W plan apochromat 63 1

Light from an 808 nm laser diode was coupled into the microscope optical train (Olympus BXFM) through a customized port and then directed and focused onto the sample surface along with the imaging light with a customized multi-bandpass filter cube and a × 63 water immersion dipping objective (Zeiss W-Plan Apochromat × 63/1.0). An additional filter placed before the camera port protected the camera sensors (Hamamatsu EM-CCD) from the laser light and allowed simultaneous sample illumination with the NIR light and low-signal level sample imaging. In a typical actuation experiment, a HAIRS-xNR sample was placed in a Petri dish filled with water or an aqueous buffer and mounted on the stage of the set-up. In this experimental configuration, the laser light could be focused down to a 10–40 μm diameter area on the sample surface.

Confocal Raman microspectroscopy analyses were performed on digestive gland and gill tissues of mussels exposed for 21 days to TiO₂ NMs using an Alpha300 R microscope (WITec) equipped with a 532‐nm laser source, a 600‐g/mm grating, and a charge‐coupled device cooled down to −61 °C. All measurements were conducted using a 63× water immersion objective (W Plan‐Apochromat 63×/1.0; Zeiss). Paraffin‐embedded tissue samples of digestive glands were cut in 5–10‐µm‐thick sections and mounted onto glass slides. After deparaffinization, water‐mounted mussel tissue was scanned with a laser power of approximately 35 mW at 532 nm. Raman spectra were collected pixel‐wise with an integration time of approximately 0.07 s. Acquired spectra were processed using the Project FOUR PLUS software (WITec).