Quantitative Second Harmonic Generation Research Articles

Quantifying in vivo collagen reorganization during immunotherapy in murine melanoma with second harmonic generation imaging.

Increased collagen linearization and deposition during tumorigenesis can impede immune cell infiltration and lead to tumor metastasis. Although melanoma is well studied in immunotherapy research, studies that quantify collagen changes during melanoma progression and treatment are lacking. We aim to image in vivo collagen in preclinical melanoma models during immunotherapy and quantify the collagen phenotype in treated and control mice. Second-harmonic generation imaging of collagen was performed in mouse melanoma tumors in vivo over a treatment time course. Animals were treated with a curative radiation and immunotherapy combination. Collagen morphology was quantified over time at an image and single-fiber level using CurveAlign and CT-FIRE software. In immunotherapy-treated mice, collagen was reorganized toward a healthy phenotype, including shorter, wider, curlier collagen fibers, with modestly higher collagen density. Temporally, collagen fiber straightness and length changed late in treatment (days 9 and 12), while width and density changed early (day 6) compared with control mice. Single-fiber collagen features calculated in CT-FIRE were the most sensitive to the changes among treatment groups compared with bulk collagen features. Quantitative second-harmonic generation imaging can provide insight into collagen dynamics in vivo during immunotherapy, with key implications in improving immunotherapy response in melanoma and other cancers.

Fluid mechanics approach to analyzing collagen fiber organization.

.Significance: The spatial organization of collagen fibers has been used as a biomarker for assessing injury and disease progression. However, quantifying this organization for complex structures is challenging.Aim: To quantify and classify complex collagen fiber organizations.Approach: Using quantitative second-harmonic generation (SHG) microscopy, we show that collagen-fiber orientation can be viewed as pseudovector fields. Subsequently, we analyze them using fluid mechanic metrics, such as energy , enstrophy , and tortuosity .Results: We show that metrics used in fluid mechanics for analyzing fluid flow can be adapted to analyze complex collagen fiber organization. As examples, we consider SHG images of collagenous tissue for straight, wavy, and circular fiber structures.Conclusions: The results of this study show the utility of the chosen metrics to distinguish diverse and complex collagen organizations. We find that the distribution of values for and increases with collagen fiber disorganization, where they divide between low and high values corresponding to uniformly aligned fibers and disorganized collagen fibers, respectively. We also confirm that the values of cluster around 1 when the fibers are straight, and the range increases up to 1.5 when wavier fibers are present.

Quantitative Polarization-Resolved Second-Harmonic-Generation Microscopy of Glycine Microneedles.

Second-harmonic generation (SHG) is a nonlinear optical process that can provide disease diagnosis through characterization of biological building blocks such as amino acids, peptides, and proteins. The second-order nonlinear susceptibility tensor χ(2) of a material characterizes its tendency to cause SHG. Here, a method for finding the χ(2) elements from polarization-resolved SHG microscopy in transmission mode is presented. The quantitative framework and analytical approach that corrects for micrometer-scale morphology and birefringence enable the determination and comparison of the SHG susceptibility tensors of β- and γ-phase glycine microneedles. The maximum nonlinear susceptibility coefficients are d33 = 15 pm V-1 for the β and d33 = 5.9 pm V-1 for the γ phase. The results demonstrate glycine as a useful biocompatible nonlinear material. This combination of the analytical model and polarization-resolved SHG transmission microscopy is broadly applicable for quantitative SHG material characterization and diagnostic imaging.

Quantitative analysis of the effect of environmental-scanning electron microscopy on collagenous tissues

Environmental-scanning electron microscopy (ESEM) is routinely applied to various biological samples due to its ability to maintain a wet environment while imaging; moreover, the technique obviates the need for sample coating. However, there is limited research carried out on electron-beam (e-beam) induced tissue damage resulting from using the ESEM. In this paper, we use quantitative second-harmonic generation (SHG) microscopy to examine the effects of e-beam exposure from the ESEM on collagenous tissue samples prepared as either fixed, frozen, wet or dehydrated. Quantitative SHG analysis of tissues, before and after ESEM e-beam exposure in low-vacuum mode, reveals evidence of cross-linking of collagen fibers, however there are no structural differences observed in fixed tissue. Meanwhile wet-mode ESEM appears to radically alter the structure from a regular fibrous arrangement to a more random fiber orientation. We also confirm that ESEM images of collagenous tissues show higher spatial resolution compared to SHG microscopy, but the relative tradeoff with collagen specificity reduces its effectiveness in quantifying collagen fiber organization. Our work provides insight on both the limitations of the ESEM for tissue imaging, and the potential opportunity to use as a complementary technique when imaging fine features in the non-collagenous regions of tissue samples.

Second Harmonic Generation Guided Raman Spectroscopy for Sensitive Detection of Polymorph Transitions.

