The surfaces of five dechorinated and five hexane-washed eggs of Canton S were examined by cryo-scanning electron microscopy (S7A–S7D Fig ). Similarly, five dechorinated eggs of each oe mutant cross were also examined by environmental scanning electron microscopy (S7E–S7H Fig ). For cryo-scanning electron microscopy, we used a Quorum system PP3010T attached to a Helios 650 (FEI Company, Eindhoven, The Netherlands). The eggs were mounted on aluminum stubs using a mixture of Tissue-Tek (Sakura Finetek Europe, Alphen aan den Rijn, The Netherlands) and colloidal graphite (Agar Scientific, Stansted, Essex, UK), frozen in nitrogen slush at −210°C and then transferred to the preparation chamber of the Quorum system. The sample was freeze-dried at −80°C for 10 min and then sputter coated with platinum at 10 mA for 25 s. After transfer on the cryostage at −140°C in the scanning electron microscope, imaging was performed at 5 keV using an Everhart-Thornley electron detector [70 (link)]. Some experiments were done under low-vacuum 300–400 Pa conditions. Fresh eggs were directly mounted on a scanning electron microscopy stub and imaged with the low-vacuum large field detector in the Quanta 250.
Colloidal graphite
Manufactured by Agar Scientific
Sourced in Netherlands
Colloidal graphite is a dispersion of graphite particles in a liquid medium, typically water or alcohol. It is a conductive and lubricating material used in various industrial and laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Helios 650, by Thermo Fisher Scientific (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Pp2000 t, by Quorum Technologies (1 mentions)
Quanta 250 feg field emission scanning electron microscope, by Thermo Fisher Scientific (1 mentions)
Tissue tek, by Agar Scientific (1 mentions)
5600lv scanning electron microscope, by JEOL (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
5 protocols using colloidal graphite
1
Scanning Electron Microscopy of Drosophila Eggs
2
Cryo-SEM Imaging of Leaf Samples
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Small leaf samples of about 5 mm × 5 mm were excised from freshly harvested leaves and immediately fixed on a plane cryo transfer shuttle with conductive mounting medium (1:1 mix of Tissue-Tek and colloidal graphite, Agar Scientific Ltd., Stansted, United Kingdom) and then processed according to [65 (link)]. In brief, after shock-freezing at -210 °C in nitrogen slush, the samples were transferred to a pre-cooled (-135 °C) cryo chamber (PP2000 T, Quorum Technologies Ltd., Laughton, United Kingdom) and sublimated for 15 min at -90 °C for 15 min. After sputtering with platinum (30 s coating at 5–10 mA in an Argon atmosphere), the specimens were transferred to the cryo-stage in the SEM chamber (T = -135 °C) and imaged (Quanta 250 FEG field emission scanning electron microscope, FEI, Brno, Czech Republic) under ultravacuum (3 10–7 mbar). Backscattered electrons were collected by an Everhart–Thornley detector at a working distance of 5 mm, and an accelerating voltage of 10 kV.
3
Cryo-SEM Visualization of Cladosporium
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Isolates of Cladosporium spp. were grown on SNA with 30 g agar/L for 3–4 d at room temperature under black light. Relevant parts of the small colonies with conidiophores and conidia were selected under a binocular, excised with a surgical blade as small agar (3 × 3 mm) blocks, and transferred to a copper cup for snap-freezing in nitrogen slush. To prevent disruption of the intricate structure of the conidiophores by liquid nitrogen, a piece of Scotch tape was placed lightly over the opening of the copper cup. Agar blocks were glued to the copper surface with frozen tissue medium (KP-Cryoblock, Klinipath, Duiven, Netherlands) mixed with 1 part colloidal graphite (Agar Scientific, Stansted, UK). Samples were examined in a JEOL 5600LV scanning electron microscope (JEOL, Tokyo, Japan) equipped with an Oxford CT1500 Cryostation for cryo-electron microscopy (cryoSEM). Electron micrographs were acquired from uncoated frozen samples, or after sputter-coating by means of a gold target for three times during 30 s. Micrographs of uncoated samples were taken at an acceleration voltage of 3 kV, and consisted out of 30 averaged fast scans (SCAN 2 mode), and at 5 kV in case of the coated samples (PHOTO mode).
4
Spore Morphology Visualization via cryoSEM
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To facilitate a better interpretation of spore morphology, particularly wall ornamentation, scanning electron microscopy (SEM) was employed. Material was selected from cultures under a binocular microscope, excised with a surgical blade as small agar blocks (3 × 3 mm), and transferred to a copper cup for snap-freezing in nitrogen slush. Agar blocks were glued to the copper surface with frozen tissue medium (KP-Cryoblock, Klinipath, Duiven, The Netherlands) mixed with one part colloidal graphite (Agar Scientific, Stansted, UK). Samples were examined in a JEOL 5600LV scanning electron microscope (JEOL, Tokyo, Japan), equipped with an Oxford CT1500 Cryostation for cryo-electron microscopy (cryoSEM).
5
Cryo-SEM imaging of wild-type and rth6 roots
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Wild-type and rth6 mutant seeds for cSEM (cryo scanning electron microscopy) were germinated for 3–4 days. Seedling roots were cut into 1–2 cm pieces with a razor blade and mounted on a specimen holder with a mixture of Tissue-Tek® O.C.T.™Compound (Sakura Finetek Europe B.V., Alphen aan den Rijn, Netherlands) and colloidal graphite (Agar Scientific, Stansted, UK) and were immediately frozen in a nitrogen slush. Specimen were then transferred into a Quorum PP3010T cryo preparation chamber (Quorum Technologies, Laughton, UK) at −140 °C the specimen holder was heated to −80 °C for 40 min were water was sublimated. Subsequently, specimen were platinum sputtered at 10 mA for 60 sec and imaged in a Zeiss SIGMA VP cryo scanning electron microscope (Zeiss, Oberkochen, Germany). Images were taken at a magnification of 100x, 1,600x and 16,000x.
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