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.      

Tensor regularized total variation for denoising of third harmonic generation images of brain tumors

Third harmonic generation (THG) microscopy shows great potential for instant pathology of brain tissue during surgery. However, the rich morphologies contained and the noise associated makes image restoration, necessary for quantification of the THG images, challenging. Anisotropic diffusion filtering (ADF) has been recently applied to restore THG images of normal brain, but ADF is hard‐to‐code, time‐consuming and only reconstructs salient edges. This work overcomes these drawbacks by expressing ADF as a tensor regularized total variation model, which uses the Huber penalty and the L₁ norm for tensor regularization and fidelity measurement, respectively. The diffusion tensor is constructed from the structure tensor of ADF yet the tensor decomposition is performed only in the non‐flat areas. The resulting model is solved by an efficient and easy‐to‐code primal‐dual algorithm. Tests on THG brain tumor images show that the proposed model has comparable denoising performance as ADF while it much better restores weak edges and it is up to 60% more time efficient.      

Slide‐free imaging of hematoxylin‐eosin stained whole‐mount tissues using combined third‐harmonic generation and three‐photon fluorescence microscopy

Intraoperative margin assessment of surgical tissues during cancer surgery is clinically important, especially in the case of tissue conserving surgery like Mohs micrographic surgery in which minimization of the surgical area is considered crucial. Frozen pathology is the gold standard of assessing excised tissues for signs of remaining cancerous lesions. The current protocol, however, is time‐consuming and labor‐intensive. Instead of the complex frozen sectioning, staining, and traditional white light microscopy imaging protocol, optically sectioned histopathological imaging of hematoxylin‐eosin stained whole‐mount skin tissues with a subfemtoliter resolution is demonstrated by using nonlinear microscopy in this study. With our proposed method, the reagents of staining and the contrast of imaging are fully consistent with the current clinical standard of frozen pathology, thus facilitating rapid intraoperative assessment of surgical tissues for future applications. Image: Slide‐free nonlinear microscopy imaging of H&E stained whole‐mount skin tissue showing the morphology of sweat glands.      

Automated pollen recognition using 3D volume images from fluorescence microscopy

Identifying and counting of pollen grains in ambient air samples is still a demanding and time-consuming task even for an experienced microscopist. This article describes a technique which may be employed to establish a fully automated system for this task. Based on a 3D volume fluorescence image of a pollen grain taken with a confocal laser scanning microscope, the described system is able to recognize the pollen taxa. The system autonomously extracts all required information for the recognition from a data base with reference objects (self-learning system) and only needs to calculate very general purpose features of the volumetric data sets (so-called gray scale invariants). This allows for easy adaptation of the system to other conditions (e.g., pollen of a special area) or even other objects than pollen (e.g., spores, bacteria etc.) just by exchanging the reference data base. When using a reference data base with the 26 most important German pollen taxa, the recognition rate is 92%. With a special database for allergic purposes recognizing only Corylus, Alnus, Betula, Poaceae, Secale, Artemisia and ``allergically non-relevant' the recognition rate is 97.4%.     

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

We present a multimodal technique for measuring the integral refractive index and the thickness of biological cells and their organelles by integrating interferometric phase microscopy (IPM) and rapid confocal fluorescence microscopy. First, the actual thickness maps of the cellular compartments are reconstructed using the confocal fluorescent sections, and then the optical path difference (OPD) map of the same cell is reconstructed using IPM. Based on the co‐registered data, the integral refractive index maps of the cell and its organelles are calculated. This technique enables rapidly measuring refractive index of live, dynamic cells, where IPM provides quantitative imaging capabilities and confocal fluorescence microscopy provides molecular specificity of the cell organelles. We acquire human colorectal adenocarcinoma cells and show that the integral refractive index values are similar for the whole cell, the cytoplasm and the nucleus on the population level, but significantly different on the single cell level.     

Rapid identification of human ovarian cancer in second harmonic generation images using radiomics feature analyses and tree‐based pipeline optimization tool

Ovarian cancer is currently one of the most common cancers of the female reproductive organs, and its mortality rate is the highest among all types of gynecologic cancers. Rapid and accurate classification of ovarian cancer plays an important role in the determination of treatment plans and prognoses. Nevertheless, the most commonly used classification method is based on histopathological specimen examination, which is time‐consuming and labor‐intensive. Thus, in this study, we utilize radiomics feature extraction methods and the automated machine learning tree‐based pipeline optimization tool (TOPT) for analysis of 3D, second harmonic generation images of benign, malignant and normal human ovarian tissues, to develop a high‐efficiency computer‐aided diagnostic model. Area under the receiver operating characteristic curve values of 0.98, 0.96 and 0.94 were obtained, respectively, for the classification of the three tissue types. Furthermore, this approach can be readily applied to other related tissues and diseases, and has great potential for improving the efficiency of medical diagnostic processes.     

Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence

The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provides detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.     

