Hc pl apo 20 0.75 imm corr cs2 objective

Three-month-old greenhouse- or in vitro-grown tree stems were used for this analysis. Basal cross sections were harvested and immediately fixed and stored in formaldehyde/acetic acid until use. Samples were thoroughly washed and mounted in water before RAMAN spectroscopy. Greenhouse-grown tree samples were hand sectioned while in vitro-grown trees were previously encased with 5% agarose to facilitate multiple samples sectioning with a Leica VT1200S vibratome. Raman microscopy was carried out on a Renishaw InVia instrument fitted with a 532 nm diode laser. Stem sections were mounted in water with a coverslip carefully placed on top and sealed with vacuum grease. A Leica HC PL APO ×20/0.75 IMM CORR CS2 objective was used with the collar set to water immersion. Raman acquisitions used a 2,400 l mm⁻¹ grating, 1,400 cm⁻¹ centre, 50% laser power (<15 mW at sample plane), pinhole engaged, regular confocal mode, 20 s pre-acquisition bleach and 0.3 s exposure with 15 accumulations. A total of 24 secondary walls were sampled for each section and then averaged using the ‘average collected datasets’ tool of Renishaw WiRE software v.4 to improve signal-to-noise ratio.

Microscopic observation was carried out with a Leica MZ 16F fluorescence binocular or a Leica TCS SP8 confocal laser scanning microscope using an HC PL APO 20 ×/0.75 IMM CORR CS2 objective. The excitation and emission of the different fluorophores were performed as described before (Westermann et al., 2020 (link)).