Multiphoton Microscopy Imaging Research Articles

Integrating structural heterogeneity, fiber orientation, and recruitment in multiscale ECM mechanics

Extracellular matrix (ECM) plays critical roles in establishing tissue structure-function relationships and controlling cell fate. However, the mechanisms by which ECM mechanics influence cell and tissue behavior remain to be elucidated since the events associated with this process span length scales from the tissue to molecular level. Entirely new methods are needed in order to better understand the multiscale mechanics of ECM. In this study, a multiscale experimental approach was established by integrating Optical Magnetic Twisting Cytometry (OMTC) with a biaxial tensile tester to study the microscopic (local) ECM mechanical properties under controlled tissue-level (global) loading. Adventitial layer of porcine thoracic artery was used as a collagen-based ECM. Multiphoton microscopy imaging was performed to capture the changes in ECM fiber structure during biaxial deformation. As visualized from multiphoton microscopy images, biaxial stretch induces gradual fiber straightening and the fiber families become evident at higher stretch levels. The OMTC measurements show that the local apparent storage and loss modulus increases with the global biaxial stretch, however there exists a complex interplay among local ECM mechanical properties, ECM structural heterogeneity, and fiber distribution and engagement. The phase lag does not change significantly with global biaxial stretch. Our results also show a much faster increase in global tissue tangent modulus compared to the local apparent complex modulus with biaxial stretch, indicating the scale dependency of ECM mechanics.

P18 - Social Behavior of Breast Cancer Stem Cell in Metastasis

My early work demonstrated that Rb tumor suppressor prevent the chemotherapeutic agent cisplatin-induced ROS stress and cell death by maintaining ATP and NAD levels as well as PARP-1 activity. Rb and Trp53 have been shown by the Sawyers and Goodrich groups in limiting cancer lineage plasticity through inhibiting Sox2 and Ezh2 pathways. My laboratory has focused on identifying gene and microRNA regulators of breast cancer stem cells in driving chemoresistance and metastasis, such as miR-30c, miR-138, and miR-206. While miR-138 targets EZH2 in breast CSCs to promote differentiation and inhibit tumor invasion, both miR-30c and miR-206 target cytoskeleton genes such as TWF1 to inhibit cancer stemness, chemoresistance and metastasis. Recently, we have identified a new social behavior of CSCs in circulating tumor cell cluster formation which greatly enhanced lung colonization and polyclonal metastasis of breast cancer. CTC clusters were less frequently detected but more metastatic than single CTCs of triple negative breast cancer patients and representative patient-derived-xenograft (PDX) models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding.

Multiphoton Imaging of Collagen, Elastin, and Calcification in Intact Soft-Tissue Samples.

Multiphoton-induced second-harmonic generation and two-photon excitation enable imaging of collagen and elastin fibers at micron-level resolution to depths of hundreds of microns, without the use of exogenous stains.These attributes can be leveraged for quantitative analysis of the 3D architecture of collagen and elastin fibers within intact, soft tissue specimens such as the artery and bladder wall. This architectureinfluences the function of intramural cells and also plays a primary role in determining tissue passive mechanical properties. Calcification deposition in soft tissues is a highly prevalent pathology in both older and diseased populations that can alter tissue properties.In this unit, we provide a protocol forsimultaneous multiphoton microscopy (MPM) imaging and analysis of 3D collagen and elastin structures with calcification, which is effective for fixed and fresh intact samples. We also provide an associated micro-CT protocolto identify regions of interest in the samples as a means to target the MPM imaging. © 2018 by John Wiley & Sons, Inc.

