Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity

<p>Abstract</p> <p>Background</p> <p>Multiphoton microscopy (MPM) offers many advantages over conventional wide-field and confocal laser scanning microscopy (CLSM) for imaging biological samples such as 3D resolution of excitation, reduced phototoxicity, and deeper tissue imaging. However, adapting MPM for critical multi-color measurements presents a challenge because of the largely overlapping two-photon absorption (TPA) peaks of common biological fluorophores. Currently, most multi-color MPM relies on the absorbance at one intermediate wavelength of multiple dyes, which introduces problems such as decreased and unequal excitation efficiency across the set of dyes.</p> <p>Results</p> <p>Here we describe an MPM system incorporating two, independently controlled sources of two-photon excitation whose wavelengths are adjusted to maximally excite one dye while minimally exciting the other. We report increased signal-to-noise ratios and decreased false positive emission bleed-through using this novel multiple-excitation MPM (ME-MPM) compared to conventional single-excitation MPM (SE-MPM) in a variety of multi-color imaging applications.</p> <p>Conclusions</p> <p>Similar to the tremendous gain in popularity of CLSM after the introduction of multi-color imaging, we anticipate that the ME-MPM system will further increase the popularity of MPM. In addition, ME-MPM provides an excellent tool to more rapidly design and optimize pairs of fluorescence probes for multi-color two-photon imaging, such as CFP/YFP or GFP/DsRed for CLSM.</p

Rapid, broadband two-photon-excited fluorescence spectroscopy and its application to red-emitting secondary reference compounds

© 2011 Optical Society of AmericaThe definitive version of this paper is available at: http://dx.doi.org/10.1364/OME.1.000551DOI: 10.1364/OME.1.000551We present a methodology for rapidly acquiring broadband two-photon absorption (2PA) spectra by means of a compact two-channel femtosecond fluorescence excitation apparatus. This technique is insensitive to differences in excitation and collection geometries as well as detection efficiencies between the sample and reference, in addition to variations in average power, pulse duration and spatial beam profile, as it utilizes sequential measurement of the sample and reference in each of the two cell positions. Our approach eliminates the need to determine the fluorescence quantum yields of the sample and reference, as it allows measurement of emission from samples at a common specified wavelength. These attributes allow for acquisition of 2PA spectra with an estimated accuracy of ± 15% (limited almost exclusively by the uncertainty in the 2PA cross section for the reference standards) over an excitation range of 550-1600 nanometers with a typical time per spectrum of ~30-60 minutes. We have applied this technique to determine the 2PA spectra of six commercially available organic dyes over a wide range of excitation wavelengths (670-1600 nm), which can be used as secondary reference standards emitting in the red and near-infrared spectral region (600-1000 nm). We have also characterized some of these compounds using the femtosecond-pulsed Z-scan method and found very good agreement with the fluorescence-based measurements

Относительная высота деревьев в изолированных популяциях сосновых древостоев

Проведено исследование относительной высоты деревьев в сосняках брусничных, черничных и сфагновых на о-ве Большом Соловецком, самом значительном по площади в Соловецком архипелаге и в Белом море. Архипелаг является объектом Всемирного наследия ЮНЕСКО. Относительная высота – важный показатель, характеризующий рост древесных растений и отражающий, на сколько сантиметров прирастает высота на сантиметр диаметра. Заложено 34 пробных площади в наиболее распространенных типах леса – сосняках брусничных, черничных и сфагновых, занимающих 82,0 % от всей территории сосняков. На каждой пробной площади подобраны учетные деревья (по 64 дерева), у которых выполнены измерения высот, диаметров, взяты керны возрастным буравом у шейки корня для определения возраста. Всего в анализе относительной высоты использовано 2176 деревьев. Средняя относительная высота в сосняках брусничных, черничных и сфагновых на о-ве Б. Соловецком составляет соответственно 62,5; 61,5 и 54,7 см/см. Такой показатель заметно меньше, чем в сосняках на материке в Архангельской области. Там значения составляют 84,9; 84,9 и 79,2 см/см для соответствующих типов леса. Применять существующие таблицы хода роста, разработанные по материалам исследования материковых деревьев, для островных сосняков нельзя. Получены уравнения для определения относительной высоты сосняков о-ва Б. Соловецкого. В качестве входного параметра предлагается использовать диаметр деревьев, а не возраст, т. к. определение возраста значительно труднее, чем диаметра на высоте груди. Для цитирования: Соболев А.Н., Феклистов П.А., Неверов Н.А., Макаров С.С. Относительная высота деревьев в изолированных популяциях сосновых древостоев // Изв. вузов. Лесн. журн. 2023. № 6. С. 102–113. https://doi.org/10.37482/0536-1036-2023-6-102-11

