Fluorodish

All experiments were performed on myelinated nerve fibers of frog Rana temporaria. All experiments on animals (we used 70 animals aged twelve months from the vivarium of the Faculty of Biology, M.V. Lomonosov Moscow State University) were carried out in accordance with the animal care regulations of the M.V. Lomonosov Moscow State University. The protocol was approved by the Bioethics Committee of the Faculty of Biology, M.V. Lomonosov Moscow State University. Prior to experiments, male frogs were maintained in containers with some water at 5°C without feeding. Animals were anesthetized using solution of propofol (50 mg/kg) administered intracelomically, which ensured the deep anesthesia condition prior to euthanasia. Anesthetized animals were decapitated with the following pithing of the brain.
Nerves were dissected and then incubated in Ringer buffer solution (RBS) consisting of 100 mM NaCl, 2 mM KCl, 1.08 mM CaCl₂, and 10 mM HEPES (pH 7.4) for at least 30 min. Single nerve fibers were obtained by splitting each nerve. Experiments were carried out at room temperature (20–22°C). Nerve fibers were placed on a glass bottom of a FluoroDish (World Precision Instruments, USA) filled with RBS and then covered by a glass coverslip [16 ].

Cumulus-free oocytes were incubated at 38.5 °C for 30 min in PZM-3 medium containing 1 µM Fura-2 AM and 0.02% pluronic F-127 to load the Ca²⁺ probe. Oocytes were then transferred into a PZM-3 drop in Fluoro dish (FD35–100, World Precision Instruments), covered with mineral oil, and observed at 38.5 °C with a Leica DMI6000 inverted microscope. The fluorescence was excited using a Fura 2 fluorescence module, and the F340/380 ratio was calculated using a Leica LAS-AF calcium imaging module. The oocytes were monitored for 5 min to record the baseline F340/380 ratio, and then, ionomycin was added to the PZM-3 drop to give a final concentration of 50 µM. After ionomycin addition, the oocytes were monitored for 20 min to record the peak F340/380 ratio. While the baseline F340/380 ratio represented the cytoplasmic calcium, the difference between the peak and baseline F340/380 ratios represented the calcium stores of an oocyte.

Wild type 72 hpf embryos were incubated with 500 µg/mL of DiR-loaded and TopFluor-PC C-NEs and embryo media (as a control condition) during 4 h at 34 °C, in 24-well plates. Afterwards, the embryos were washed with PBS and fixed with formaldehyde overnight at 4 ºC. Later, they were washed again with PBS and maintained at 4 ºC before visualization. For sample preparation, a Fluorodish (World Precision Instruments, Sarasota, FL, USA) was covered with a layer of agar gel (1% w/v in distilled water) and the zebrafish embryos were placed on top of it. The embryos were observed using a Confocal Microscope Leica TCS SP8 with a HC PL Apo 10x/0.4 objective, and scanned every 10 µm acquiring a total of 27 planes in z-axis direction with a 7.5 × magnification. Images were analyzed using Leica Application Suite X software.

Fluorescent microscopy uptake studies were conducted using FITC-labeled, AS1411-conjugated nanodroplets. Images were acquired using an EVOS FL digital fluorescence microscope (Advanced Microscopy Group, Mill Creek, WA, USA). Human MDA-MB-231 breast cancer cells were plated for 48 hr at a density of 4,000 cells/cm² in glass cell culture dishes (FluoroDish, World Precision Instruments, Sarasota, FL USA). AS1411-conjugated fluorescent nanodroplet emulsions were added to cells at various doses (4%, 2%, 1%, 0.4%, 0.2% and 0% v/v PFP) and incubated for various amounts of time (0, 1, 4, 24, 48, and 72 hr) at 0.4% v/v PFP. Slides were washed with HBSS, fixed with 3.5% paraformaldehyde, stained with 0.05% Hoechst 33342 for 5 minutes at room temperature to detect nuclei, washed twice, and mounted (ClearMount, Invitrogen, Frederick, MD, USA) for at least 3 hours prior to imaging. All images were acquired with identical microscope settings (60% brightness for FITC and 10% brightness for Hoechst). Fluorescence intensity of FITC in cells was quantified using ImageJ.

Live cell microscopy experiments were undertaken according to the method of Weiss et al. [38 (link)]. Briefly, highly synchronous late-stage schizonts were diluted 1:25 in media ± drug treatment and allowed to settle to produce a monolayer onto a 35 mm FluoroDish (World Precision Instruments, Sarasota, FL, USA). At 37 °C on a Zeiss Axio Observer.Z1 fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with humidified gas chamber (90 % N₂, 5 % O₂ and 5 % CO₂), selected schizonts were observed until they ruptured and released their merozoites. Time-lapse videos of the invading merozoites were recorded with a high-resolution AxioCam MRm camera (Zeiss). ImageJ was used to perform image analysis and GraphPad Prism used to perform statistical analyses using a Chi-squared test.