Feature characterization of scarring and non‐scarring types of alopecia by multiphoton microscopy

Non-invasive visualization of hair follicles is important for proper diagnosis and management of alopecia; however, histological assessment remains the gold standard. Laser imaging technologies have made possible noninvasive in vivo evaluation of skin and hair follicle. The aim of this study was to evaluate the ability of multiphoton microscopy (MPM) to non-invasively identify morphological features that can distinguish scarring from non-scarring alopecia. MPM images were obtained from areas on the scalp affected by alopecia. Investigators blinded to the diagnosis analyzed hair follicle and shaft sizes. Patients were recruited and imaged at the UC Irvine Health Medical Center and the University of California, Irvine Beckman Laser Institute. Patients with androgenetic alopecia (AGA) and alopecia areata (AA), and scarring alopecia, in particular frontal fibrosing alopecia (FFA) were recruited and imaged from July 2016 to July 2017. We imaged 5 normal scalp subjects and 12 patients affected by non-scarring (7 subjects) and scarring (5 subjects) alopecia. In normal and non-scarring alopecia patients, MPM identified presence of sebaceous glands associated with hair follicles. MPM images of scarring alopecia were characterized by the presence of inflammatory cells surrounding hair follicles. Measurements of hair follicle diameter sizes were found to be significantly smaller in scarring alopecia patients compared to normal (P < 0.001) and compared to non-scarring alopecia patients (P = 0.046); non-scarring hair follicles were also significantly smaller than normal hair follicles (P = 0.043). This study shows that MPM imaging can non-invasively identify morphological features that distinguish scarring from non-scarring alopecia. Further studies are needed to validate this technique and evaluate its potential to be used as an aid for guiding treatment. Lasers Surg. Med. 51:95-103, 2019. © 2018 Wiley Periodicals, Inc.

Using elongated microparticles to enhance tailorable nanoemulsion delivery in excised human skin and volunteers

This study demonstrates, for the first time, clinical testing of elongated silica microparticles (EMP) combined with tailorable nanoemulsions (TNE) to enhance topical delivery of hydrophobic drug surrogates. Likewise, this is the first report of 6-carboxyfluorescein (a model molecule for topically delivered hydrophobic drugs) AM1 & DAMP4 (novel short peptide surfactants) used in volunteers. The EMP penetrates through the epidermis and stop at the dermal-epidermal junction (DEJ). TNE are unusually stable and useful because the oil core allows high drug loading levels and the surface properties can be easily controlled. At first, we chose alginate as a crosslinking agent between EMP and TNE. We initially incorporated a fluorescent lipophilic dye, DiI, as a hydrophobic drug surrogate into TNE for visualization with microscopy. We compared four different coating approaches to combine EMP and TNE and tested these formulations in freshly excised human skin. The delivery profile characterisation was imaged by dye- free coherent anti-Stoke Raman scattering (CARS) microscopy to detect the core droplet of TNE that was packed with pharmaceutical grade lipid (glycerol) instead of DiI. These data show the EMP penetrating to the DEJ followed by controlled release of the TNE. Freeze-dried formulations with crosslinking resulted in a sustained release profile, whereas a freeze-dried formulation without crosslinking showed an immediate burst-type release profile. Finally, we tested the crosslinked TNE coated EMP formulation in volunteers using multiphoton microscopy (MPM) and fluorescence-lifetime imaging microscopy (FLIM) to document the penetration depth characteristics. These forms of microscopy have limitations in terms of image acquisition speed and imaging area coverage but can detect fluorescent drug delivery through the superficial skin in volunteers. 6-Carboxyfluorescein was selected as the fluorescent drug surrogate for the volunteer study based on the similarity of size, charge and hydrophobicity characteristics to small therapeutic drugs that are difficult to deliver through skin. The imaging data showed a 6-carboxyfluorescein signal deep in volunteer skin supporting the hypothesis that EMP can indeed enhance the delivery of TNE in human skin. There were no adverse events recorded at the time of the study or after the study, supporting the use of 6-carboxyfluorescein as a safe and detectable drug surrogate for topical drug research. In conclusion, dry formulations, with controllable release profiles can be obtained with TNE coated EMP that can effectively enhance hydrophobic payload delivery deep into the human epidermis.

Visualize and quantify the structural alteration of the rat spinal cord injury based on multiphoton microscopy.

