Glioblastoma (GBM) is one of the most aggressive human cancers with a median survival of less than two years. A distinguishing pathological feature of GBM is a high degree of inter- and intratumoral heterogeneity. Intertumoral heterogeneity of GBM has been extensively investigated on genomic, methylomic, transcriptomic, proteomic and metabolomics levels, however only a few studies describe intratumoral heterogeneity because of the lack of methods allowing to analyze GBM samples with high spatial resolution. Here, we applied TOF-SIMS (Time-of-flight secondary ion mass spectrometry) for the analysis of single cells and clinical samples such as paraffin and frozen tumor sections obtained from 57 patients. We developed a technique that allows us to simultaneously detect the distribution of proteins and metabolites in glioma tissue with 800 nm spatial resolution. Our results demonstrate that according to TOF-SIMS data glioma samples can be subdivided into clinically relevant groups and distinguished from the normal brain tissue. In addition, TOF-SIMS was able to elucidate differences between morphologically distinct regions of GBM within the same tumor. By staining GBM sections with gold-conjugated antibodies against Caveolin-1 we could visualize border between zones of necrotic and cellular tumor and subdivide glioma samples into groups characterized by different survival of the patients. Finally, we demonstrated that GBM contains cells that are characterized by high levels of Caveolin-1 protein and cholesterol. This population may partly represent a glioma stem cells. Collectively, our results show that the technique described here allows to analyze glioma tissues with a spatial resolution beyond reach of most of other omics approaches and the obtained data may be used to predict clinical behavior of the tumor.

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Relaxation processes in a 4 dimethylaminochalcone molecule after excitation with a light pulse of duration 70 fs were studied. During 0.4-1 ps after excitation, an absorbance of an excited state S 1 with a maximum at ~460 nm is formed in both polar and nonpolar media. Subsequent relaxation processes depend on the polarity of the medium. In nonpolar hexane, the 4 dimethylaminochalcone molecule transits to the triplet state having an absorption maxi mum at 570 nm (lifetime longer than 600 ps) for 20 ps. In polar aprotic acetonitrile, the absorbance at 460 nm decreases slowly (during hundreds of picoseconds), indicating that the molecules return to the ground state. The induced emission from the level S 1 in a region of 520-550 nm and fluorescence from the same level with a maximum at 537 nm are also observed in acetonitrile. Thus, a reason for a sharp decrease in the fluorescence yield on going from polar to nonpolar media was found. The mechanism of fluorescence quenching of 4 dimethylaminochalcone in nonpolar media is confirmed by the data on phosphorescence. The phosphorescence of 4 dimethylaminochalcone is observed at -196 °C in nonpolar sol vents, indicating a triplet excited state, while no phosphorescence is revealed in polar solvents.Key words: fluorescent probe, 4 dimethylaminochalcone, femtosecond dynamics, polarity of medium, triplet state.4 Dimethylaminochalcone (DMAC) as a fluorescent probe is used for studying biological membranes and lipo proteins. 1-6 It is poorly soluble in water and, hence, binds to lipids in the presence of mem branes or lipoproteins. 1 In polar solvents, the quantum yield of fluores cence can reach 0.26; however, the fluorescence of DMAC in water is almost completely quenched due to the forma tion of a hydrogen bond (other protic solvents quench DMAC flluorescence in the same manner). 3,4 When bind ing to proteins or lipids, a DMAC molecule is shielded from water, and the fluorescence yield can increase to 0.1 and higher. However, if the molecule has "drown" in the lipid depth and got into a nonpolar environment, the yield decreases to 0.01 and lower. Thus, it seems clearly why polar protic solvents quench fluorescence, while rea sons for fluorescence quenching in nonpolar media are unknown. In this work, we attempted to elucidate this problem. Experimental4 Dimethylaminochalcone was synthesized and kindly pre sented by B. M. Krasovitskii (Institute for Single Crystals, Na tional Academy of Sciences, Kharkov, Ukraine). 7 Acetonitrile, 95% EtOH, methylcyclohexane, and hexane (Aldrich) were used.The femtosecond dynamics of transient absorption spectra of DMAC was studied on an experimental system based on a femtosecond CPM dye laser designed at the N. N. Semenov Institute of Chemical Physics of the Russian Academy of Sci ences. 8 The "pump-probe" spectroscopy technique was used. The second harmonic from the main sequence of pulses gener ated by the system was used as pump pulses. The bearing wave length of a pump pulse was 308 nm, and the duration was 70 fs. Probe pulses were carr...

Quantum-chemical calculations of ground and excited states for membrane fluorescent probe 4-dimethylaminochalcone (DMAC) in vacuum were performed. Optimized geometries and dipole moments for lowest-lying singlet and triplet states were obtained. The nature of these electronic transitions and the relaxation path in the excited states were determined; changes in geometry and charge distribution were assessed. It was shown that in vacuum the lowest existed level is of (n, π*) nature, and the closest to it is the level of (π, π*) nature; the energy gap between them is narrow. This led to an effective (1)(π, π*) →(1)(n, π*) relaxation. After photoexcitation the molecule undergoes significant transformations, including changes in bond orders, pyramidalization angle of the dimethylamino group, and planarity of the molecule. Its dipole moment rises from 5.5 Debye in the ground state to 17.1 Debye in the (1)(π, π*) state, and then falls to 2 Debye in the (1)(n, π*) state. The excited (1)(n, π*) state is a short living state; it has a high probability of intersystem crossing into the (3)(π, π*) triplet state. This relaxation path explains the low quantum yield of DMAC fluorescence in non-polar media. It is possible that (3)(π, π*) is responsible for observed DMAC phosphorescence.

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