Label-free macroscopic fluorescence lifetime imaging of brain tumors

Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival time. Real-time pathology assisted, or image guided surgical procedures that eliminate tumors promise to improve the clinical outcome and prolong the lives of patients. Our work is focused on the development of a rapid and sensitive assay for intraoperative diagnostics of glioma and identification of optical markers essential for differentiation between tumors and healthy brain tissues. We utilized fluorescence lifetime imaging (FLIM) of endogenous fluorophores related to metabolism of the glioma from freshly excised brains tissues. Macroscopic time-resolved fluorescence images of three intracranial animal glioma models and surgical samples of patients\u27 glioblastoma together with the white matter have been collected. Several established and new algorithms were applied to identify the imaging markers of the tumors. We found that fluorescence lifetime parameters characteristic of the glioma provided background for differentiation between the tumors and intact brain tissues. All three rat tumor models demonstrated substantial differences between the malignant and normal tissue. Similarly, tumors from patients demonstrated statistically significant differences from the peritumoral white matter without infiltration. While the data and the analysis presented in this paper are preliminary and further investigation with a larger number of samples is required, the proposed approach based on the macroscopic FLIM has a high potential for diagnostics of glioma and evaluation of the surgical margins of gliomas

Towards targeted colorectal cancer biopsy based on tissue morphology assessment by compression optical coherence elastography

Identifying the precise topography of cancer for targeted biopsy in colonoscopic examination is a challenge in current diagnostic practice. For the first time we demonstrate the use of compression optical coherence elastography (C-OCE) technology as a new functional OCT modality for differentiating between cancerous and non-cancerous tissues in colon and detecting their morphological features on the basis of measurement of tissue elastic properties. The method uses pre-determined stiffness values (Young’s modulus) to distinguish between different morphological structures of normal (mucosa and submucosa), benign tumor (adenoma) and malignant tumor tissue (including cancer cells, gland-like structures, cribriform gland-like structures, stromal fibers, extracellular mucin). After analyzing in excess of fifty tissue samples, a threshold stiffness value of 520 kPa was suggested above which areas of colorectal cancer were detected invariably. A high Pearson correlation (r =0.98; p <0.05), and a negligible bias (0.22) by good agreement of the segmentation results of C-OCE and histological (reference standard) images was demonstrated, indicating the efficiency of C-OCE to identify the precise localization of colorectal cancer and the possibility to perform targeted biopsy. Furthermore, we demonstrated the ability of C-OCE to differentiate morphological subtypes of colorectal cancer – low-grade and high-grade colorectal adenocarcinomas, mucinous adenocarcinoma, and cribriform patterns. The obtained ex vivo results highlight prospects of C-OCE for high-level colon malignancy detection. The future endoscopic use of C-OCE will allow targeted biopsy sampling and simultaneous rapid analysis of the heterogeneous morphology of colon tumors

Multiparametric optical bioimaging reveals the fate of epoxy crosslinked biomeshes in the mouse subcutaneous implantation model

Biomeshes based on decellularized bovine pericardium (DBP) are widely used in reconstructive surgery due to their wide availability and the attractive biomechanical properties. However, their efficacy in clinical applications is often affected by the uncontrolled immunogenicity and proteolytic degradation. To address this issue, we present here in vivo multiparametric imaging analysis of epoxy crosslinked DBPs to reveal their fate after implantation. We first analyzed the structure of the crosslinked DBP using scanning electron microscopy and evaluated proteolytic stability and cytotoxicity. Next, using combination of fluorescence and hypoxia imaging, X-ray computed microtomography and histology techniques we studied the fate of DBPs after subcutaneous implantation in animals. Our approach revealed high resistance to biodegradation, gradual remodeling of a surrounding tissue forming the connective tissue capsule and calcification of crosslinked DBPs. These changes were concomitant to the development of hypoxia in the samples within 3 weeks after implantation and subsequent induction of angiogenesis and vascularization. Collectively, presented approach provides new insights on the transplantation of the epoxy crosslinked biomeshes, the risks associated with its applications in soft-tissue reconstruction and can be transferred to studies of other types of implants

Toxicological Analysis of Hepatocytes Using FLIM Technique: In Vitro versus Ex Vivo Models

The search for new criteria indicating acute or chronic pathological processes resulting from exposure to toxic agents, testing of drugs for potential hepatotoxicity, and fundamental study of the mechanisms of hepatotoxicity at a molecular level still represents a challenging issue that requires the selection of adequate research models and tools. Microfluidic chips (MFCs) offer a promising in vitro model for express analysis and are easy to implement. However, to obtain comprehensive information, more complex models are needed. A fundamentally new label-free approach for studying liver pathology is fluorescence-lifetime imaging microscopy (FLIM). We obtained FLIM data on both the free and bound forms of NAD(P)H, which is associated with different metabolic pathways. In clinical cases, liver pathology resulting from overdoses is most often as a result of acetaminophen (APAP) or alcohol (ethanol). Therefore, we have studied and compared the metabolic state of hepatocytes in various experimental models of APAP and ethanol hepatotoxicity. We have determined the potential diagnostic criteria including the pathologically altered metabolism of the hepatocytes in the early stages of toxic damage, including pronounced changes in the contribution from the bound form of NAD(P)H. In contrast to the MFCs, the changes in the metabolic state of hepatocytes in the ex vivo models are, to a greater extent, associated with compensatory processes. Thus, MFCs in combination with FLIM can be applied as an effective tool set for the express modeling and diagnosis of hepatotoxicity in clinics

