Ultrafluar

An optical microscope (Carl-Zeiss Axio-10) equipped with ×5 (0.13 NA), ×10 (0.25 NA), ×20 (0.4 NA), ×50 (0.75 NA), and ×100 (0.85 NA) objective lens is used to obtain the reflection and transmission optical micrographs with different magnifications. The transmittance spectra under normal incidence were collected by an Olympus microscope (BX-51) with a spectrometer through a ×100 objective (MPlan-FLN, 0.9 NA). The extinction spectra were measured in transmission mode using a 2030 PV ultraviolet–visible–near-infrared range microspectrophotometer (CRAIC Technology Inc.) equipped with a xenon light source (80 W) and an optical objective (ZEISS Ultrafluar, ×10, 0.2 NA). Both the incident and collected light were normal to the quartz substrate, thus providing linearly polarized light excitation in plane with the surface of the nanostructures.

The tissue sections were placed without specific mounting medium on circular quartz slides for DUV imaging (diameter 12.7 mm, thickness 0.17 mm, ESCO optics, New Jersey, USA). A monochromatic beam was used at 280 nm through a 40X objective (Ultrafluar, Zeiss, Germany) and the recovery of the fluorescence was carried out between 300 and 540 nm with 0.5 nm spectral resolution. The spectrum was chosen until 530 nm to explore the characteristic NADH band^{19 (link)}. Single point exploration was performed to separately analyze fiber and connective tissue. A total of 230 spectra were acquired per region (fiber cytoplasm and connective tissue) and per animal, using 3 dogs per group (GR, GRMD, GRMDT). Spectral data were analyzed using The Unscrambler® X (CAMO Software Process AS) multivariate dataset. The spectra were grouped in a same matrix. Working matrices were created by gathering samples from the same dog groups. Six hundred ninety spectra per dog group were pre-processed according to the unit vector normalization method in order to be able to compare each spectrum one by one and perform Principal Component Analysis (PCA) on the variance between spectra. While the score plots allowed comparison of the DUV spectra, the corresponding loading plots revealed the main characteristic emission bands behind the clustering of the spectra.

This analysis was performed on four post-metamorphic juveniles with mean weight ± SD and total length ± SD of 3.6 ± 0.8 g and 7.5 ± 0.5 cm, respectively. Individual fish were dark adapted for 8 h, following which the fish was euthanized, and the retina extracted under infrared illumination. Small pieces of retina were teased apart and prepared for viewing with the dichroic microspectrophotometer (DMSP) as per previous studies^{42 (link)–44 (link)}. The DMSP is a computer-controlled, wavelength-scanning, single-beam photometer that simultaneously records average and polarized transmitted light fluxes through microscopic samples^{42 (link),44 (link)}. The DMSP was equipped with ultrafluar (Zeiss) objectives: 32/0.4 for the condenser and 100/1.20 for the objective. With the aid of reference measurements recorded through cell-free areas, individual photoreceptor outer segments were illuminated sideways with a measuring beam of rectangular cross section of ca. 2 × 0.6 μm. Absolute absorbance spectra were computed in 2 nm increments from the obtained transmittances (each spectrum consisted of an average of 8 scans). The solid spectra (fits) were derived from experimental data by Fourier filtering^{43 (link)}.