Confocal laser scanning microscope (CLSM)
To document the autofluorescence of the tooth material, two clean
radulae and some individual mature teeth were arranged on object glass
slides, following the procedure of Michels & Gorb (2012). Each radula
was surrounded by a stack of reinforcement rings. The rings were filled
with glycerin (greater than or equal to 99.5%, free of water, Carl Roth
GmbH & Co. KG, Karlsruhe, Germany) and subsequently covered by a glass
cover slip. Following the protocol of Krings et al. (2022d, 2023),
samples were visualized employing a Zeiss LSM 700 confocal laser
scanning microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Four
stable solid-state lasers with wavelengths of 405 nm, 488 nm, 555 nm,
and 639 nm were used. Bandpass or longpass emission filters (420–480
nm, greater than or equal to 490 nm, greater than or equal to 560 nm, or
greater than or equal to 640 nm) were applied. After scanning, images of
autofluorescence were superimposed (with maximum intensity projection)
using the software Zeiss Efficient Navigation (Zen) (Carl Zeiss
MicroImaging GmbH). Finally, the color blue was assigned to the
autofluorescence signal received from the laser with wavelength 405 nm,
green to 488 nm, red (50% saturation) to 555 nm and red (50%
saturation) to 639 nm.