The development of imaging technique to visualize and quantify the structural alteration of the spinal cord injury (SCI) may lead to better understanding and treatments of the injuries. In this work, multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) was tentatively applied to quantitatively visualize the cellular microstructures of SCI to demonstrate the feasibility and superiority of MPM in SCI imaging. High-contrast MPM images of normal and injured spinal cord tissue were obtained for comparison. Moreover, the changes of injured spinal cord were characterized by the quantitative analysis of the MPM images. These results showed that MPM combined with quantitative method has the ability to identify the characteristics of spinal cord injury including the changes in the contents of nerve fibers and extracellular matrix. With the advancement of MPM, we believe that this technique has great potential to provide the histological diagnosis for the monitoring and evaluation of SCI.

Evaluation of Glomerular Hemodynamic Function by Empagliflozin in Diabetic Mice Using In Vivo Imaging

Improvement of glomerular hyperfiltration via tubuloglomerular feedback (TGF) is considered to be one of the possible pathways for renal protection with SGLT2 inhibition (SGLT2i) in diabetic kidney disease (DKD). However, the mechanisms that regulate renal function in response to SGLT2i have not been fully elucidated. We explored the renal protective mechanisms of SGLT2i with empagliflozin (EMPA), with a focus on glomerular hemodynamic effects and TGF using in vivo multiphoton microscope (MPM) imaging techniques. C57BL/6 mice and spontaneously diabetic Ins2 Akita (AKITA) mice were used. The mice were treated with EMPA (20mg/kg/day) and insulin for four weeks, and single-nephron GFR (SNGFR) was measured using MPM. An nNOS inhibitor (7-nitroindazole, 20mg/kg/day) or a COX2 inhibitor (SC58236, 6mg/L), or an adenosine A1 receptor (A1aR) antagonist (8-cyclopentyl-1,3-dipropylxanthine, 1mg/kg/day) were administered to elucidate mechanisms of TGF signaling and SNGFR regulation. The urinary excretion of adenosine, nitric oxide metabolites (NOx) and the prostanoid PGE2 was also quantified. SNGFR in the AKITA group was higher compared to controls (C57BL/6; 4.9±1.3 nl/min vs. AKITA; 15.8±6.8 nl/min) and decreased in AKITA/EMPA to 8.0±3.3 nl/min (p&amp;lt;0.01). In vivo imaging also revealed concomitant afferent arteriolar dilation (p&amp;lt;0.01) and increased glomerular permeability of albumin in the AKITA group. EMPA ameliorated these changes (p&amp;lt;0.01). Urinary adenosine excretion in the AKITA/EMPA group was higher compared to AKITA (AKITA; 3.4±1.4 nmol/day, AKITA/EMPA; 11.2±3.0 nmol/day, p&amp;lt;0.05), while NOx and PGE2 did not differ. A1aR antagonism, but not nNOS or COX2 inhibition, blocked the effect of EMPA on renal function. EMPA increased urinary adenosine excretion and reduced hyperfiltration via afferent arteriolar constriction, effects that were abolished by A1aR blockade. The adenosine/A1aR system plays a pivotal role in the regulation of SNGFR via TGF mechanisms in DKD. Disclosure K. Kidokoro: Research Support; Spouse/Partner; Sanwa Kagaku Kenkyusho Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Chugai Pharmaceutical Co., Ltd., Pfizer Inc., MSD K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Sanofi K.K., Takeda Pharmaceutical Co., Astellas Pharma Inc., TORII PHARMACEUTICAL Co.. Y. Sogawa: None. D. Cherney: Consultant; Self; AbbVie Inc.. Other Relationship; Self; AstraZeneca, Boehringer Ingelheim GmbH, Eli Lilly and Company. Consultant; Self; Sanofi. Other Relationship; Self; Merck &amp; Co., Inc.. Consultant; Self; Mitsubishi Tanabe Pharma Corporation. Other Relationship; Self; Janssen Pharmaceuticals, Inc.. V. Kulasingam: None. H. Nagasu: None. M. Satoh: None. T. Sasaki: None. N. Kashihara: None.

Novel hemodynamic structures in the human glomerulus.

