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

Novel highly emissive tetracyanotetraphenylporphyrazine ytterbium complex for optoelectronic and biophotonic applications.

International audienceThe prepn. is described of a novel highly emissive ytterbium complex with a proposed unusual structure obtained by reaction of tricyanovinylbenzene (TCNVB) with bis(indenyl)ytterbium(II) in THF. The reaction occurs under extremely mild conditions, the tetraphenyltetracyanoporphyrazine macrocycle being assembled in high yield from TCNVB building-blocks by Yb3+-template synthesis. The anal., spectral and electrochem. investigations of the obtained ytterbium complex indicate its existence in the form of a binuclear adduct with Yb(TCNVB)3 species in which a one doubly reduced TCNVB mol. bridges two Yb3+ cations. The formation of a disordered polynuclear coordination polymer network including a macrocyclic structure and metal cations bridged through the nitrile nitrogen atoms is proposed. The complex is readily sol. and is compatible with a variety of polymeric matrixes giving doped polymeric glasses and films which are highly luminescent in the biol. relevant optical window covering the visible and near IR range (640-1000 nm). In addn., doped polymeric glasses and films highly emissive at the telecommunication wavelength (1540 nm) including the novel ytterbium complex and originally not luminescent erbium chelate in an equimolar ratio have been obtained. The compd. is found to be an extraordinarily strong sensitizer of near-IR Er3+ emission. Use of the Yb complex as a fluorescent marker for biomedical in vitro investigations has been demonstrate

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

The Role of Plasma Membrane Viscosity in the Response and Resistance of Cancer Cells to Oxaliplatin

Maintenance of the biophysical properties of membranes is essential for cell survival upon external perturbations. However, the links between a fluid membrane state and the drug resistance of cancer cells remain elusive. Here, we investigated the role of membrane viscosity and lipid composition in the responses of cancer cells to oxaliplatin and the development of chemoresistance. Plasma membrane viscosity was monitored in live colorectal cancer cells and tumor xenografts using two-photon excited fluorescence lifetime imaging microscopy (FLIM) using the fluorescent molecular rotor BODIPY 2. The lipid profile was analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS). It was found that the plasma membrane viscosity increased upon oxaliplatin treatment, both in vitro and in vivo, and that this correlated with lower phosphatidylcholine and higher cholesterol content. The emergence of resistance to oxaliplatin was accompanied by homeostatic adaptation of the membrane lipidome, and the recovery of lower viscosity. These results suggest that maintaining a constant plasma membrane viscosity via remodeling of the lipid profile is crucial for drug resistance in cancer

Measurement of Patient-Derived Glioblastoma Cell Response to Temozolomide Using Fluorescence Lifetime Imaging of NAD(P)H

Personalized strategies in glioblastoma treatment are highly necessary. One of the possible approaches is drug screening using patient-derived tumor cells. However, this requires reliable methods for assessment of the response of tumor cells to treatment. Fluorescence lifetime imaging microscopy (FLIM) is a promising instrument to detect early cellular response to chemotherapy using the autofluorescence of metabolic cofactors. Here, we explored FLIM of NAD(P)H to evaluate the sensitivity of patient-derived glioma cells to temozolomide (TMZ) in vitro. Our results demonstrate that the more-responsive cell cultures displayed the longest mean fluorescence lifetime τm after TMZ treatment due to an increase in the protein-bound NAD(P)H fraction α2 associated with a shift to oxidative phosphorylation. The cell cultures that responded poorly to TMZ had generally shorter τm, i.e., were more glycolytic, and showed no or insignificant changes after treatment. The FLIM data correlate well with standard measurements of cellular drug response—cell viability and proliferation index and clinical response in patients. Therefore, FLIM of NAD(P)H provides a highly sensitive, label-free assay of treatment response directly on patient-derived glioblastoma cells and can become an innovative platform for individual drug screening for patients

Visualization of intracellular hydrogen peroxide with HyPer, a genetically encoded fluorescent probe

The fluorescent sensor HyPer allows monitoring of intracellular H O levels with a high degree of sensitivity and specificity. Here, we provide a detailed protocol of ratiometric imaging of HO produced by cells during phagocytosis, including instructions for experiments on different commercial confocal systems, namely, Leica SP2, Leica SP5, and Carl Zeiss LSM, as well as wide-field Leica 6000 microscope. The general experimental scheme is easily adaptable for imaging HO production by various cell types under a variety of conditions

FLIM of NAD(P)H in Lymphatic Nodes Resolves T-Cell Immune Response to the Tumor

Assessment of T-cell response to the tumor is important for diagnosis of the disease and monitoring of therapeutic efficacy. For this, new non-destructive label-free methods are required. Fluorescence lifetime imaging (FLIM) of metabolic coenzymes is a promising innovative technology for the assessment of the functional status of cells. The purpose of this work was to test whether FLIM can resolve metabolic alterations that accompany T-cell reactivation to the tumors. The study was carried out on C57Bl/6 FoxP3-EGFP mice bearing B16F0 melanoma. Autofluorescence of the immune cells in fresh lymphatic nodes (LNs) was investigated. It was found that fluorescence lifetime parameters of nicotinamide adenine dinucleotide (phosphate) NAD(P)H are sensitive to tumor development. Effector T-cells in the LNs displayed higher contribution of free NADH, the form associated with glycolysis, in all tumors and the presence of protein-bound NADPH, associated with biosynthetic processes, in the tumors of large size. Flow cytometry showed that the changes in the NADH fraction of the effector T-cells correlated with their activation, while changes in NADPH correlated with cell proliferation. In conclusion, FLIM of NAD(P)H in fresh lymphoid tissue is a powerful tool for assessing the immune response to tumor development