Dsm 960

At the end of the experimental period, images of cryo-fractured transversal peach tree leaf sections were obtained with a digital scanning electron microscope (SEM) (Zeiss DSM 960, Oberkochen, Germany) as described elsewhere (Ojeda-Barrios et al., 2012 (link)). Sections of fresh peach leaf tissue (2.5 × 2.5 mm leaf pieces) were mounted on aluminum stubs, cryo-fixed in slush N₂, cryo-transferred to a vacuum chamber at −180°C, and fractured using a stainless steel spike. Once inside the microscope, the samples underwent superficial etching under vacuum and were overlaid with gold. Fractured samples were observed at low temperature using secondary and back-scattered (BSE) electrons.
Semi-quantitative Fe analysis in the peach tree transversal leaf sections was carried out using microprobe analysis with an Energy Dispersive X-ray (EDX) system (Pentaflet, Oxford, UK), using only smooth surfaces (Hess et al., 1975 (link)). Semi-quantitative analysis was carried out using standard ZAF (atomic number, absorption and fluorescence) correction procedures with Link Isis (Oxford, UK) v.3.2 software. Eight points of analysis per leaf tissue and three leaves per treatment were analyzed.

Leaf pieces were mounted on aluminum stubs with adhesive (Gurr®, optimum cutting temperature control; BDH, Poole, UK), cryo-fixed in slush nitrogen (−196°C), cryo-transferred to a vacuum chamber at −180°C, and then fractured using a stainless steel spike. Once inside the microscope, the samples underwent superficial etching under vacuum (−90°C, 120 s, 2 kV), and then were overlaid with gold for observation and microanalysis. This freeze-fracture procedure leads to cell rupture only at the fracture plane, whereas the general internal leaf structure is well-preserved. Fractured samples were observed at low temperature with a digital scanning electron microscope (Zeiss DSM 960, Oberkochen, Germany) using secondary and back-scattered electrons. Secondary electron images (1024 × 960 pixels) were obtained at 133 eV operating at a 35° take-off angle, an accelerating voltage of 15 kV, a working distance of 25 mm and a specimen current of 1–5 nA. Microscopy was run in the Institute of Agricultural Sciences-CSIC (ICA-CSIC), Madrid, Spain.

Bagasse morphology was analyzed by SEM before and after undergoing pretreatments. Samples from surfaces or transversal sections (obtained by fracture in liquid N₂) were oven dried and coated with Au in a SCD 050 sputter coater (Oerlikon-Balzers, Balzers, Liechtenstein) Sample imaging was carried out using the scanning electron microscopes, models DSM 960 (Zeiss, Oberkochen, Germany) or JSM 5900LV (Jeol, Tokyo, Japan).
Sample features such as lumen diameters and cell wall thickness were manually measured using the program Axio Vision 4.8 (Carl Zeiss, Oberkochen, Germany). Averaged values of the lumen diameters were obtained by measuring about 350 lumens from different regions of raw bagasse and treated samples (four images by sample). Since most of the lumens have a distorted circumferential aspect, two diameters were measured: the maximum and the minimum axis, approximately perpendicular to each other, so that a mean diameter could be obtained for each lumen.

Specimens (from 3 week old mice) were fixed with 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.3 for 3 hours at room temperature and postfixed for two hours in 2% osmium tetroxide in 0.1 sodium phosphate buffer. After dehydration in graded ethanol, samples for scanning electron microscopy (SEM) were dried in a critical-point dryer (Polaron, Watford, UK), mounted on stubs, and coated with gold-palladium in a cool sputter coater (Fisons Instruments Uckfield, UK). The specimens were examined using a scanning electron microscope DSM 960 (Zeiss Oberkochen, Germany).