Autosamdri 815b

The morphology of P. yuhuli AB48 was described using light and scanning electron microscopy (SEM) techniques. For brightfield images, a Zeiss Axio Observer 7 microscope with an attached Calibiri 7 light source and an Axiocam 702 mono camera was used. For the detection of autofluorescence, a mRF12 filter was used. The samples imaged using SEM were isolated from suspension and filtered onto 0.2 µm Nucleopore filters where they were fixed with 4% formaldehyde/2% glutaraldeyde in 0.05 M, pH 7.4 sodium cacodylate. The cells were washed 3x with fixative-free buffer, then post-fixed with 1% buffered OsO₄ before multiple washes with distilled, deionized water. Following the washes, the samples were stacked in a stainless-steel filter holder, and taken through a staged, microwave dehydration using 1 min at power-level 3 (∼180W) in 30, 50, and 70% ethanol. The samples were kept at 70% ethanol overnight at −20°C. Following overnight dehydration, the samples were treated for 1 min PL3 at 80, 90, and 95% ethanol and finally for 3 min PL3 at 100% dry ethanol for dehydration, twice. The final dehydration step was performed for 10 min at room temperature in 100% dry ethanol. The samples were then critically point-dried (Tousimis Auto-Sam Dri 815B) mounted to aluminum SEM stubs using double-sided tabs, sputter coated with 10 nm gold (Cressington 208HR), and imaged on a Hitachi S2600 VP-SEM.

After helium ion irradiation, the whole NBPT CNMs were transferred onto another substrate for further investigations again with the HIM. For the transfer of NBPT CNMs onto a SiO₂/Si substrate the samples were spin-coated with a layer of poly(methyl methacrylate) (PMMA) for stabilization and baked on a hotplate at 90 °C for 5 min. The separation of the PMMA/CNM/Au layer from the mica substrate was achieved by carefully dipping the sample into water. Subsequently, the Au layer was completely etched by a gold etchant (5 wt % I₂ and 10 wt % KI in water). Afterwards, the PMMA/CNM layer was transferred to a Si substrate with an oxide layer with the thickness of 300 nm and the sample was immersed into acetone for 40 min for the dissolution of the PMMA layer. For the fabrication of freestanding NBPT CNMs on a TEM grid the same process was carried out, except for the drying process being conducted in a critical-point dryer (CPD, Autosamdri-815B, Tousimis, USA) to yield intact and suspended CNMs.

hESC-CMs cultured on the quartz nanopillars were fixed with 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and post-fixed in 1% osmium tetroxide in the same buffer at 4 °C for 1 h. Chemicals were from Electron Microscopy Sciences. After rinsing, the sample was dehydrated in a graded ethanol series and dried in a liquid CO₂ critical point dryer (Autosamdri 815b, Tousimis, Rockville, MD, USA). The sample was then sputter-coated with 4 nm Au/Pd and imaged by electron microscopy (Strata 235DB, FEI, Hillsboro, OR, USA).

Collections of inflorescence material were killed and fixed in formalin–acetic acid–alcohol (FAA) for approximately 1 week followed by storage in 70% ethanol. Some material was dehydrated through an ethanol series to 100% ethanol, transferred to Histoclear before embedding in Paraplast using standard protocols. The wax blocks were sectioned at 10 μm thickness on a rotary microtome (Leica RM2155) and the resulting sections were stained in 0.5% (w/v) solution of toluidine blue before mounting on microscope slides in DPX mountant. Images were captured using a Zeiss Axiocam HRc camera attached to a Leica DMLB microscope.
Other material was dehydrated through an alcohol series into acetone and transferred to a critical-point drier (Tousimis Autosamdri 815B). Dried material was then sputter-coated with platinum in a sputter coater (Emitech K550). The material was examined on a Hitachi S-4700 II cold-field emission scanning electron microscope.

Cells of Virgibacillus sp. strain 21D were filtered onto 0.1 μm polycarbonate Whatman filters (Nucleopore) before fixation with 2.5% glutaraldehyde in 0.1 M cacodylate buffer. Filters were washed in 0.1 M cacodylate buffer and subsequently post-fixed in osmium tetroxide. Samples were washed with an ethanol gradient (20–100% ethanol) and subject to critical point drying (Autosamdri-815B, Tousimis). Filters were coated with a 5 nm layer of Au/Pb using a K575X sputter coater (Quorum) and images were acquired using a Quanta 600 FEG (Thermo Scientific) scanning electron microscope at an acceleration voltage of 5 kV.