Using a micropipette, cells of Nematodinium sp. and P. kofoidii were individually transferred into a droplet of filtered seawater. Cells were frozen immediately to minimize fixation artifacts, using a Leica EM HPM100 high-pressure freezer (Leica). Subsequently, freeze substitution was used to remove the aqueous content of the cells and replace it with an acetone solution containing 5% water, 1% osmium tetroxide, and 0.1% uranyl acetate, at −80°C for 48 hours, −20°C for 6 hours, then graded back to 4°C for 13 hours. The prepared samples were washed twice in 100% acetone. Two cells were recovered by micropipette. Each cell was placed on a separate ThermoNox coverslip, where it adhered to a patch of poly-l -lysine. In preparation for FIB-SEM, cells were infiltrated with a 1:1 mix of acetone and Embed 812 resin for 2 hours, then 100% resin overnight. A second ThermoNox coverslip was applied, sandwiching each cell in a thin layer of resin between the coverslips. Resin was polymerized at 65°C for 24 hours. Afterward, the top coverslip was removed with a razorblade to expose the resin face overlying the cell.
Leica em hpm100
Manufactured by Leica camera
The Leica EM HPM100 is a high-performance cryo-ultramicrotome designed for sectioning frozen samples for electron microscopy. It features a precision-engineered cutting system and a temperature-controlled environment to ensure the integrity of the sample during the sectioning process.
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3 protocols using leica em hpm100
1
High-Pressure Freezing and Freeze Substitution of Nematodinium and Polykrikos Cells
2
High-Pressure Freezing for Ultrastructural Analysis
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C6 and C14 culture (100 ml) were collected at 1,000 × g using a Stat Spin Microprep 2 table top centrifuge. Cells were transferred to aluminium platelets (150 µm depth) containing 1-hexadecene94 . Platelets were frozen using a Leica EM HPM100 high-pressure freezer (Leica). Frozen samples were transferred to a Leica EM AFS2 automatic freeze substitution unit and substituted at −90 °C in a solution containing anhydrous acetone and 0.1% tannic acid for 24 h, and in anhydrous acetone, 2% OsO4 and 0.5% anhydrous glutaraldehyde (Electron Microscopical Science) for a further 8 h. After further incubation over 20 h at −20 °C, samples were warmed to +4 °C and subsequently washed with anhydrous acetone. Samples were embedded at room temperature in Agar 100 (Epon 812 equivalent) at 60 °C for 24 h. Thin sections (80 nm) were counterstained using Reynolds lead citrate solution for 7 s and examined using a Talos L120C microscope (Thermo Fisher).
3
Ultrastructural Analysis of Fungal Hyphae and Conidia
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Hyphae and conidia from PDA cultures grown for 7 days were used for TEM observations. Samples were frozen in a high-pressure freezer (Leica EM HPM100; Leica). Fast-frozen samples were then immersed into a freezing tube containing osmic acid (2%) and placed into the freeze substitution device (Leica EM AFS; Leica) at –90°C for 3 days. They were then slowly warmed to 4°C. Following freezing substitution, samples were rinsed four times with 100% acetone at room temperature. Dehydrated specimens were slowly infiltrated with SPI Pon 812 resin by placing them in mixtures of acetone and resin of different grades (25, 50, 75, and 100% [v/v]). The liquid resin was then polymerized at 60°C for 48 h. Ultrathin sections were cut using an ultramicrotome (Leica EM UC6; Leica) equipped with a diamond knife and placed on a TEM grid. The emulsion was observed using TEM (FEI Tacnai Spirit) at 100 kV. Four cultures of each strain were observed.
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