Flame s vis nir es spectrometer

Excitation-emission spectra of the urine samples were recorded using a FLAME-S-VIS-NIR-ES spectrometer (Ocean Optics, Largo, FL, USA) with a light source LLS-365 and an INSTMA-20 slit (slit width: 200 µm). One mL urine was transferred into a quartz cuvette with a 1 cm path length (Carl Zeiss, Oberkochen, Germany). The reflection probe (QR400-7-VIS-BX, premium 400 µm, VIS/NIR) (Ocean Optics, Largo, FL, USA) was positioned against the cuvette and a black background was used. The OceanView program (Ocean Optics, Largo, FL, USA) was set with an integration time of 10 ms and an average of 128 scans. Using an excitation wavelength of 365 nm, the fluorescence spectra of urinary AGEs were recorded at a 400–620 nm emission range. After background correction, the fluorescence signal of each urine sample was measured. Normalized fluorescence spectra were prepared by dividing the relative fluorescence intensity at each wavelength by the (maximum) relative fluorescence intensity at the (corresponding) peak wavelength. As the urinary concentration of AGEs depends on the urine volume, the relative fluorescence intensity (expressed in arbitrary units) was adjusted for the urinary creatinine concentration.

We measured the spectral reflectance of the eyes of A. irradians using a modified Olympus CX-31 microscope (Center Valley, PA, USA). We replaced the right eyepiece of the microscope with a custom-made adapter to which we attached a Y-shaped reflection probe (QR400-7-UV-VIS; Ocean Optics, Dunedin, FL, USA) that supplied light from a 20 W tungsten halogen lamp with an emission range of 360–2400 nm (HL-2000-HP-FHSA; Ocean Optics) and carried reflected light back to a Flame-S-VIS-NIR-ES spectrometer (Ocean Optics) that we operated using Ocean View software (Ocean Optics). We focused light onto samples using an Olympus 10X PlanC N UIS2 objective. To standardize our measurements, we used a reflectance standard made of Spectralon (WS-1-SL; Ocean Optics). Because fixing the eyes of scallops appears to alter their colour slightly, we measured the spectral reflectance of fresh, unfixed eyes. We compared our reflectance measurements from the eyes of scallops with measurements taken from the unpigmented mantle tissue immediately adjacent to the eyes.