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In vivo SHG imaging of the collagen networks in a normal mouse ear at (a) zone 1, (b) zone 2, (c) zone 3, (d) zone 4, (e) zone 5, (f) zone 6, (g) zone 7, (h) zone 8, and (i) zone 9. Fields of view: 240 μm × 240 μm. These images were acquired for control analysis.
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Using in vivo second harmonic generation (SHG) and third harmonic generation (THG) microscopies, we tracked the course of collagen remodeling over time in the same melanoma microenvironment within an individual mouse. The corresponding structural and morphological changes were quantitatively analyzed without labeling using an orientation index (OI)...
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... implanting the melanoma cells in the mice ears, we obtained SHG sectioning images of the collagen fibers in vivo at nine different zones as a normal control (Fig. 4). The way we define the zones is described in the supplementary information. In each zone, we arbitrarily chose three locations for tomographic imaging. At each location, ten sectioning images at different depths were ...
Context 2
... 7b). Except for the decay of correlation, we further developed a new index Var GLCM to evaluate the fluctuation of GLCM traces. To calculate this index, over all pixel distances, we summed the square of correlation fluctuation relative to the slowly varying background. Similar to the results of GLCM, it shows a large (6167%) regional variation ( Fig. S4) and HA injection didn't deviate the value away from the range of baseline ones (Fig. ...
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Aims
Patients with tetralogy of Fallot (TOF) are often affected by right ventricular fibrosis, which has been associated with arrhythmias. This study aimed to assess fibrosis distribution in right ventricular outflow tract (RVOT) myocardium of TOF patients to evaluate the utility of single histology-section analyses, and to explore the possibility...
Citations
... 29,[32][33][34] Prior work has used SHG imaging to probe collagen changes primarily in breast 6,[35][36][37][38][39][40][41][42] and pancreatic cancer [43][44][45][46][47][48] with some analyses in melanoma. 22,49,50 We aim to expand this analysis to melanoma in the context of radiotherapy and immunotherapy treatment. ...
... [29][30][31][32]58 Though SHG imaging has enabled insights into collagen trends in breast and pancreatic cancer, few studies have performed SHG of collagen in melanoma. 22,49,50 To our knowledge, this is the first study to quantify in vivo mouse melanoma tumor collagen reorganization during immunotherapy treatment using label-free SHG imaging. We quantified six collagen morphology features during combination radiotherapy and immunotherapy or PBS vehicle using CurveAlign and CT-FIRE software. ...
... 5,20 The decrease in straightness or alignment in our treated mouse tumors was also consistent with in vivo collagen SHG of mouse healthy ears (less aligned) compared to mouse ears bearing B16-F10 melanoma (more aligned). 49 Additionally, shorter and wider collagen fibers restrict tumor cell invasion, via decreased β1 integrins and metalloproteinase associated genes as well as increased E-cadherin on tumor cells, in human and rat breast cancers. 59 The difference in collagen phenotype between our PBS vehicle tumors and treated mouse tumors may be due to the treatment itself or changes occurring during tumor regression. ...
Significance: 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.
Aim: Image in vivo collagen in preclinical melanoma models during immunotherapy and quantify the collagen phenotype in treated and control mice.
Approach: 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.
Results: In immunotherapy-treated mice, collagen 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 (Day 9 and 12) while width and density changed early (Day 6) compared to control mice. Single fiber level collagen analysis was most sensitive to the changes between treatment groups compared to image level analysis.
Conclusions: 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.
... With the present state of RCM devices and technology, this approach is currently restricted to accessible diseases and cancers on the skin and mucosa. Complementary multimodal approaches 60 such as dynamic optical coherence tomography or optical frequency domain imaging for imaging vasculature, lymphatics and tissue viability 61,62 , multiphoton microscopy for better contrast and collagen delineation [63][64][65] , photoacoustic microscopy for functional vascular imaging 66 and fluorescence lifetime imaging for probing immune cell specificity and activation states 67 will further enhance in vivo TiME visualization and enhance current TiME phenotyping in the future. ...
... It has been suggested that alterations in collagen density, fiber orientation as well as its molecular architecture correlates with phenotypic changes in different malignant tumors (59)(60)(61)(62)(63). In fact, second harmonic generation imaging of tumor biopsies has revealed unique collagen organizational signatures between normal tissues and tumors (59)(60)(61)(62)(63). Biophysical alterations in collagen structure can alter signaling that controls cell adhesion, migration and proliferation, and selective targeting of certain cryptic collagen elements can inhibit angiogenesis, tumor growth and metastasis in animal models (3,9,10,31,64). ...
... It has been suggested that alterations in collagen density, fiber orientation as well as its molecular architecture correlates with phenotypic changes in different malignant tumors (59)(60)(61)(62)(63). In fact, second harmonic generation imaging of tumor biopsies has revealed unique collagen organizational signatures between normal tissues and tumors (59)(60)(61)(62)(63). Biophysical alterations in collagen structure can alter signaling that controls cell adhesion, migration and proliferation, and selective targeting of certain cryptic collagen elements can inhibit angiogenesis, tumor growth and metastasis in animal models (3,9,10,31,64). ...
