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Surgeries achieving maximal tumor resection remain the major effective treatment of pancreatic cancer. Rapid and precise intraoperative diagnosis of pancreatic tissues is critical for optimum surgical outcomes but is challenging for the current staining-based histological methods. We demonstrated that label-free coherent nonlinear optical microscopy with combined stimulated Raman scattering (SRS) and second harmonic generation (SHG) could reveal key diagnostic features of both normal and cancerous human pancreatic tissues. Adjacent pairs of tissue sections from resection margins of 37 patients were imaged by SRS and hematoxylin and eosin staining for direct comparison, demonstrating high diagnostic concordance (Cohen's kappa, κ > 0.97) between them. Fresh unprocessed tissues showed well-preserved histoarchitectures including pancreatic ducts, islets, acini, and nerves. Moreover, the area ratios of collagen fibers were analyzed and found to correlate with the drainage pancreatic amylase level (odds ratio = 28.0, p = 0.0017). Our results indicated that SRS/SHG histology provides potential for rapid intraoperative diagnosis of pancreatic cancer as well as a predictive value of postoperative pancreatic fistula.

Precise evaluation of breast tumor malignancy based on tissue calcifications has important practical value in the disease diagnosis, as well as the understanding of tumor development. Traditional X-ray mammography provides the overall morphologies of the calcifications but lacks intrinsic chemical information. In contrast, spontaneous Raman spectroscopy offers detailed chemical analysis but lacks the spatial profiles. Here, we applied hyperspectral stimulated Raman scattering (SRS) microscopy to extract both the chemical and morphological features of the microcalcifications, based on the spectral and spatial domain analysis. A total of 211 calcification sites from 23 patients were imaged with SRS, and the results were analyzed with a support vector machine (SVM) based classification algorithm. With optimized combinations of chemical and geometrical features of microcalcifications, we were able to reach a precision of 98.21% and recall of 100.00% for classifying benign and malignant cases, significantly improved from the pure spectroscopy or imaging based methods. Our findings may provide a rapid means to accurately evaluate breast tumor malignancy based on fresh tissue biopsies.

Maximal resection of tumor while preserving the adjacent healthy tissue is particularly important for larynx surgery, hence precise and rapid intraoperative histology of laryngeal tissue is crucial for providing optimal surgical outcomes. We hypothesized that deep-learning based stimulated Raman scattering (SRS) microscopy could provide automated and accurate diagnosis of laryngeal squamous cell carcinoma on fresh, unprocessed surgical specimens without fixation, sectioning or staining. Methods: We first compared 80 pairs of adjacent frozen sections imaged with SRS and standard hematoxylin and eosin histology to evaluate their concordance. We then applied SRS imaging on fresh surgical tissues from 45 patients to reveal key diagnostic features, based on which we have constructed a deep learning based model to generate automated histologic results. 18,750 SRS fields of views were used to train and cross-validate our 34-layered residual convolutional neural network, which was used to classify 33 untrained fresh larynx surgical samples into normal and neoplasia. Furthermore, we simulated intraoperative evaluation of resection margins on totally removed larynxes. Results: We demonstrated near-perfect diagnostic concordance (Cohen's kappa, κ > 0.90) between SRS and standard histology as evaluated by three pathologists. And deep-learning based SRS correctly classified 33 independent surgical specimens with 100% accuracy. We also demonstrated that our method could identify tissue neoplasia at the simulated resection margins that appear grossly normal with naked eyes. Conclusion: Our results indicated that SRS histology integrated with deep learning algorithm provides potential for delivering rapid intraoperative diagnosis that could aid the surgical management of laryngeal cancer.

Wenshuo Wang,1,* Huan Liu,1,* Yuntao Lu,1,* Xiaole Wang,2,* Bohan Zhang,3 Shuo Cong,1 Yun Zhao,1 Minbiao Ji,3 Hongyue Tao,4 Lai Wei1,5 1Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai 200030, China; 2Department of Radiology, Second People’s Hospital of Nantong City, Nantong 226002, Jiangsu, China; 3State Key Laboratory of Surface Physics and Department of Physics, Fudan University, Shanghai 200433, China; 4Department of Radiology, Zhongshan Hospital, Fudan University, Shanghai 200040, China; 5Department of Cardiac Surgery, Shanghai Public Health Clincal Center, Shanghai 201508, China *These authors contributed equally to this work Background: Hydrogen sulfide (H2S) has shown promising therapeutic benefits in reversing a variety of pathophysiological processes in cardiovascular system, including myocardial ischemia–reperfusion (IR) injury. However, the achievement of controlled and sustained release of H2S has been a technical bottleneck that limits the clinical application of the gas molecule.Methods: The current study describes the development of mesoporous iron oxide nanoparticles (MIONs) which were loaded with diallyl trisulfide (DATS), a H2S donor compound, and calibrated by stimulated Raman scattering/transient absorption.Results: The synthesized MIONs were characterized with excellent mesoporosity and a narrow size distribution, which enabled them to slow down the release of H2S to a suitable rate and prolong the plateau period. The controlled-release feature of DATS-MIONs resulted in little adverse effect both invitro and invivo, and their protective effect on the heart tissue that underwent IR injury was observed in the mouse model of myocardial ischemia. The rapid biodegradation of DATS-MIONs was induced by Kupffer cells, which were specialized macrophages located in the liver and caused limited hepatic metabolic burden.Conclusion: The sustained-release pattern and excellent biocompatibility make DATS-MIONs a promising H2S donor for research and medical purposes. Keywords: steady release, porous structure, biocompatibility, biodegeneration