Second harmonic generation (SHG) was integrated with Raman spectroscopy for the analysis of pharmaceutical materials. Particulate formulations of clopidogrel bisulfate were prepared in two crystal forms (Form I and Form II). Image analysis approaches enable automated identification of particles by bright field imaging, followed by classification by SHG. Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with 99.95% confidence in a total measurement time of ∼10 ms per particle. Complementary measurements by Raman and synchrotron XRD are in excellent agreement with the classifications made by SHG, with measurement times of ∼1 min and several seconds per particle, respectively. Coupling these capabilities with at-line monitoring may enable real-time feedback for reaction monitoring during pharmaceutical production to favor the more bioavailable but metastable Form I with limits of detection in the ppm regime.

Application of quantitative second-harmonic generation microscopy to posterior cruciate ligament for crimp analysis studies.

We use second-harmonic generation (SHG) microscopy to quantitatively characterize collagen fiber crimping in the posterior cruciate ligament (PCL). The obtained SHG images are utilized to define three distinct categories of crimp organization in the PCL. Using our previously published spatial-frequency analysis, we develop a simple algorithm to quantitatively distinguish the various crimp patterns. In addition, SHG microscopy reveals both the three-dimensional structural variation in some PCL crimp patterns as well as an underlying helicity in these patterns that have mainly been observed using electron microscopy. Our work highlights how SHG microscopy could potentially be used to link the fibrous structural information in the PCL to its mechanical properties.

Quantitative SHG imaging in osteoarthritis model mice, implying a diagnostic application.

Osteoarthritis (OA) restricts the daily activities of patients and significantly decreases their quality of life. The development of non-invasive quantitative methods for properly diagnosing and evaluating the process of degeneration of articular cartilage due to OA is essential. Second harmonic generation (SHG) imaging enables the observation of collagen fibrils in live tissues or organs without staining. In the present study, we employed SHG imaging of the articular cartilage in OA model mice ex vivo. Consequently, three-dimensional SHG imaging with successive image processing and statistical analyses allowed us to successfully characterize histopathological changes in the articular cartilage consistently confirmed on histological analyses. The quantitative SHG imaging technique presented in this study constitutes a diagnostic application of this technology in the setting of OA.

Graphics processing unit-based quantitative second-harmonic generation imaging.

We adapt a graphics processing unit (GPU) to dynamic quantitative second-harmonic generation imaging. We demonstrate the temporal advantage of the GPU-based approach by computing the number of frames analyzed per second from SHG image videos showing varying fiber orientations. In comparison to our previously reported CPU-based approach, our GPU-based image analysis results in ∼10× improvement in computational time. This work can be adapted to other quantitative, nonlinear imaging techniques and provides a significant step toward obtaining quantitative information from fast in vivo biological processes.

Application of quantitative second-harmonic generation microscopy to dynamic conditions

We present a quantitative second-harmonic generation (SHG) imaging technique that quantifies the 2D spatial organization of collagen fiber samples under dynamic conditions, as an image is acquired. The technique is demonstrated for both a well-aligned tendon sample and a randomly aligned, sparsely distributed collagen scaffold sample. For a fixed signal-to-noise ratio, we confirm the applicability of this method for various window sizes (pixel areas) as well as with using a gridded overlay map that allows for correlations of fiber orientations within a given image. This work has direct impact to in vivo biological studies by incorporating simultaneous SHG image acquisition and analysis.

Quantifying collagen structure in breast biopsies using second-harmonic generation imaging

Quantitative second-harmonic generation imaging is employed to assess stromal collagen in normal, hyperplastic, dysplastic, and malignant breast tissues. The cellular scale organization is quantified using Fourier transform-second harmonic generation imaging (FT-SHG), while the molecular scale organization is quantified using polarization-resolved second-harmonic generation measurements (P-SHG). In the case of FT-SHG, we apply a parameter that quantifies the regularity in collagen fiber orientation and find that malignant tissue contains locally aligned fibers compared to other tissue conditions. Alternatively, using P-SHG we calculate the ratio of tensor elements (d15/d31, d22/d31, and d33/d31) of the second-order susceptibility χ2 for collagen fibers in breast biopsies. In particular, d15/d31 shows potential differences across the tissue pathology. We also find that trigonal symmetry (3m) is a more appropriate model to describe collagen fibers in malignant tissues as opposed to the conventionally used hexagonal symmetry (C6). This novel method of targeting collagen fibers using a combination of two quantitative SHG techniques, FT-SHG and P-SHG, holds promise for breast tissue analysis and applications to characterizing cancer in a manner that is compatible with clinical practice.