Thermokarst Landscape Development Detected by Multiple-Geospatial Data in Churapcha, Eastern Siberia

Thermokarst is a typical process that indicates widespread permafrost degradation in yedoma landscapes. The Lena-Aldan interfluvial area in Central Yakutia in eastern Siberia is now facing extensive landscape changes with surface subsidence due to thermokarst development during the past few decades. To clarify the spatial extent and rate of subsidence, multiple spatial datasets, including GIS and remote sensing observations, were used to analyze the Churapcha rural locality, which has a typical yedoma landscape in Central Yakutia. Land cover classification maps for 1945 and 2009 provide basic information on anthropogenic disturbance to the natural landscape of boreal forest and dry grassland. Interferometric synthetic aperture radar (InSAR) with ALOS-2/PALSAR-2 data revealed activated surface subsidence of 2 cm/yr in the disturbed area, comprising mainly abandoned agricultural fields. Remote sensing with an unmanned aerial system also provided high-resolution information on polygonal relief formed by thermokarst development at a disused airfield where InSAR analysis exhibited extensive subsidence. It is worth noting that some historically deforested areas have likely recovered to the original landscape without further thermokarst development. Spatial information on historical land-use change is helpful because most areas with thermokarst development correspond to locations where land was used by humans in the past. Going forward, the integrated analysis of geospatial information will be essential for assessing permafrost degradation

Excited state absorption : a key phenomenon for the improvement of biphotonic based optical limiting at telecommunication wavelengths

International audienceSpectroscopic properties, two-photon absorption (TPA) and excited state absorption (ESA), of two organic cyanine dyes and of a ruthenium based organometallic cyanine are compared in order to rationalize their similar ns-optical power limiting (OPL) efficiency in the telecommunication wavelength range. The TPA contribution to the ns-OPL behavior is higher for both organic cyanines, while the main process is a TPA-induced ESA in the case of the organometallic system, in which the ruthenium induces a broadening of the NIR-ESA band and resulting in a strong spectral overlap between TPA and ESA spectra

Cysteine residues 244 and 458-459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions

Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines' thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na,K-ATPase and alter responses of the enzyme to hypoxia or upon treatment with cardiotonic steroids

Synthesis and properties of the heterospin (S1 = S2 = 1/2) radical-ion salt bis(mesitylene)molybdenum(I) [1,2,5]thiadiazolo[3,4-c][1,2,5]thiadiazolidyl