Gout is a common metabolic disease with growing burden, caused by monosodium urate (MSU) microcrystal deposition. In situ and chemical-specific histological identification of MSU is crucial in the diagnosis and management of gout, yet it remains inaccessible for current histological methods. Methods: Stimulated Raman scattering (SRS) microscopy was utilized to image MSU based on its fingerprint Raman spectra. We first tested SRS for the diagnosis capability of gout and the differentiation power from pseudogout with rat models of acute gout arthritis, calcium pyrophosphate deposition disease (CPDD) and comorbidity. Then, human synovial fluid and surgical specimens (n=120) were were imaged with SRS to obtain the histopathology of MSU and collagen fibers. Finally, quantitative SRS analysis was performed in gout tissue of different physiological phases (n=120) to correlate with traditional histopathology including H&E and immunohistochemistry staining. Results: We demonstrated that SRS is capable of early diagnosis of gout, rapid detection of MSU in synovial fluid and fresh unprocessed surgical tissues, and accurate differentiation of gout from pseudogout in various pathophysiological conditions. Furthermore, quantitative SRS analysis revealed the optical characteristics of MSU deposition at different pathophysiological stages, which were found to matched well with corresponding immunofluorescence histochemistry features. Conclusion: Our work demonstrated the potential of SRS microscopy for rapid intraoperative diagnosis of gout and may facilitate future fundamental researches of MSU-based diseases.

Hexagonal boron nitride (h-BN) is an important member of two-dimensional (2D) materials with a large direct bandgap, and has attracted growing interest in ultraviolet optoelectronics and nanoelectronics. Compared with graphene and graphite, h-BN has weak Raman effect because of the far off-resonance excitation; hence, it is difficult to exploit Raman spectroscopy to characterize important properties of 2D h-BN, such as thickness, doping, and strain effects. Here, we applied stimulated Raman scattering (SRS) to enhance the sensitivity of the E2g Raman mode of h-BN. We showed that SRS microscopy achieves rapid high resolution imaging of h-BN with a pixel dwell time 4 orders of magnitude smaller than conventional spontaneous Raman microscopy. Moreover, the near-perfect linear dependence of signal intensity on h-BN thickness and isotropic polarization dependence allow convenient determination of the flake thickness with SRS imaging. Our results indicated that SRS microscopy provides a promising tool for high-speed quantification of h-BN and holds the potential for vibrational imaging of 2D materials.

The degradation of hemoglobin in brain tissues results in the deposition of hemosiderin, which is a major form of iron-storage protein and closely related to neurological disorders such as epilepsy. Optical detection of hemosiderin is vitally important yet challenging for the understanding of disease mechanisms, as well as improving surgical resection of brain lesions. Here, we provide the first label-free microscopy study of sensitive hemosiderin detection in both an animal model and human brain tissues. Methods: We applied spectrally and temporally resolved femtosecond pump-probe microscopy, including transient absorption (TA) and stimulated Raman scattering (SRS) techniques, to differentiate hemoglobin and hemosiderin in brain tissues. The label-free imaging results were compared with Perls' staining to evaluate our method for hemosiderin detection. Results: Significant differences between hemoglobin and hemosiderin transient spectra were discovered. While a strong ground-state bleaching feature of hemoglobin appears in the near-infrared region, hemosiderin demonstrates pure excited-state absorption dynamics, which could be explained by our proposed kinetic model. Furthermore, simultaneous imaging of hemoglobin and hemosiderin can be rapidly achieved in both an intracerebral hemorrhage (ICH) rat model and human brain surgical specimens, with perfect correlation with Perls' staining. Conclusion: Our results suggest that rapid, label-free detection of hemosiderin in brain tissues could be realized by femtosecond pump-probe microscopy. Our method holds great potential in providing a new tool for intraoperative detection of hemosiderin during brain surgeries.