ДИАЛЕКТИКА РОЗВИТКУ ІННОВАЦІЙНОЇ ДІЯЛЬНОСТІ ТА ІНСТИТУЦІЙНОГО СЕРЕДОВИЩА: ТЕОРЕТИЧНИЙ АСПЕКТ

The main ideas of such notions as “innovation performance”, “innovation activity”,“ innovative capacity”, “institutional environment” were specified in the work. The peculiar features of the notions in course of institutionalization of innovation economy were taken into account which made it possible structure the research and make specification of the given notions. Besides, the peculiarities and regularities of development of innovation performance and institutional environment in their dialectical unity and objective contradiction were revealed.В процессе исследования уточнены сущностные характеристики понятий: «инновационная деятельность», «инновационная активность», «инновационный потенциал», «институциональная среда» с учетом их специфики в процессе институционализации инновационной экономики, что позволило структурировать научное исследование и провести детализацию данных понятий. Кроме того, выявлены особенности и закономерности развития инновационной деятельности и институциональной среды в их диалектическом единстве и объективном противоречии.У процесі дослідження уточнено сутнісні характеристики понять: «інноваційна діяльність», «інноваційна активність», «інноваційний потенціал», «інституційне середовище» з урахуванням їх специфіки в процесі інституціоналізації інноваційної економіки, що дозволило структурувати наукове дослідження і провести деталізацію даних понять. Крім того, виявлено особливості та закономірності розвитку інноваційної діяльності та інституційного середовища в їх діалектичній єдності і об'єктивному протиріччі

Novel PEG-organized biocompatible fluorescent nanoparticles doped with an ytterbium cyanoporphyrazine complex for biophotonic applications

International audienceThe preparation and properties are described of two types of novel PEG-organized nanoparticles including silica-modified uniform disk-shaped nanoparticles doped with a fluorescent ytterbium cyanoporphyrazine complex; a large enhancement of red emission for both types of nanoparticles is observed in physiological liquids owing to their binding to biomolecule

FLIM for Evaluation of Difference in Metabolic Status between Native and Differentiated from iPSCs Dermal Papilla Cells

iPSCs and their derivatives are the most promising cell sources for creating skin equivalents. However, their properties are not fully understood. In addition, new approaches and parameters are needed for studying cells in 3D models without destroying their organization. Thus, the aim of our work was to study and compare the metabolic status and pH of dermal spheroids created from dermal papilla cells differentiated from pluripotent stem cells (iDP) and native dermal papilla cells (hDP) using fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). For this purpose, fluorescence intensities of NAD(P)H and FAD, fluorescence lifetimes, and the contributions of NAD(P)H, as well as the fluorescence intensities of SypHer-2 and BCECF were measured. iDP in spheroids were characterized by a more glycolytic phenotype and alkaline intra-cellular pH in comparison with hDP cells. Moreover, the metabolic activity of iDP in spheroids depends on the source of stem cells from which they were obtained. So, less differentiated and condensed spheroids from iDP-iPSDP and iDP-iPSKYOU are characterized by a more glycolytic phenotype compared to dense spheroids from iDP-DYP0730 and iDP-hES. FLIM and fluorescent microscopy in combination with the metabolism and pH are promising tools for minimally invasive and long-term analyses of 3D models based on stem cells

Laser-assisted fabrication and in vitro verification of functionalized surface for cells biointegration

International audienceThe paper investigates how the surface relief of an implant affects cell behavior. Currently, most implant manufacturers claim the key impact biocompatibility factor to be surface micro-roughness. We suppose that the interaction between cells and implants also depends on such relief peculiarities as continuous or discontinuous topography, subcellular distance between peaks and presence of porous oxide layer. We have developed the laser processing conditions that provide three different reliefs: «open grooves», «grid» and «close grooves». Along with the micro-roughness characteristics the reliefs differ with their deepness and period of grooves. The surface composition analysis results have shown a sandwich structure consisting of Ti → TiO → Ti 2 O 3 Nx → TiO 2 (anatase) → TiO 2 (rutile). The wettability study has demonstrated superhydrophilicity (CA is 0 •) for all reliefs. The quantitative and qualitative analysis of hMSCs proliferation and osteogenic differentiation was performed for 20 days. In vitro study has revealed the topography affects the spatial orientation of cells. The shape and size of the cell nuclei vary with different topographies. We have found continuous «open grooves» structures with the subcellular to cellular period are beneficial for cells' life-sustaining activity. Discontinuous «grid» structures with individual slots might not provide cells with mobility with the least external mechanical effect compared to «open grooves»

A Collagen Basketweave from the Giant Squid Mantle as a Robust Scaffold for Tissue Engineering

The growing applications of tissue engineering technologies warrant the search and development of biocompatible materials with an appropriate strength and elastic moduli. Here, we have extensively studied a collagenous membrane (GSCM) separated from the mantle of the Giant squid Dosidicus Gigas in order to test its potential applicability in regenerative medicine. To establish the composition and structure of the studied material, we analyzed the GSCM by a variety of techniques, including amino acid analysis, SDS-PAGE, and FTIR. It has been shown that collagen is a main component of the GSCM. The morphology study by different microscopic techniques from nano- to microscale revealed a peculiar packing of collagen fibers forming laminae oriented at 60–90 degrees in respect to each other, which, in turn, formed layers with the thickness of several microns (a basketweave motif). The macro- and micromechanical studies showed high values of the Young’s modulus and tensile strength. No significant cytotoxicity of the studied material was found by the cytotoxicity assay. Thus, the GSCM consists of a reinforced collagen network, has high mechanical characteristics, and is non-toxic, which makes it a good candidate for the creation of a scaffold material for tissue engineering