The authors are grateful to the Presidium of the Russian Academy of Sciences (Project 8.14), the Royal Society (RS International Joint Project 2010/R3), the Leverhulme Trust (Project IN-2012-094), the Siberian Branch of the Russian Academy of Sciences (Project 13), the Ministry of Education and Science of the Russian Federation (Project of Joint Laboratories of Siberian Branch of the Russian Academy of Sciences and National Research Universities), and the Russian Foundation for Basic Research (Projects 13-03-00072 and 15-03-03242) for financial support of various parts of this work. N.A.S. thanks the Council for Grants of the President of Russian Federation for postdoctoral scholarship (grant MK-4411.2015.3). B.E.B. is grateful for an EaStCHEM Hirst Academic Fellowship. A.V.Z. thanks the Foundation named after D. I. Mendeleev, Tomsk State University, for support of his work.Low-temperature interaction of [1,2,5]thiadiazolo[3,4-c][1,2,5]thiadiazole (1) with MoMes2 (Mes = mesitylene / 1,3,5-trimethylbenzene) in tetrahydrofuran gave the heterospin (S1 = S2 = 1/2) radical-ion salt [MoMes2]+[1]– (2) whose structure was confirmed by single-crystal X-ray diffraction (XRD). The structure revealed alternating layers of the cations and anions with the Mes ligands perpendicular, and the anions tilted by 45°, to the layer plane. At 300 K the effective magnetic moment of 2 is equal to 2.40 μB (theoretically expected 2.45 μB) and monotonically decreases with lowering of the temperature. In the temperature range 2−300 K, the molar magnetic susceptibility of 2 is well-described by the Curie-Weiss law with parameters C and θ equal to 0.78 cm3⋅K⋅mol–1 and −31.2 K, respectively. Overall, the magnetic behavior of 2 is similar to that of [CrTol2]+[1]– and [CrCp*2]+[1]–, i.e. changing the cation [MAr2]+ 3d atom M = Cr (Z = 24) with weak spin-orbit coupling (SOC) to a 4d atom M = Mo (Z = 42) with stronger SOC does not affect macroscopic magnetic properties of the salts. For the XRD structure of salt 2, parameters of the Heisenberg spin-Hamiltonian were calculated using the broken-symmetry DFT and CASSCF approaches, and the complex 3D magnetic structure with both the ferromagnetic (FM) and antiferromagnetic (AF) exchange interactions was revealed with the latter as dominating. Salt 2 is thermally unstable and slowly loses the Mes ligands upon storage at ambient temperature. Under the same reaction conditions, interaction of 1 with MoTol2 (Tol = toluene) proceeded with partial loss of the Tol ligands to afford diamagnetic product.PostprintPostprintPeer reviewe

All-ceramic or metal-ceramic tooth-supported fixed dental prostheses (FDPs)? A systematic review of the survival and complication rates. Part II: Multiple-unit FDPs

Objective To assess the 5-year survival of metal-ceramic and all-ceramic tooth-supported fixed dental prostheses (FDPs) and to describe the incidence of biological, technical and esthetic complications. Methods Medline (PubMed), Embase and Cochrane Central Register of Controlled Trials (CENTRAL) searches (2006-2013) were performed for clinical studies focusing on tooth-supported FDPs with a mean follow-up of at least 3 years. This was complemented by an additional hand search and the inclusion of 10 studies from a previous systematic review [1]. Survival and complication rates were analyzed using robust Poisson's regression models to obtain summary estimates of 5-year proportions. Results Forty studies reporting on 1796 metal-ceramic and 1110 all-ceramic FDPs fulfilled the inclusion criteria. Meta-analysis of the included studies indicated an estimated 5-year survival rate of metal-ceramic FDPs of 94.4% (95% CI: 91.2-96.5%). The estimated survival rate of reinforced glass ceramic FDPs was 89.1% (95% CI: 80.4-94.0%), the survival rate of glass-infiltrated alumina FDPs was 86.2% (95% CI: 69.3-94.2%) and the survival rate of densely sintered zirconia FDPs was 90.4% (95% CI: 84.8-94.0%) in 5 years of function. Even though the survival rate of all-ceramic FDPs was lower than for metal-ceramic FDPs, the differences did not reach statistical significance except for the glass-infiltrated alumina FDPs (p = 0.05). A significantly higher incidence of caries in abutment teeth was observed for densely sintered zirconia FDPs compared to metal-ceramic FDPs. Significantly more framework fractures were reported for reinforced glass ceramic FDPs (8.0%) and glass-infiltrated alumina FDPs (12.9%) compared to metal-ceramic FDPs (0.6%) and densely sintered zirconia FDPs (1.9%) in 5 years in function. However, the incidence of ceramic fractures and loss of retention was significantly (p = 0.018 and 0.028 respectively) higher for densely sintered zirconia FDPs compared to all other types of FDPs. Conclusions Survival rates of all types of all-ceramic FDPs were lower than those reported for metal-ceramic FDPs. The incidence of framework fractures was significantly higher for reinforced glass ceramic FDPs and infiltrated glass ceramic FDPs, and the incidence for ceramic fractures and loss of retention was significantly higher for densely sintered zirconia FDPs compared to metal-ceramic FDPs