Tcs sp5ii laser scanning microscope

The presence of HMs in tissue of Silene leaves was demonstrated using the fluorescent indicator Phen Green SK diacetate salt (C₃₇H₂₁Cl₂N₃O₈, N-(6-Methoxy-8-Quinolyl)-p-Toluenesulfonamide, Life Technologies, USA). It is cell permeable and sensitive to a range of transition elements including Pb²⁺. The series of handmade leaf sections were stained in 50 μM fluorescent probe for 1 h at room temperature. As a control, unstained sections were used. After incubation, sections were washed three times by PBS buffer and observed under Leica TCS SP5II laser scanning microscope (Leica Microsystems CMS, Wetzlar, Germany). The excitation/emission range of 488/505–530 nm was chosen to visualize the presence of metals.

Overall accumulation of ROS was monitored microscopically using widely applied reagents—CellROX® Oxidative Stress Reagents (Life Technologies, USA). These cell-permeate dyes are non-fluorescent while in a reduced state and exhibit strong fluorogenic signal upon oxidation by ROS. The signals from CellROX® Deep Red and CellROX® Orange Reagents are localized in the cytoplasm, whereas from CellROX® Green Reagent in the nucleus and mitochondria. Stock solution of proper dye in DMSO was diluted by 0.1 M PBS buffer (pH = 6.9) to final concentration of 5 mM. The series of handmade leaf sections were stained in each dye for 30 min at 37 °C. As a control, unstained sections were used. After incubation, sections were washed three times by PBS buffer and observed under Leica TCS SP5II laser scanning microscope (Leica Microsystems CMS, Wetzlar, Germany). The excitation/emission range of 644/665–677 nm, 545/565–600 nm, and 485/510–530 nm were chosen for Red, Orange, and Green Reagents, respectively.

In situ labeling of glutathione (GSH) was carried out with monochlorobimane (MCB) as fluorescent marker. Stock solutions of 100 mM MCB (Molecular Probes, USA) were prepared in dimethyl sulfoxide (DMSO) and stored at − 20 °C. Aliquots were diluted in deionized water to a final concentration of 100 μM. In order to deplete ATP levels and thereby inhibit vacuolar sequestration of glutathione S-bimane conjugate (GSB), sodium azide (Sigma-Aldrich, Taufkirchen, Germany) was added to the dye solution at a final concentration of 5 mM. The series of handmade leaf sections were incubated for 15 min in the vacuum and then for 75 min at room temperature. As a control, unstained sections were used. After incubation, sections were washed three times by DMSO buffer and transferred to a drop of deionized water on a microscope slide. GSB fluorescence was imaged using Leica TCS SP5II laser scanning microscope (Leica Microsystems CMS, Wetzlar, Germany). The excitation/emission range of 405/522–555 nm was applied (Hartmann et al. 2003 (link)).