2 mm sections of the recently fully expanded mid-region of leaves 4 and 7 were fixed (1 h) in 0.5% glutaraldehyde in 0.1 M sodium cacodylate buffer and incubated (3 h) in 0.2 M disodium EDTA, pH 9.0 in a water bath at 55°C. After incubation, samples were rinsed (20 min) in water and subsequently digestion buffer (20 min; 0.15 M sodium hydrogen phosphate, 0.04 M citric acid, pH 5.3). Leaf tissues were then incubated (1 h) at 45°C in 2% pectinase in digestion buffer and rinsed (30 min) in digestion buffer. Bundle sheath cells could be distinguished from mesophyll cells by an elongated shape (Figures 2 B and 2D). Isolated bundle sheath cells were viewed with Nomarski optics and bundle sheath cell volume and chloroplast size were quantified with ImageJ [58 (link)] from images captured on a Zeiss Axioplan equipped with Olympus cellSens imaging software.
Axioplan
Manufactured by Olympus
The Axioplan is a high-performance optical microscope designed for advanced laboratory applications. It features a robust and ergonomic design, providing a stable platform for precise observations and analysis. The Axioplan's core function is to enable high-quality imaging and examination of samples across a wide range of scientific disciplines.
Automatically generated - may contain errors
3 protocols using axioplan
1
Isolation and Characterization of Bundle Sheath Cells
2
Petrographic Microscopy of Polished Core Samples
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Transmitted light microscopy of polished thin sections from the core samples listed in table S2 was conducted on Zeiss Axioplan and Olympus BX41 petrographic microscopes. Image capture for the Zeiss Axioplan used a Nikon DS-Fi1 camera (2/3 inch, 5.24 megapixel CCD, 2560 × 1920 recording pixels) with Nikon NIS-Elements F3.0 software. Image capture for the Olympus BX41 used an Olympus DP25 camera (2/3 inch, 5.24 megapixel CCD, 2560 × 1920 recording pixels) with cell^B v2.8 software.
3
Phytoplankton Quantification and Identification
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Samples for qualitative and quantitative phytoplankton analyses were taken from the subsurface layer (about 50 cm depth) in each lake. Living specimens were examined at the Antarctic Esperanza station using light microscopy for their identification based on morphology and consulting specialized taxonomical literature for each algal group (e.g. Ettl 1983; Komárek and Fott 1983; Starmach 1985; Komárek and Anagnostidis 1999; Komárek and Anagnostidis 2005) . Samples for quantitative analyses were fixed with 1% acidified Lugol's iodine solution, and counts were performed with an inverted microscope following the Utermöhl (1958) method. After 1998, samples for the quantification of pico and nanoplankton by epifluorescence were also collected and fixed with filtered 10% cold glutaraldehyde (1% final concentration) and immediately filtered through 0.2 and 0.6-m pore-size polycarbonate filters respectively. Counts were carried out in Zeiss Axioplan or Olympus BX40 microscopes following the procedure described in previous publications (e.g. Allende & Pizarro 2006; Izaguirre et al. 2016) . Specific biovolumes were calculated using appropriate geometric formulae according to Hillebrand et al. (1999) .
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