To investigate human glomerular structure under conditions of physiological perfusion, we have analyzed fresh and perfusion-fixed normal human glomeruli at physiological hydrostatic and oncotic pressures using serial resin section reconstruction, confocal, multiphoton, and electron microscope imaging. Afferent and efferent arterioles (21.5 ± 1.2 µm and 15.9 ± 1.2 µm diameter), recognized from vascular origins, lead into previously undescribed wider regions (43.2 ± 2.8 µm and 38.4 ± 4.9 µm diameter) we have termed vascular chambers (VCs) embedded in the mesangium of the vascular pole. Afferent VC (AVC) volume was 1.6-fold greater than efferent VC (EVC) volume. From the AVC, long nonbranching high-capacity conduit vessels (n = 7) (Con; 15.9 ± 0.7 µm diameter) led to the glomerular edge, where branching was more frequent. Conduit vessels have fewer podocytes than filtration capillaries. VCs were confirmed in fixed and unfixed specimens with a layer of banded collagen identified in AVC walls by multiphoton and electron microscopy. Thirteen highly branched efferent first-order vessels (E1; 9.9 ± 0.4 µm diameter) converge on the EVC, draining into the efferent arteriole (15.9 ± 1.2 µm diameter). Banded collagen was scarce around EVCs. This previously undescribed branching topology does not conform to the branching of minimum energy expenditure (Murray’s law), suggesting that even distribution of pressure/flow to the filtration capillaries is more important than maintaining the minimum work required for blood flow. We propose that AVCs act as plenum manifolds possibly aided by vortical flow in distributing and balancing blood flow/pressure to conduit vessels supplying glomerular lobules. These major adaptations to glomerular capillary structure could regulate hemodynamic pressure and flow in human glomerular capillaries.

Automated classification of multiphoton microscopy images of ovarian tissue using deep learning.

Histopathological image analysis of stained tissue slides is routinely used in tumor detection and classification. However, diagnosis requires a highly trained pathologist and can thus be time-consuming, labor-intensive, and potentially risk bias. Here, we demonstrate a potential complementary approach for diagnosis. We show that multiphoton microscopy images from unstained, reproductive tissues can be robustly classified using deep learning techniques. We fine-train four pretrained convolutional neural networks using over 200 murine tissue images based on combined second-harmonic generation and two-photon excitation fluorescence contrast, to classify the tissues either as healthy or associated with high-grade serous carcinoma with over 95% sensitivity and 97% specificity. Our approach shows promise for applications involving automated disease diagnosis. It could also be readily applied to other tissues, diseases, and related classification problems.

Intraoperative assisting diagnosis of esophageal submucosal cancer using multiphoton microscopy

Multiphoton microscopy (MPM) based on two-photon excited fluorescence and second harmonic generation can achieve high-resolution images of biological tissues at the cellular and subcellular levels. In this work, we used MPM imaging of intraoperative hematoxylin and eosin (H&E)-stained frozen sections (FSs) of esophagus to explore whether MPM can provide complementary information to the auxiliary diagnosis of esophageal submucosal cancer during the intraoperative period. It was found that MPM has the ability not only to clearly reveal biological tissue microstructure and its morphological changes, but can reveal information not distinguishable in H&E-stained images. The complementary information of nonlinear spectral analysis, orientation and morphology changes in the collagen showed that MPM has important accessory diagnostic value for the differential diagnosis of submucosal carcinoma of the esophagus during the intraoperative period.

Spatial and temporal identification of cerebral infarctions based on multiphoton microscopic imaging.

Ischemic stroke is a leading cause of death and permanent disability worldwide. Middle cerebral artery occlusion (MCAO) of variable duration times could be anticipated to result in varying degrees of injury that evolve spatially over time. Therefore, investigations following strokes require information concerning the spatiotemporal dimensions of the ischemic core as well as of perilesional areas. In the present study, multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) was applied to image such pathophysiological events. The ischemic time-points for evaluation were set at 6, 24, 48, and 72 hours after MCAO. Our results demonstrated that MPM has the ability to not only identify the normal and ischemic brain regions, but also reveal morphological changes of the cortex and striatum at various times following permanent MCAO. These findings corresponded well with the hematoxylin and eosin (H&E) stained tissue images. With the technologic progression of miniaturized imaging devices, MPM can be developed into an effective diagnostic and monitoring tool for ischemic stroke.

