Tcs sp2 fluorescence microscope

Skin penetration of Rhodamine 123 was assessed by confocal laser scanning microscopy (CLSM) at Osmania University, Hyderabad, after application to Goat skin in-vitro of systems containing probe and nanoemulsion containing, 0.03% rhodamine, were used for the experiments. Nanoemulsion gel was applied homogeneously and nonocclusively to skin. The experiments were run in Franz diffusion cells and the receiver contained phosphate buffer pH 5.5 solution. After 24 h, the skin was removed and washed with phosphate buffer. The skin was then rapidly frozen by liquid nitrogen and a skin surface perpendicular rectangular piece was taken from the site of drug application with the help of a sharp blade. This tissue was fixed on the sample holder with the help of a Tissue frozen medium gel. The skin perpendicular sections (dermis to horny layer) of (250 μm) full thickness were cut with the help of cryomicrotome (Gung, Leica, Germany). The treated area was cut out and tested for probe penetration. The full skin thickness was optically scanned at 15–30 nm increments through the Z-axis of a Leica DMIRE2 confocal laser-scanning microscope (CLSM; Germany) attached to a Leica TCS SP2 fluorescence microscope. Optical excitation was carried out with a 488 nm argon laser-beam and fluorescence emission was detected above 555 nm.

Fluorescein isothiocyanate (FITC) labeling of mature normal RBCs (nRBCs) was performed as described previously.^{19 (link)} The FITC-labeled nRBCs (FITC-nRBCs) were cultured in P. falciparum complete culture medium before they were used in FITC-Dextran transfer assays. For light and fluorescence microscopy, parasite-infected RBCs were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) in PBS and were examined at the indicated time points using a Leica DM2000 fluorescence microscope (Leica, Bensheim, Germany). For indirect immunofluorescence assay, thin blood smears were incubated with a mixture of anti-Ago2 mouse monoclonal antibody (ab 57113, Abcam, Cambriage, MA, USA) and anti-parasite cytoplasm rat monoclonal antibody 5H2C6V (maintained in our lab). Normal mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used as a negative control. FITC-conjugated goat anti-mouse IgG and AlexaFluor 594-conjugated donkey anti-rat IgG were used as secondary antibodies. The slides were mounted with mounting medium containing DAPI (ZSGB-bio, Beijing, China) and were analyzed using a confocal Leica TCS-SP2 fluorescence microscope (Leica). The transfer potential of FITC-Dextran directly toward RBCs was evaluated with 5 × 10⁷ nRBCs cultured in medium containing 0.5 mg/mL FITC-Dextran for 24 or 72 h and then analyzed by flow cytometry (Supplementary Figure S1).

X-ray diffraction (XRD) pattern was recorded on a Bruker AXN X-ray diffractometer using Cu Kα radiation (λ = 1.5418 Å). Images of scanning electron microscopy (SEM) were obtained using a JEOL 6400 microscope set to 20 kV. Fourier transform infrared (FTIR) spectra were obtained using the KBr pellet technique on a JASCO FTIR 4100 apparatus at 5000 psi hydraulic pressure. Field emission transmission electron microscopy (FETEM, JEOL 2010) images were obtained at an accelerating voltage of 200 kV. A Nova 4000 e surface area analyzer was used to perform N₂ adsorption-desorption studies. The Barrett–Joyner–Halenda (BJH) technique was used to calculate the pore size from the adsorption branch. On a Malvern Zetasizer Nano-ZS (Malvern Instruments), the surface charge and particle size in suspension were measured. Thermogravimetric analysis (TGA) was performed on a Perkin-Elmer Pyris Diamond thermogravimetric analyzer with a heating rate of 10 °C/min under N₂ atmosphere. Images of fluorescence microscopy were captured using a Leica (TCS-SP2) fluorescence microscope.