Quantitative second-harmonic generation microscopy for imaging porcine cortical bone: Comparison to SEM and its potential to investigate age-related changes

We propose the use of second-harmonic generation (SHG) microscopy for imaging collagen fibers in porcine femoral cortical bone. The technique is compared with scanning electron microscopy (SEM). SHG microscopy is shown to have excellent potential for bone imaging primarily due its intrinsic specificity to collagen fibers, which results in high contrast images without the need for specimen staining. Furthermore, this technique's ability to quantitatively assess collagen fiber organization is evaluated through an exploratory examination of bone structure as a function of age, from very young to mature bone. In particular, four different age groups: 1month, 3.5months, 6months, and 30months, were studied. Specifically, we employ the recently developed Fourier transform-second harmonic generation (FT-SHG) imaging technique for the quantification of the structural changes, and observe that as the bone develops, there is an overall reduction in porosity, the number of osteons increases, and the collagen fibers become comparatively more organized. It is also observed that the variations in structure across the whole cross-section of the bone increase with age. The results of this work show that quantitative SHG microscopy can serve as a valuable tool for evaluating the structural organization of collagen fibers in ex vivo bone studies.

Ex vivo imaging and quantification of liver fibrosis using second-harmonic generation microscopy

Conventionally, liver fibrosis is diagnosed using histopathological techniques. The traditional method is time-consuming in that the specimen preparation procedure requires sample fixation, slicing, and labeling. Our goal is to apply multiphoton microscopy to efficiently image and quantitatively analyze liver fibrosis specimens bypassing steps required in histological preparation. In this work, the combined imaging modality of multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) was used for the qualitative imaging of liver fibrosis of different METAVIR grades under label-free, ex vivo conditions. We found that while MAF is effective in identifying cellular architecture in the liver specimens, it is the spectrally distinct SHG signal that allows the characterization of the extent of fibrosis. We found that qualitative SHG imaging can be used for the effective identification of the associated features of liver fibrosis specimens graded METAVIR 0 to 4. In addition, we attempted to associate quantitative SHG signal to the different METAVIR grades and found that an objective determination of the extent of disease progression can be made. Our approach demonstrates the potential of using multiphoton imaging in rapid classification of ex vivo liver fibrosis in the clinical setting and investigation of liver fibrosis-associated physiopathology in animal models in vivo.

MULTIPHOTON MICROSCOPY: A NEW APPROACH, IN PHYSIOLOGICAL STUDIES AND PATHOLOGICAL DIAGNOSIS FOR OPHTHALMOLOGY

Multiphoton microscopy (MPM), with the advantages of improved penetration depth, decreased photo-damage, and optical sectioning capability, has become an indispensable tool for biomedical imaging. The combination of multiphoton fluorescence (MF) and second-harmonic generation (SHG) microscopy is particularly effective in imaging tissue structures of the ocular surface. This work is intended to be a review of advances that MPM has made in ophthalmic imaging. The MPM not only can be used for the label-free imaging of ocular structures, it can also be applied for investigating the morphological alterations in corneal pathologies, such as keratoconus, infected keratitis, and corneal scar. Furthermore, the corneal wound healing process after refractive surgical procedures such as conductive keratoplasty (CK) can also be studied with MPM. Finally, qualitative and quantitative SHG microscopy is effective for characterizing corneal thermal denaturation. With additional development, multiphoton imaging has the potential to be developed into an effective imaging technique for in vivo studies and clinical diagnosis in ophthalmology.

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.

Measurement of muscle disease by quantitative second-harmonic generation imaging

Determining the health of muscle cells by in vivo imaging could impact the diagnosis and monitoring of a large number of congenital and acquired muscular or cardiac disorders. However, currently used technologies are hampered by insufficient resolution, lack of specificity, or invasiveness. We have combined intrinsic optical second-harmonic generation from sarcomeric myosin with a novel mathematical treatment of striation pattern analysis, to obtain measures of muscle contractile integrity that correlate strongly with the neuromuscular health of mice suffering from genetic, acquired, and age-related decline in skeletal muscle function. Analysis of biopsies from a pilot group of human volunteers suggests a similar power in quantifying sarcopenic changes in muscle integrity. These results provide the first strong evidence that quantitative image analysis of sarcomere pattern can be correlated with physiological function, and they invite the application of SHG imaging in clinical practice, either in biopsy samples or via microendoscopy.

Quantitative second-harmonic generation microscopy in collagen.

The second-harmonic signal in collagen, even in highly organized samples such as rat tail tendon fascicles, varies significantly with position. Previous studies suggest that this variability may be due to the parallel and antiparallel orientation of neighboring collagen fibrils. We applied high-resolution second-harmonic generation microscopy to confirm this hypothesis. Studies in which the focal spot diameter was varied from approximately 1 to approximately 6 microm strongly suggest that regions in which collagen fibrils have the same orientation in rat tail tendon are likely to be less than approximately 1 microm in diameter. These measurements required accurate determination of the focal spot size achieved by use of different microscope objectives; we developed a technique that uses second-harmonic generation in a quartz reference to measure the focal spot diameter directly. We also used the quartz reference to determine a lower limit (dXXX > 0.4 pm/V) for the magnitude of the second-order nonlinear susceptibility in collagen.