Fn s2n

A JSM-7401F field emission scanning electron microscope was used to image LNPs and LNPs photonic crystal samples. An accelerating voltage of 1–2 kV and a working distance of 2–15 mm were used during measurement. Some samples were coated for 60–120 s with gold using a JFC-1200 fine coater before the SEM study. The gold particles added by sputtering are about 5–15 nm in size and they were added to increase the contrast in the images. Annular dark field (ADF) scanning transmission electron microscopy (STEM) images were obtained using a ThermoFisher Themis Z double aberration-corrected TEM operated at 300 kV with a convergence angle of 21 mrad and a dwell time of 3 µs. The material was sectioned to 200 nm thickness using ultramicrotomy using a Leica Ultracut UCT with a 45° diamond knife (Diatome) after the crystalline lignin nanoparticles first had been embedded in Agar low viscosity resin to facilitate sectioning.
A Nikon FN-S2N (Japan) microscope was used to image the rectangular platelets of LNPs and record a movie of the assembly and rearrangement of LNPs during evaporation. A Dino-Lite Edge 3.0 digital microscope was used to image the photonic crystals of LNPs.

A male moth was restrained in a plastic pipette tip with only the head protruding from the aperture, and a tungsten wire serving as a reference electrode was inserted into the abdomen. Single sensillum recordings (SSRs) were performed under a light microscope (Nikon FN-S2N) with 750x magnification, using tungsten electrodes (Clark Instruments Ltd). The recording electrode was attached to an AC/DC 10× gain probe (INR-02; Syntech), and its tip was inserted at the base of a pheromone-sensitive long trichoid sensillum using a micromanipulator (Märzhauser PM-10) until extracellular electrical contact with olfactory sensory neurons (OSNs) was established. The signal was amplified, digitized (IDAC-4 USB; Syntech) and visualized with AutoSpike 3.7 software (Syntech). A stream of charcoal filtered humidified air was continuously flushed over the antenna (1 L.min⁻¹), through a glass tube (1.0 mm i.d.), which terminated 2.0 cm from the antenna. During stimulation, a 0.5-s air pulse (1.0 L min⁻¹) controlled by a stimulus controller (CS-55; Syntech) was passed through the stimulus pipette, which was inserted into a hole in the glass tube. Compounds were tested with an inter-stimulus interval of at least 1 min. The response of OSNs was expressed as the number of spikes during the stimulation period after stimulus onset minus the number of spikes before stimulus onset.

One day before the experiment, D-luciferin potassium salt (Wako) was added to the culture medium (final concentration, 0.1 mM). On the day of the experiment, cultured cortical slices were placed in an incubation chamber (Tokai Hit UK-A16U, 35°C, 5% CO₂) on a microscope stage. After identifying DsRed2- or EGFP-positive cells, the luciferase signals were captured by an EMCCD camera (Andor, iXon3) attached to an upright microscope (Nikon, FN-S2N) through a 20x objective lens (NA, 0.5). The signal to noise ratio was increased by 4 × 4 binning, gain 1,000, and 5- or 10- min exposure.
After confirming that the luciferase signals were stable on the microscope stage, the recording was started. The signals were recorded for 3 h before pharmacological, electrophysiological, and optogenetic stimulation, and the slices were further observed for more than 12 h. The luciferase signals were taken at 30-min intervals.