Structural alterations of collagen impact signaling that helps control tumor progression and the responses to therapeutic intervention. Integrins represent a class of receptors that include members that mediate collagen signaling. However, a strategy of directly targeting integrins to control tumor growth has demonstrated limited activity in the clinical setting. New molecular understanding of integrins have revealed that these receptors can regulate both pro‑ and anti‑tumorigenic functions in a cell type‑dependent manner. Therefore, designing strategies that block pro‑tumorigenic signaling, without impeding anti‑tumorigenic functions, may lead to development of more effective therapies. In the present study, evidence was provided for a novel signaling cascade in which β3‑integrin‑mediated binding to a secreted RGDKGE‑containing collagen fragment stimulates an autocrine‑like signaling pathway that differentially governs the activity of both YAP and (protein kinase‑A) PKA, ultimately leading to alterations in the levels of immune checkpoint molecule PD‑L1 by a proteasome dependent mechanism. Selectively targeting this collagen fragment, reduced nuclear YAP levels, and enhanced PKA and proteasome activity, while also exhibiting significant antitumor activity in vivo. The present findings not only provided new mechanistic insight into a previously unknown autocrine‑like signaling pathway that may provide tumor cells with the ability to regulate PD‑L1, but our findings may also help in the development of more effective strategies to control pro‑tumorigenic β3‑integrin signaling without disrupting its tumor suppressive functions in other cellular compartments.
... With the present state of RCM devices and technology, this approach is currently restricted to accessible diseases and cancers on the skin and mucosa. Complementary multimodal approaches 60 such as dynamic optical coherence tomography or optical frequency domain imaging for imaging vasculature, lymphatics and tissue viability 61,62 , multiphoton microscopy for better contrast and collagen delineation [63][64][65] , photoacoustic microscopy for functional vascular imaging 66 and fluorescence lifetime imaging for probing immune cell specificity and activation states 67 will further enhance in vivo TiME visualization and enhance current TiME phenotyping in the future. ...
Response to immunotherapies can be variable and unpredictable. Pathology- based phenotyping of tumors into ‘hot’ and ‘cold’ is static, relying solely on T-cell infiltration in single-time single-site biopsies, resulting in suboptimal treatment response prediction. Dynamic vascular events (tumor angiogenesis, leukocyte trafficking) within tumor immune microenvironment (TiME) also influence anti-tumor immunity and treatment response. Here, we report dynamic cellular-level TiME phenotyping in vivo that combines inflammation profiles with vascular features through non-invasive reflectance confocal microscopic imaging. In skin cancer patients, we demonstrate three main TiME phenotypes that correlate with gene and protein expression, and response to toll-like receptor agonist immune-therapy. Notably, phenotypes with high inflammation associate with immunostimulatory signatures and those with high vasculature with angiogenic and endothelial anergy signatures. More- over, phenotypes with high inflammation and low vasculature demonstrate the best treatment response. This non-invasive in vivo phenotyping approach integrating dynamic vasculature with inflammation serves as a reliable pre- dictor of response to topical immune-therapy in patients.
... With the present state of RCM devices and technology, this approach is currently restricted to accessible diseases and cancers on the skin and mucosa. Complementary multimodal approaches 60 such as dynamic optical coherence tomography or optical frequency domain imaging for imaging vasculature, lymphatics and tissue viability 61,62 , multiphoton microscopy for better contrast and collagen delineation [63][64][65] , photoacoustic microscopy for functional vascular imaging 66 and fluorescence lifetime imaging for probing immune cell specificity and activation states 67 will further enhance in vivo TiME visualization and enhance current TiME phenotyping in the future. ...
... The ICG-R15 retention rate would thus be overestimated. Tingting et al. used photoacoustic tomography to study the feasibility of ICG dosimetry in partial hepatectomy rabbits [25]. The animal model is still not in the context of HCC. ...
... The laser-scanned inverted microscope system (Leica, DMI 3000M) was excited by a home-build 1250 nm femtosecond Cr: forsterite laser. Details about the light source and the imaging system have been reported elsewhere [25]. The mode-locked output power was 570 mW. ...
Using in vivo multiphoton fluorescent dosimetry, we demonstrate that the clearance dynamics of Indocyanine Green (ICG) in the blood can quickly reveal liver function reserve. In normal rats, the ICG retention rate was below 10% at the 15-minute post-administration; While in the rat with severe hepatocellular carcinoma (HCC), the 15-minute retention rate is over 40% due to poor liver metabolism. With a 785 nm CW laser, the fluorescence dosimeter can evaluate the liver function reserve at a 1/10 clinical dosage of ICG without any blood sampling. In the future, this low-dosage ICG 15-minute retention dosimetry can be applied for the preoperative assessment of hepatectomy or timely perioperative examination.