The study of amyotrophic lateral sclerosis (ALS) and potential interventions would be facilitated if motor axon degeneration could be more readily visualized. Here we demonstrate that stimulated Raman scattering (SRS) microscopy could be used to sensitively monitor peripheral nerve degeneration in ALS mouse models and ALS autopsy materials. Three-dimensional imaging of pre-symptomatic SOD1 mouse models and data processing by a correlation-based algorithm revealed that significant degeneration of peripheral nerves could be detected coincidentally with the earliest detectable signs of muscle denervation and preceded physiologically measurable motor function decline. We also found that peripheral degeneration was an early event in FUS as well as C9ORF72 repeat expansion models of ALS, and that serial imaging allowed long-term observation of disease progression and drug effects in living animals. Our study demonstrates that SRS imaging is a sensitive and quantitative means of measuring disease progression, greatly facilitating future studies of disease mechanisms and candidate therapeutics., Sensitive and label-free imaging methods to visualize nerve degeneration are currently lacking. Here authors show that stimulated Raman scattering (SRS) microscopy can be used to monitor peripheral nerve degeneration in mouse models of amyotrophic lateral sclerosis (ALS) and in postmortem tissue from ALS patients.

Stimulated Raman scattering (SRS) microscopy that is capable of both high speed imaging and rapid spectroscopy will be advantageous for detailed chemical analysis of heterogeneous biological specimens. We have developed a system based on spectral focusing SRS technology, with the integration of a rapid scanning optical delay line (RSODL), which allows continuous tuning of SRS spectra by scanning a galvo mirror. We demonstrated SRS spectral measurements of dimethyl sulfoxide solution at low concentrations, and multi-color imaging of rice pollens and HeLa cells with line-by-line delay tuning to reduce motion artifacts, as well as fast acquisition of SRS spectra at specific regions of interest.

There is limited understanding of the effects of major oncogenic pathways and their combinatorial actions on lipid composition and transformation during hepatic tumorigenesis. Here, we report a negative correlation of Wnt/Myc activity with steatosis in human hepatocellular carcinoma (HCC) and perform in vivo functional studies using three conditional transgenic zebrafish models. Double-transgenic zebrafish larvae conditionally expressing human CTNNB1mt and zebrafish tcf7l2 or murine Myc together with krasv12 in hepatocytes led to severe hepatomegaly and significantly attenuated accumulation of lipid droplets and cell senescence triggered by krasv12 expression alone. UPLC-MS–based, nontargeted lipidomic profiling and transcriptome analyses revealed that Wnt/Myc activity promotes triacylglycerol to phospholipid transformation and increases unsaturated fatty acyl groups in phospholipids in a Ras-dependent manner. Small-scale screenings suggested that supplementation of certain free fatty acids (FA) or inhibition of FA desaturation significantly represses hepatic hyperplasia of double-transgenic larvae and proliferation of three human HCC cells with and without sorafenib. Together, our studies reveal novel Ras-dependent functions of Wnt signaling in remodeling the lipid metabolism of cancerous hepatocytes in zebrafish and identify the SCD inhibitor MK8245 as a candidate drug for therapeutic intervention. Significance: These findings identify FA desaturation as a significant downstream therapeutic target for antagonizing the combinatorial effects of Wnt and Ras signaling pathways in hepatocellular carcinoma. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/19/5548/F1.large.jpg. Cancer Res; 78(19); 5548–60. ©2018 AACR.

Conventional far-field microscopy cannot directly resolve the sub-diffraction spatial distribution of localized surface plasmons in metal nanostructures. Using BaTiO3 microspheres as far-field superlenses by collecting the near-field signal, we can map the origin of enhanced two-photon photoluminescence signal from the gap region of gold nanosphere dimers and gold nanorod dimers beyond the diffraction limit, on a conventional far-field microscope. As the angle θ between dimer’s structural axis and laser polarisation changes, photoluminescence intensity varies with a cos4θ function, which agrees quantitatively with numerical simulations. An optical resolution of about λ/7 (λ: two-photon luminescence central wavelength) is demonstrated at dimer’s gap region.

© 2017 American Society for Microbiology. L-2-Hydroxyglutarate aciduria (L-2-HGA) is an autosomal recessive neurometabolic disorder caused by a mutation in the L-2-hydroxyglutarate dehydrogenase (L2HGDH) gene. In this study, we generated L2hgdh knockout (KO) mice and observed a robust increase of L-2-hydroxyglutarate (L-2-HG) levels in multiple tissues. The highest levels of L-2-HG were observed in the brain and testis, with a corresponding increase in histone methylation in these tissues. L2hgdh KO mice exhibit white matter abnormalities, extensive gliosis, microglia-mediated neuroinflammation, and an expansion of oligodendrocyte progenitor cells (OPCs). Moreover, L2hgdh deficiency leads to impaired adult hippocampal neurogenesis and late-onset neurodegeneration in mouse brains. Our data provide in vivo evidence that L2hgdh mutation leads to L-2-HG accumulation, leukoencephalopathy, and neurodegeneration in mice, thereby offering new insights into the pathophysiology of L-2-HGA in humans.

