Oceanview

The optical measurements were based on reflectometry with a simple and compact setup, which consisted of a tungsten-halogen lamp light source (LS-1, Ocean Insight Tokyo, Japan), optical fiber (R400-7, UV-VIS, Ocean Insight) and a spectrometric part. In this study, two spectrometric setups were used: one was composed of a commercially available spectrometer (FLAME UV-VIS, Ocean Insight) and operation software (Ocean view, Ocean Insight) and the other of a slit, grating (Go spectro, KLV Co. Ltd., Tokyo, Japan), CMOS sensor equipped with a smartphone (iPhone 11, Apple Inc., Los Altos, CA, USA) and application software (Go spectro). In the smartphone-based spectroscopy, the color spectrum of LED light obtained by the CCD sensor was used to calibrate the wavelength in the measured color spectrum. The reflection spectra of the IPCF were measured and normalized to that of the IPCF after blocking treatment with EA, described as functionalized-IPCF (F-IPCF). The maximum reflection intensity was defined as R, and the label-free detection of the target molecules was based on the decrease in the reflection intensity, $\Delta R$ . The reflectometry was conducted in air after each incubation process.

A white-LED was used as the excitation source as previously described. For RRS experiments, light scattered at 90° was measured using a Spark-VIS spectral sensor (Ocean Optics, Inc.) whose entrance aperture was placed near the sample in a custom-made cuvette holder (Supplementary Fig. S8b). Spectra were averaged five times (integration time 500 ms), smoothed with a 30-point first-degree Savitzky-Golay filter and the background was substracted with OceanView (Ocean Optics, Inc.) before analysis. RRS ratios are reported as the mean ± standard deviation of three measurements.

Mitochondrial ROS emission from a permeabilized fiber bundle was determined using the Amplex Red reagent (Life Technologies, Carlsbad, Californa) (7, 22, 23). Permeabilized fibers were incubated in Buffer Z along with Amplex Ultra Red (AR) (Thermo Fisher Scientific, Waltham, MA, Cat# A36006). Hydrogen peroxide reacts with AR and horseradish peroxidase (HRP) to produce resorufin (excitation wavelength 563 nm, emission 587 nm), in a 1:1 ratio. Fluorescence was quantified using Maya LSL Spectrometer (Ocean Optics, Dunedin Florida) with the appropriate excitation source. Data was collected Ocean View (Ocean Optics, Dunedin, Florida) and analyzed using Pasco Capstone Software (Roseville, California). Baseline fluorescence (respiration buffer, permeabilized fibers, HRP, and AR) for 8 min and then H₂O₂ production was increased by the addition of succinate (final concentration of 10 mM). The slopes at baseline and succinate-stimulated were measured, converted to H₂O₂ concentrations based on H₂O₂-derived standard curve and corrected by dry tissue mass.

Floral colours were measured using an Ocean Optics spectrophotometer (Ocean Optics Inc., USB-4000+, USA) with a UV-VIS-NIR light source (Ocean Optics Inc., DH-2000-BAL, USA) and a quartz fiber-optic probe (Ocean Optics Inc., Lab-grade Reflection Probes, USA) relative to a 100% white standard and 0% black dark reference. The flower reflectance spectra were measured from 300 to 700 nm and then processed by the software OCEAN VIEW (Ocean Optics Inc., USA), and this range encompasses the visible spectrum of key pollinators. We sampled 3-5 flowers from each respective species from all of the sampling sites. All the flower colour data are available in Tai et al., 2020b (link) (Dryad database).

Radiation-induced changes in the irradiated PVA films enriched with silver nanoparticles with and without dye additives were characterized using UV-Vis spectra obtained using the spectrophotometer Ocean Optics with USB 4000 (Ocean Optics, Inc., Dunedin, FL, USA). Light source—HL-2000-LL by Ocean Insight—long lifetime tungsten halogen with wavelength range of 360 nm–2.4 µm and power of 4.75 W. Variations in the optical characteristics of the films were analyzed in the range between 350 and 900 nm using “Ocean View” spectroscopy software (version 1.6.7 by Ocean Optics).

To measure the absorbance spectrum of RTA-treated Au nanostructures, we placed the 2 cm × 2 cm substrate into a customized spectroscopy setup (Figure S1). The system consisted of (1) a broad-spectrum light source, connected to an illumination fiber (UV-VIS 600, Ocean Optics, Dunedin, New Zealand) to excite the Au nanostructures; (2) a spectrometer (HR-4000, Ocean Optics) connected to a detection fiber (UV-VIS 600, Ocean Optics) under the substrate to collect the transmitted light; (3) an X-Y axis motorized stage to move the substrate and (4) a peristaltic pump (BT100-1F, LongerPump, Hebei, China) to load samples into the microchannel (μ-Slide VI^0.4, Ibidi) attached to the Au nanostructures.
All spectral data was averaged across 20 spectrum scans with 10 milliseconds integration time using spectrum process software (OceanView, Ocean Optics). The peak intensity and wavelength of the absorbance spectrum were further analyzed by Origin 2015 software. Here, we used an 8-degree (x⁸) polynomial curve with R² > 0.98 to fit the absorbance spectrum (Figure S2). This fitting function allowed us to determine the peak wavelength and the quadratic coefficient, which corresponds to the full width at half maximum (FWHM). To quantify the surface morphology of the nanostructures, we used AFM (Multi-Mode 8, Bruker, Camarillo, CA, USA) to scan the substrate.

Floral colors were measured using an Ocean Optics spectrophotometer (Ocean Optics Inc., USB-4000+, United States) with a UV-VIS-NIR light source (Ocean Optics Inc., DH-2000-BAL, United States) and a quartz fiber-optic probe (Ocean Optics Inc., Lab-grade Reflection Probes, United States). The reflectance spectra were measured from 300 to 700 nm (see Supplementary Method S1) and then processed by the software OCEAN VIEW (Ocean Optics Inc., United States). Additional details for the measurement of reflectance spectra were given in Supplementary Method S1 and Supplementary Figure S1 for the details of marker point comparison to show the evidence of bees are the primary pollinator of flowering plants at low, middle, and high altitudes in Taiwan.