... The five modes comprise opticalresolution optoacoustic microscopy (OAM) for imaging microvasculature, two-photon excitation fluorescence (2PEF) modality for capturing skin morphology based on skin autofluorescence, second and third harmonic generation (SHG and THG) modes for visualizing the extracellular collagen matrix and lipids/ sebaceous glands, respectively, and brightfield (BF) microscopy for providing an anatomical reference. For the herein used athymic nude Hsd Foxn1 mouse model, we expect Co5M's OA signals to be dominated by hemoglobin [53][54][55][56][57] , SHG signals by the extracellular collagen matrix 21,58-62 , THG signals by lipid bilayers (cell membranes, especially keratinocytes of the epidermis) and local lipid accumulations such as in sebaceous glands 21,[58][59][60][61] , and 2PEF signals by elastin, FAD, NADH, and keratin 21,59,62,63 . Co5M's modalities access that by intrinsic biological contrast, which is analyzed to outline the spatial characteristics of the observed biological moieties. ...
The non-invasive investigation of multiple biological processes remains a methodological challenge as it requires capturing different contrast mechanisms, usually not available with any single modality. Intravital microscopy has played a key role in dynamically studying biological morphology and function, but it is generally limited to resolving a small number of contrasts, typically generated by the use of transgenic labels, disturbing the biological system. We introduce concurrent 5-modal microscopy (Co5M), illustrating a new concept for label-free in vivo observations by simultaneously capturing optoacoustic, two-photon excitation fluorescence, second and third harmonic generation, and brightfield contrast. We apply Co5M to non-invasively visualize multiple wound healing biomarkers and quantitatively monitor a number of processes and features, including longitudinal changes in wound shape, microvascular and collagen density, vessel size and fractality, and the plasticity of sebaceous glands. Analysis of these parameters offers unique insights into the interplay of wound closure, vasodilation, angiogenesis, skin contracture, and epithelial reformation in space and time, inaccessible by other methods. Co5M challenges the conventional concept of biological observation by yielding multiple simultaneous parameters of pathophysiological processes in a label-free mode.
... Aside from tumor vasculature and oxygenation, many other components can be evaluated with PAI including collagen, MMPs, and immune cell infiltration. Preclinical studies of fibrosis and lipids has mainly focused on other diseases including Crohn's disease [268,269] and atherosclerosis [270,271] , but show excellent promise for its application in the TME. The monitoring of MMP production or activity in the TME provides a potential predictive biomarker of metastasis and their production has been monitored preclinically with PAI [272][273][274][275] . ...
Standard small molecule and nanoparticulate chemotherapies are used for cancer treatment; however, their effectiveness remains highly variable. One reason for this variable response is hypothesized to be due to nonspecific drug distribution and heterogeneity of the tumor microenvironment, which affect tumor delivery of the agents. Nanoparticle drugs have many theoretical advantages, but due to variability in tumor microenvironment (TME) factors, the overall drug delivery to tumors and associated antitumor response are low. The nanotechnology field would greatly benefit from a thorough analysis of the TME factors that create these physiological barriers to tumor delivery and treatment in preclinical models and in patients. Thus, there is a need to develop methods that can be used to reveal the content of the TME, determine how these TME factors affect drug delivery, and modulate TME factors to increase the tumor delivery and efficacy of nanoparticles. In this review, we will discuss TME factors involved in drug delivery, and how biomedical imaging tools can be used to evaluate tumor barriers and predict drug delivery to tumors and antitumor response.
... -Influence of collagen structure in cancer progression [33,47]. ...
Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy.
... Until now, non-linear optical modalities, such as Multiphoton Excitation Fluorescence (MPEF), Second Harmonic Generation (SHG) and Third Harmonic Generation (THG) have been applied to detect responses to neoadjuvant therapies for cancer [13][14][15] and to define surgical margins in tissues 10,13,16 . Commonly to other non-linear imaging techniques, THG not only enables recording of labelfree images without causing photobleaching and photoxicity effects on the biological samples, but it also allows quantitative analysis [5][6][7]17,18 . Of note, THG signal can be enhanced by the presence of multilayered structures detected in membranes, lipid bodies 19 and inhomogeneities. ...
The ability to distinguish and grade malignant cells during surgical procedures in a fast, non-invasive and staining-free manner is of high importance in tumor management. To this extend, Third Harmonic Generation (THG), Second Harmonic Generation (SHG) and Fourier-Transform Infrared (FTIR) spectroscopy were applied to discriminate malignant from healthy cells in human breast tissue biopsies. Indeed, integration of non-linear processes into a single, unified microscopy platform offered complementary structural information within individual cells at the submicron level. Using a single laser beam, label-free THG imaging techniques provided important morphological information as to the mean nuclear and cytoplasmic area, cell volume and tissue intensity, which upon quantification could not only distinguish cancerous from benign breast tissues but also define disease severity. Simultaneously, collagen fibers that could be detected by SHG imaging showed a well structured continuity in benign tumor tissues, which were gradually disoriented along with disease severity. Combination of THG imaging with FTIR spectroscopy could provide a clearer distinction among the different grades of breast cancer, since FTIR analysis showed increased lipid concentrations in malignant tissues. Thus, the use of non-linear optical microscopy can be considered as powerful and harmless tool for tumor cell diagnostics even during real time surgery procedures.