Rapid 3D fluorescence imaging of individual optically trapped living immune cells

We demonstrate an approach to rapidly characterize living suspension cells in 4 dimensions while they are immobilized and manipulated within optical traps. A single, high numerical aperture objective lens is used to separate the imaging plane from the trapping plane. This facilitates full control over the position and orientation of multiple trapped cells using a spatial light modulator, including directed motion and object rotation, while also allowing rapid 4D imaging. This system is particularly useful in the handling and investigation of the behavior of non‐adherent immune cells. We demonstrate these capabilities by imaging and manipulating living, fluorescently stained Jurkat T cells. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

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)     

Quantitative second harmonic generation imaging of cartilage damage

Cartilage damage was studied using non-invasive multiphoton-excited autofluorescence and quantitative second harmonic generation (SHG) microscopy. Two cryopreservation techniques based upon freezing and vitrification methods, respectively, were employed to determine whether or not the collagen fiber structure of full thickness porcine articular cartilage was affected by cryopreservation and whether the level of collagen damage could be determined quantitatively in non-processed (non-fixed, non-sliced, non-stained) tissues. Multiphoton-induced autofluorescence imaging revealed the presence of chondrocytes, as well as collagenous structures in all fresh, vitrified and frozen cryopreserved cartilage samples. SHG imaging of the frozen cryopreserved specimens showed a dramatic loss of mean gray value intensities when compared to both fresh and vitrified tissues (P < 0.05), indicating structural changes of the extracellular matrix, in particular the deformation and destruction of the collagen fibers in the analyzed articular cartilage. A 0.9974 correlation coefficient was observed between the metabolic cell activity assessed by the alamarBlue technique, and retention of collagen structure between the three experimental groups. These studies suggest that multiphoton-induced autofluorescence imaging combined with quantitative SHG signal profiling may prove to be useful tools for the investigation of extracellular matrix changes in preserved cartilage, giving insights on the structural quality prior to implantation.     

Structural alterations in cell organelles induced by photodynamic treatment with chlorin p6-histamine conjugate in human oral carcinoma cells probed by 3D fluorescence microscopy

We have explored the intracellular cell organelle's structural alterations after photodynamic treatment with chlorin p₆-histamine conjugate (Cp₆-his) in human oral cancer cells. Herein, the cells were treated with Cp₆-his (10 μm) and counterstained with organelle-specific fluorescence probes to find the site of intracellular localization using confocal microscopy. For photodynamic therapy (PDT), the cells were exposed to ~30 kJ/m² red light (660 ± 20 nm) to induce ~90% cytotoxicity. We used the three-dimensional (3D) image reconstruction approach to analyze the photodynamic damage to cell organelles. The result showed that Cp₆-his localized mainly in the endoplasmic reticulum (ER) and lysosomes but not in mitochondria and Golgi apparatus (GA). The 3D model revealed that in necrotic cells, PDT led to extensive fragmentation of ER and fragmentation and swelling of GA as well. Results suggest that the indirect damage to GA occurred due to loss of connection between ER and GA. Moreover, in damaged cells with no sign of necrosis, the perinuclear ER appeared condensed and surrounded by several small clumps at the peripheral region of the cell, and the GA was observed to form a single condensed structure. Since these structural changes were associated with apoptotic cell death, it is suggested that the necrotic and apoptotic death induced by PDT with Cp₆-his is determined by the severity of damage to ER and indirect damage to GA. The results suggest that the indirect damage to cell organelle apart from the sites of photosensitizer localization and the severity of damage at the organelle level contribute significantly to the mode of cell death in PDT.     

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 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.     

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.     

Quantitative assessment of microenvironment characteristics and metabolic activity in glioma via multiphoton microscopy

Tumor microenvironment and metabolic activity in gliomas are the important biomarkers to evaluate the progression of gliomas. Many evidences have suggested that the targeting of metabolic activity and tumor microenvironment simultaneously can be more effective to take the tumor therapy. Therefore, the noninvasive, accurate assessment of tumor microenvironment and metabolic activity is quite important in clinical practice. Multiphoton microscopy (MPM), based on two‐photon‐excited fluorescence and second harmonic generation was performed on unstained glioma tissues. With our combined image analysis approaches, our research findings indicate that MPM is able to qualitatively and quantitatively describe the microenvironment characteristics in gliomas, such as collage deposition in extracellular matrix, lymphocyte infiltration and tumor angiogenesis, etc. Meanwhile, the metabolic activity can also be quantitatively evaluated by optical redox ratio, NADH and FAD intensity. With the microendoscope and fiberscope are portable, MPM technique can be used to perform in‐vivo studies and clinical examinations in gliomas.      

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.     

Quantitative IR microscopy and spectromics open the way to 3D digital pathology

Currently, only mass‐spectrometry (MS) microscopy brings a quantitative analysis of chemical contents of tissue samples in 3D. Here, the reconstruction of a 3D quantitative chemical images of a biological tissue by FTIR spectro‐microscopy is reported. An automated curve‐fitting method is developed to extract all intense absorption bands constituting IR spectra. This innovation benefits from three critical features: (1) the correction of raw IR spectra to make them quantitatively comparable; (2) the automated and iterative data treatment allowing to transfer the IR‐absorption spectrum into a IR‐band spectrum; (3) the reconstruction of an 3D IR‐band matrix (x, y, z for voxel position and a 4^th dimension with all IR‐band parameters). Spectromics, which is a new method for exploiting spectral data for tissue metadata reconstruction, is proposed to further translate the related chemical information in 3D, as biochemical and anatomical tissue parameters. An example is given with oxidative stress distribution and the reconstruction of blood vessels in tissues. The requirements of IR microscopy instrumentation to propose 3D digital histology as a clinical routine technology is briefly discussed.

     

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.      

Label‐free identification and characterization of human pluripotent stem cell‐derived cardiomyocytes using second harmonic generation (SHG) microscopy

Pluripotent stem cell‐derived cardiomyocytes (PSC‐CMs) are a potentially unlimited source of cardiomyocytes (CMs) for cardiac transplantation therapies. The establishment of pure PSC‐CM populations is important for this application, but is hampered by a lack of CM‐specific surface markers suitable for their identification and sorting. Contemporary purification techniques are either non‐specific or require genetic modification. We report a second harmonic generation (SHG) signal detectable in PSC‐CMs that is attributable to sarcomeric myosin, dependent on PSC‐CM maturity, and retained while PSC‐CMs are in suspension. Our study demonstrates the feasibility of developing a SHG‐activated flow cytometer for the non‐invasive purification of PSC‐CMs. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)