Noninvasive assessment of skin structure by combined photothermal radiometry and optical spectroscopy: coregistration with multiphoton microscopy.

We are combining two optical techniques, pulsed photothermal radiometry (PPTR) and diffuse reflectance spectroscopy (DRS), for noninvasive assessment of the structure and composition of human skin in vivo. The analysis involves simultaneous multidimensional fitting of the measured PPTR signals and DRS spectra with predictions of a numerical model of light transport (Monte Carlo) in a four-layer model optical model of human skin, accounting for the epidermis, papillary and reticular dermis, and subcutis. The assessed epidermal thickness values were tested by coregistration with a multiphoton microscope, which provides vertical sectioning capability based on two-photon excited fluorescence and second-harmonic generation in selected skin components. The comparison shows that these values correspond well to the maximal epidermal thicknesses measured in the multiphoton microscopy images, the rete ridges.

Diagnosing pituitary adenoma in unstained sections based on multiphoton microscopy.

If we can find a new method that can achieve rapid diagnosis of adenoma during operation, it will help surgeon shorten the operation time and enhance the treatment efficacy. This study discusses the feasibility of multiphoton microscopy (MPM) in diagnosing pituitary adenoma. MPM, based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) is performed for the diagnosis of pituitary adenoma in unstained sections. Our results show that MPM can reveal the variation of reticulin fiber by SHG signals of collagen, combined with the measurement of area of acinus, thickness of collagen fiber and collagen percentage. MPM can further reflect the change of meshwork in normal pituitary and hyperplasia quantitatively. And the characteristics of typical growth patterns of pituitary adenoma are demonstrated by the overlay of SHG and TPEF images. What's more, we can identify the boundary of normal pituitary, hyperplasia and adenoma from MPM images. And the experiment also results verify the feasibility of this method in frozen sections. These results indicated that MPM can make a diagnosis of pituitary adenoma by the morphological changes without routine pathological processing including hematoxylin-eosin (H&E) staining and other special staining. Therefore, this technique is expected to help diagnosis of pituitary adenoma during operation.

Phospholipid Magnesome-a nasal vesicular carrier for delivery of drugs to brain.

The goal of this work was to investigate a new nasal carrier for enhanced drug delivery to brain, we call Phospholipid Magnesome. The system contains soft phospholipid vesicles, composed of phospholipid, water, propylene glycol, magnesium salt, and the mucoadhesive polymer, alginate. The carrier was characterized by various methods: electron microscopy, calorimetry, and dynamic light scattering. The ability of the carrier's vesicles to entrap various molecules was studied by CLSM and ultracentrifugation combined with HPLC quantification. Mucoadhesivity of the carrier was tested in vitro using porcine nasal mucosa. The delivery of rohdamine 6G, insulin, and epidermal growth factor was estimated by two methods, multiphoton microscopy and near infrared (NIR) imaging. Pharmacodynamic effects of nasal treatment with oxytocin and insulin incorporated in Phospholipid Magnesome were evaluated in animal models. Results show that the system is composed of soft multilamellar nanosized vesicles with the ability to entrap both lipophilic and hydrophilic molecules. The mucoadhesivity test results indicate a prolonged contact time of the drug with the nasal membrane as compared to control. Multiphoton microscopy and NIR imaging of brain show effective delivery of the tested molecules to brain following nasal administration in Phospholipid Magnesome relative to controls. Moreover, the results of the pharmacodynamic study measuring the antinociceptive effect of oxytocin administrated nasally to an animal model indicate the efficiency of the Phospholipid Magnesome as compared to three control systems. Further, nasal administration of insulin resulted in a strong and prolonged hypoglycemic effect for the drug incorporated in the new carrier but not for control systems. Based on the results of the histopathological test, the carrier is safe for local administration on the nasal membrane. In conclusion, the results of this study suggest that Phospholipid Magnesome nasal carrier is able to improve drug effects, probably by a combined mechanism, absorption enhancement, and prolongation of mucosal contact.

