rapid initial assessment of ovarian cancer samples.     

Spatial orientation mapping of fibers using polarization‐sensitive second harmonic generation microscopy

In this work, we present a non‐invasive approach to determine azimuth and elevation angles of collagen fibers capable of generating second harmonic signal. The azimuth angle was determined using the minimum of second harmonic generation (SHG) signal while rotating the plane of polarization of excitation light. The elevation angle was estimated from the ratio of the minimal SHG intensity to the intensity when laser polarization and fiber directions were parallel to each other using experimentally determined calibration curve. Pixel‐resolution images of collagen fiber spatial orientation in tendon from bovine leg, chicken leg, and chicken skin were acquired using our approach of SHG polarization‐resolved microscopy. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Monitoring aging-associated structural alterations in Caenorhabditis elegans striated muscles via polarization-dependent second-harmonic generation measurements

The in-vivo elucidation of the molecular mechanisms underlying muscles dysfunction due to aging via non-invasive label free imaging techniques is an important issue with high biological significance. In this study, polarization-dependent second-harmonic generation (PSHG) was used to evaluate structural alterations in the striated muscles during Caenorhabditis elegans lifespan. Young and old wild-type animals were irradiated. The obtained results showed that it was not feasible to detect differences in the structure of myosin that could be correlated with the aging of the worms, via the implementation of the classical, widely used, cylindrical symmetry model and the calculation of the SHG anisotropy values. A trigonal symmetry model improved the extracted results; however, the best outcome was originated from the application of a general model. Myosin structural modifications were monitored via the estimation of the difference in spectral phases derived from discrete Fourier transform analysis. Age classification of the nematodes was achieved by employing both models, proving the usefulness of the usage of PSHG microscopy as a potential diagnostic tool for the investigation of muscle diseases.     

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.     

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.      

Visualization photofragmentation‐induced rhodamine B release from gold nanorod delivery system by combination two‐photon luminescence imaging with correlation spectroscopy

Plasmon‐enhanced gold nanorod (AuNR) with high photothermal conversion efficiency is a promising light‐controllable nanodrug delivery system for cancer therapy. Understanding the mechanism for the light‐controllable drug release of AuNR delivery systems is important for the development of nanomedicine. In this study, the rhodamine B (RB) released from AuNR‐RB nanodelivery system was quantitated and visualized by using two‐photon luminescence (TPL) imaging combined with correlation spectroscopy. The photofragmentation of AuNR induced by femtosecond pulsed laser was revealed by TPL correlation spectroscopy when the laser energy was above the thermal damage threshold of AuNR, and the RB released from this nanodrug delivery system was visualized by TPL imaging. Furthermore, the photofragmentation‐induced release of RB from AuNR‐RB nanodelivery system was visualized in living MCF‐7 breast cancer cells by TPL imaging combined with correlation spectroscopy. These results provided a novel optical approach to quantify the release of drugs from gold nanocarriers in complex biological media.     

Analyzing the feasibility of discriminating between collagen types I and II using polarization‐resolved second harmonic generation

According to previous studies, the nonlinear susceptibility tensor ratio χ₃₃/χ₃₁ obtained from polarization‐resolved second harmonic generation (P‐SHG) under the assumption of cylindrical symmetry can be used to distinguish between fibrillar collagen types. Discriminating between collagen fibrils of types I and II is important in tissue engineering of cartilage. However, cartilage has a random organization of collagen fibrils, and the assumption of cylindrical symmetry may be incorrect. In this study, we simulated the P‐SHG response from different collagen organizations and demonstrated a possible method to exclude areas where cylindrical symmetry is not fulfilled and where fibrils are located in the imaging plane. The χ₃₃/χ₃₁‐ratio for collagen type I in tendon and collagen type II in cartilage was estimated to be 1.33 and 1.36, respectively, using this method. These ratios are now much closer than what has been reported previously in the literature, and the larger reported differences between collagen types can be explained by variation in the structural organization.