Calcium Transients and Transmitter Secretion in Different Parts of Frog Nerve Endings in Different Conditions of Calcium Ion Influx

© 2020, Springer Science+Business Media, LLC, part of Springer Nature. Experiments on frog neuromuscular preparations were performed to study the characteristics of the calcium response and the quantum secretion of acetylcholine in different pats of extended nerve terminals in different conditions of calcium influx. A calcium-sensitive fluorescent dye was used to analyze Ca2+ influx (Ca2+ transients) into the proximal and distal parts of nerve endings in conditions of increased K+ ion content, in response to blockers of N- and L-type calcium channels, and on blockade of calcium-activated potassium channels. These studies showed that at a uniform distribution density of voltage-gated calcium channels along nerve endings, the proximal-to-distal decrement in calcium transients and quantum secretion intensity persisted in conditions of additional opening of voltage-gated calcium channels by potassium depolarization, on “thinning” of these channels using specific blockers, but changed on blockade of calcium-activated potassium channels

Enhancement of the Temporal Resolution of Fluorescent Signals Acquired by the Confocal Microscope

© Microscopy Society of America 2020. Here, we describe a method of acquisition of fast fluorescent signals with the help of the laser scanning confocal microscope (LSCM). Our method permits an increase in the temporal resolution of acquired signals. The method is based on LSCM recordings of fast fluorescent signals with the shortest achievable time sweep, which are performed with the help of a proprietary algorithm. A series of recordings is made in multiple steps; at each step, the fluorescent signal is incremented by a time interval smaller than the time sweep of the frame of LSCM. The size of the increment determines the achievable time resolution. The convolution of the recorded images results in a signal with the temporal resolution determined by the chosen time increment. This method was applied to register the change in fluorescence (calcium transient) of calcium dye preloaded into peripheral nerve endings by electrical stimulation of the motor nerve. Calculated parameters of the calcium transient were identical to the parameters obtained earlier with the help of a high-speed camera and photodiode. We conclude that the method described here can be applied for the registration of fast fluorescent signals by LSCM with a high spatial and temporal resolution

Structure impact on photodynamic therapy and cellular contrasting functions of colloids constructed from dimeric Au(I) complex and hexamolybdenum clusters

Electrostatically driven self-assembly of [Au2L2]2+ (L is cyclic PNNP ligand) with [{Mo6I8}(L')6]2− (L' = I−, CH3COO−) in aqueous solutions is introduced as facile route for combination of therapeutic and cellular contrasting functions within heterometallic colloids (Mo6-Au2). The nature of L' affects the size and aggregation behavior of crystalline Mo6-Au2 aggregates, which in turn affect the luminescence of the cluster units incorporated into Mo6-Au2 colloids. The spin trap facilitated electron spin resonance spectroscopy technique indicates that the level of ROS generated by Mo6-Au2 colloids is also affected by their size. Both (L' = I−, CH3COO−) Mo6-Au2 colloids undergo cell internalization, which is enhanced by their assembly with poly-DL-lysine (PL) for L' = CH3COO−, but remains unchanged for L' = I−. The colloids PL-Mo6-Au2 (L' = CH3COO−) are visualized as huge crystalline aggregates both outside and inside the cell cytoplasm by confocal microscopy imaging of the incubated cells, while the smaller sized (30–50 nm) PL-Mo6-Au2 (L' = I−) efficiently stain the cell nuclei. Quantitative colocalization analysis of PL-Mo6-Au2 (L' = CH3COO−) in lysosomal compartments points to the fast endo-lysosomal escape of the colloids followed by their intracellular aggregation. The cytotoxicity of PL-Mo6-Au2 differs from that of Mo6 and Au2 blocks, predominantly acting through apoptotic pathway. The photodynamic therapeutic effect of the PL-Mo6-Au2 colloids on the cancer cells correlates with their intracellular trafficking and aggregation

Mitochondria-targeted mesoporous silica nanoparticles noncovalently modified with triphenylphosphonium cation: Physicochemical characteristics, cytotoxicity and intracellular uptake

Novel nanocomposite system based on mesoporous silica nanoparticles (MSNs) noncovalently modified with hexadecyltriphenylphosphonium bromide (HTPPB) has been prepared, thoroughly characterized and used for encapsulation of model cargo Rhodamine B (RhB). The high encapsulation efficacy of this dye by HTPPB-modified mesoporous particles was demonstrated by spectrophotometry and thermography techniques. The bioavailability of MSN@HTPPB was testified. Cytotoxicity assay revealed that a marked suppression of M−HeLa cancer cells (epithelioid carcinoma of the cervix) occurs at concentration of 0.06 μg/mL, while the higher viability of Chang liver normal cell line was preserved in the concentration range of 0.98–0.06 μg/mL. Hemolysis assay demonstrated that only 2% of red blood cells are destructed at ~ 30 μg/mL concentration. This allows us to select the most harmless compositions based on MSN@HTPPB with minimal side effects toward normal cells and recommend them for the development of antitumor formulations. Fluorescence microscopy technique testified satisfactory penetration of HTPPB-modified carriers into M−HeLa cells. Importantly, modification of the MSN with HTPPB is shown to promote efficient delivery to mitochondria. To the best of our knowledge, it is one of the first successful examples of noncovalent surface modification of the MSNs with lipophilic phosphonium cation that improves targeted delivery of loads to mitochondria