Label-free imaging for T staging of gastric carcinoma by multiphoton microscopy.

Gastric cancer is one of the most common malignancies worldwide. The accurate diagnosis of tumor invasion depth is critical for therapeutic strategy and prognosis. Without fluorescent labelling, multiphoton microscopy (MPM) imaging could directly reveal tissue architecture based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). In this study, we aimed to explore the feasibility of MPM imaging to assess the gastric tumor morphology and infiltration. Unstained slides of 18 fresh gastric tissues with different T staging were examined by multiphoton microscopy. Morphological and quantitative analyses were both conducted. The nuclear area was defined as the area of nuclear boundary. Collagen content was defined as the ratio of SHG pixels to all pixels. Gastric normal and tumor tissues under different T stages visually presented with cellular and subcellular features on fluorescent imaging. The nuclear areas of normal and cancerous cells were 32.01 ± 2.89 and 58.41 ± 6.06μm2 (P < 0.001), respectively. Collagen content was quantified as 0.087 ± 0.012 in normal mucosa but 0.020 ± 0.007 in cancerous mucosa (P < 0.001). All results were in accord with the paired H&E-stained slides. Our findings suggested the convincing potential of MPM for judging T staging of gastric cancer. Without staining intervention, TPEF and SHG of MPM imaging could objectively and quantitatively indicate the subcellular and molecular changes during carcinogenesis. With the advancement of deep penetration, self-focus imaging and three-dimensional (3D) visualization, label-free MPM imaging compacted with endoscopy could be further introduced to realize the real-time in vivo assessment of tumor invasion clinically.

Two-photon probes for pH: Detection of human colon cancer using two-photon microscopy.

607 Background: In cancer cells, lysosomal pH decreases along with a concomitant increase in lysosomal volume and cathepsin expression levels. Lysosomes also play crucial roles in cancer progression following their release into the extracellular space. Since cancer cells invade a tissue by secreting degradative enzymes, the extracellular pH of tumor tissues becomes acidic. However, to date, there has been no report on the use of multi-photon microscopy (MPM) probes to image human colon cancer tissues. Methods: We have developed multi-photon (MP) pH-sensitive probes (BH-2 and BHEt-1) that exhibit absorption and emission maxima at 370 and 466 nm, and TP absorption cross-section values of 51 and 61 GM (1 GM = 10−50 cm4 s/photon), respectively, at 750 nm and pH 3.0 in a universal buffer (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M KCl)/1,4-dioxane (7/ 3) solution. Results: The TPM images of CCD-18co (a normal colon cell line) and HCT116 cells (a colon cancer cell line) labeled with BH-2 were too dim to be distinguished. When the same cells were labeled with BHEt-1, however, the MPM image of the HCT116 cells was much brighter than that of CCD-18co cells, and the relative proportion of the acidic vesicles (Pacid) of the former was 5-fold larger than that of latter. BHEt-1 could also differentiate HepG2 cells (a human liver cancer cell line) from LX-2 cells (a human hepatic stellate cell line) with a 6-fold larger P acid value. Human colon cancer tissues labeled with BHEt-1 showed similar results, demonstrating much brighter MPM images and 6-fold larger Pacid values compared to normal tissue. Conclusions: These results suggest the potential utility of BHEt-1 for molecular image analysis of colon cancer tissues using MPM.

Structural analysis of processed corneas

Background: Disruption of the microstructure in corneal stroma can lead to the loss of transparency. The lack of a characterization method for the microstructure prevents such scaffolds to be implemented in tissue transplantation. The non-invasive, three-dimensional (3D) rendering multiphoton microscopy (MPM) poses the potential to solve this problem. Methods: MPM images and data analyses were performed with three kinds of samples with known and different quality. Isosurfaces (ISOs) were constructed for the evaluation of void volume and collagen distribution. Results: The differences in the microstructures of these samples were revealed with clear indications and links to their behaviours in rehydration and possible transparency. According to this analysis, the scaffold with the highest void space ratio amongst the three presented the highest successful rates to be thoroughly rehydrated. Conclusions: Such a method can be developed for assessing the quality of tissue engineered corneas, or donated corneas, and be useful as a powerful research tool in cornea related research.