The interaction of charged species in aqueous solution has important implications for chemical, biological, and environmental processes. We have used 2DIR spectroscopy to study the equilibrium dynamics of thiocyanate chemical exchange between free ion (NCS(-)) and contact ion pair configurations (MNCS(+)), where M(2+) = Mg(2+) or Ca(2+). Detailed studies of the influence of anion concentration and anion speciation show that the chemical exchange observed with the 2DIR measurements results from NCS(-) exchanging with other anion species in the first solvation shell surrounding Mg(2+) or Ca(2+). The presence of chemical exchange in the 2DIR spectra provides an indirect, but robust, determinant of contact ion pair formation. We observe preferential contact ion pair formation between soft Lewis base anions and hard Lewis acid cations. This observation cannot be easily reconciled with Pearson's acid-base concept or Collins' Law of Matching Water Affinities. The anions that form contact ion pairs also correspond to the ions with an affinity for water and protein surfaces, so similar physical and chemical properties may control these distinct phenomena.

Conventional histopathology with hematoxylin & eosin (H&E) has been the gold standard for histopathological diagnosis of a wide range of diseases. However, it is not performed in vivo and requires thin tissue sections obtained after tissue biopsy, which carries risk, particularly in the central nervous system. Here we describe the development of an alternative, multicolored way to visualize tissue in real-time through the use of coherent Raman imaging (CRI), without the use of dyes. CRI relies on intrinsic chemical contrast based on vibrational properties of molecules and intrinsic optical sectioning by nonlinear excitation. We demonstrate that multicolor images originating from CH(2) and CH(3) vibrations of lipids and protein, as well as two-photon absorption of hemoglobin, can be obtained with subcellular resolution from fresh tissue. These stain-free histopathological images show resolutions similar to those obtained by conventional techniques, but do not require tissue fixation, sectioning or staining of the tissue analyzed.

Differentiating tumor from normal brain is a major barrier to achieving optimal outcome in brain tumor surgery. New imaging techniques for visualizing tumor margins during surgery are needed to improve surgical results. We recently demonstrated the ability of stimulated Raman scattering (SRS) microscopy, a nondestructive, label-free optical method, to reveal glioma infiltration in animal models. We show that SRS reveals human brain tumor infiltration in fresh, unprocessed surgical specimens from 22 neurosurgical patients. SRS detects tumor infiltration in near-perfect agreement with standard hematoxylin and eosin light microscopy (κ = 0.86). The unique chemical contrast specific to SRS microscopy enables tumor detection by revealing quantifiable alterations in tissue cellularity, axonal density, and protein/lipid ratio in tumor-infiltrated tissues. To ensure that SRS microscopic data can be easily used in brain tumor surgery, without the need for expert interpretation, we created a classifier based on cellularity, axonal density, and protein/lipid ratio in SRS images capable of detecting tumor infiltration with 97.5% sensitivity and 98.5% specificity. Quantitative SRS microscopy detects the spread of tumor cells, even in brain tissue surrounding a tumor that appears grossly normal. By accurately revealing tumor infiltration, quantitative SRS microscopy holds potential for improving the accuracy of brain tumor surgery.

Brain tumor surgery is one of the key factors in prolonging survival in patients with low- and high-grade gliomas. However, resections of these infiltrative lesions have historically been limited by the inability to accurately detect tumor margins. New methods in microscopy and dye injection have enabled more complete resections, but continue to lack biochemical specificity or high-resolution image acquisition. Stimulated Raman scattering microscopy represents an improvement over past techniques in the ability to differentiate intraparenchymal tissues on the basis of biochemical attributes, and is available for use in real-time, a feature that facilitates its translation to the surgical setting.

Rapid histology of brain tissues with sufficient diagnostic information has the great potential to aid neurosurgeons during operations. Stimulated Raman Scattering (SRS) microscopy is an emerging label-free imaging technique, with the intrinsic chemical resolutions to delineate brain tumors from normal tissues without the need of time-consuming tissue processing. Growing number of studies have shown SRS as a “virtual histology” tool for rapid diagnosis of various types of brain tumors. In this review, we focus on the basic principles and current developments of SRS microscopy, as well as its applications for brain tumor imaging.

Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

Correction to: Laboratory Investigation (2012); doi: 10.1038/labinvest.2012.109; advance online publication, 20 August 2012. In this article, the name of the eighth author is incorrect. The correct name is Ying Wei.