Young and Especially Senescent Endothelial Microvesicles Produce NADPH: The Fuel for Their Antioxidant Machinery.

In a previous study, we demonstrated that endothelial microvesicles (eMVs) have a well-developed enzymatic team involved in reactive oxygen species detoxification. In the present paper, we demonstrate that eMVs can synthesize the reducing power (NAD(P)H) that nourishes this enzymatic team, especially those eMVs derived from senescent human umbilical vein endothelial cells. Moreover, we have demonstrated that the molecules that nourish the enzymatic machinery involved in NAD(P)H synthesis are blood plasma metabolites: lactate, pyruvate, glucose, glycerol, and branched-chain amino acids. Drastic biochemical changes are observed in senescent eMVs to optimize the synthesis of reducing power. Mitochondrial activity is diminished and the glycolytic pathway is modified to increase the activity of the pentose phosphate pathway. Different dehydrogenases involved in NADPH synthesis are also increased. Functional experiments have demonstrated that eMVs can synthesize NADPH. In addition, the existence of NADPH in eMVs was confirmed by mass spectrometry. Multiphoton confocal microscopy images corroborate the synthesis of reducing power in eMVs. In conclusion, our present and previous results demonstrate that eMVs can act as autonomous reactive oxygen species scavengers: they use blood metabolites to synthesize the NADPH that fuels their antioxidant machinery. Moreover, senescent eMVs have a stronger reactive oxygen species scavenging capacity than young eMVs.

Multiphoton microscopy: A novel diagnostic method for solid tumors in a prospective pediatric oncologic cohort, an experimental study

BackgroundThe prognosis of solid pediatric tumors strongly correlates with accurate staging and complete local control. Currently, surgeons rely on macroscopic cues and intraoperative cryosection to determine resection borders. Multiphoton Microscopy (MPM) is a real time technique that allows imaging of tissue without time-consuming tissue processing. PurposeThis pilot study evaluates the diagnostic potential of MPM in pediatric solid tumors compared to routine histopathology. MethodsSlides of pediatric tumor samples (nephroblastoma and neuroblastoma [n = 2]; ganglioneuroma, pleuropulmonary blastoma, hepatocellular carcinoma [n = 1]) were prepared to allow direct comparison of MPM with conventional light microscopy. Additionally, we applied MPM to native tumor tissue blocks to evaluate direct visualization of malignant cells through the tumor capsule. Images were interpreted by an attending surgical pathologist. Detectability of tumor-specific features was compared between MPM and conventional histology. ResultsA total of 7 tumors from 7 recruited patients were analyzed. All MPM images were accurate in diagnosing typical criteria of each particular neoplasm. In addition, MPM clearly visualized tumors through the capsule without sectioning or labeling procedures. The quality of MPM was sufficient to make the diagnosis and visualize typical entity-specific architectural changes. ConclusionMPM is comparable to conventional histopathology in the diagnosis of pediatric solid tumors without the need for fixation or staining. It therefore has tremendous potential for future real-time intraoperative diagnostics and as an alternative to conventional frozen section histopathology. Level of evidenceIII.

Cellular Detection of G-Quadruplexes by Optical Imaging Methods.

G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration). Here, we listed a repertoire of protocols that we have developed over the past years to visualize quadruplexes in cells. These achievements were made possible thanks to the discovery of a novel family of versatile quadruplex-selective fluorophores, the twice-as-smart quadruplex ligands named TASQ (for template-assembled synthetic G-quartet). The versatility of this probe allows for multiple imaging techniques in both fixed and live cells, including the use of the multiphoton microscopy, confocal microscopy, and real-time fluorescent image collection. © 2017 by John Wiley & Sons, Inc.