The catalyst-coated membranes were prepared by sputter deposition. The electrodeposited palladium foils were placed in a Leica EM MED020 coating system at a working distance of 6 cm, the chamber was sealed, and a vacuum applied to achieve a base pressure of 1 × 10–4 mbar. After the base pressure was reached, argon was continuously flowed into the chamber to maintain a pressure of 1 10–2 mbar, the plasma was ignited, and voltage was adjusted to maintain a constant sputter current of 30 mA. Following a 30 s presputter, the target shutter was opened and 10-nm platinum was sputtered onto the electrodeposited palladium catalyst. The sputtering rate was 0.2 nm/s, as determined by in situ quartz crystal microbalance. The foils were used for NADH regeneration and enzymatic reduction/reductive amination experiments without any further processing. The catalyst-coated membranes were used for up to 20 reactions. The entire catalyst layer of Pt-coated Pd black was removed with concentrated nitric acid, then the foil was cleaned (described above), replated, and reused. Each palladium foil was used for ~5 cleaning cycles before being discarded.
Em med020 coating system
Manufactured by Leica
The EM MED020 is a coating system designed for the preparation of samples for electron microscopy. It provides a controlled environment for the deposition of thin conductive or protective coatings on the surface of the sample, enhancing its conductivity and preserving its structure for high-resolution imaging.
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3 protocols using em med020 coating system
1
Sputter-Deposited Pt-Coated Pd Catalyst
2
Open Microcavity Fabrication Protocol
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Methyl salicylate (99%), methylcyclohexane (99%), benzonitrile (99%), toluene (99%), and polyvinyl acetate (MW: 100 000) were purchased from Sigma-Aldrich and used without further purification. CaF 2 windows (diameter: 25.0 mm, thickness: 5.0 mm) were purchased from Crystran LTD, while a mirror mount with a piezoelectric adjuster and a voltage controller were purchased from Thorlabs, Inc.
Open microcavity preparation 54, 55 The Ag films, typically 10 nm thick (unless otherwise explicitly stated), were deposited on a CaF 2 substrate by a high vacuum sputter coater (Leica EM MED 020 coating system). The thickness of the metal film was controlled using a quartz microbalance film thickness monitoring system (EM QSG100) as a reference during sputtering. The microcavity was realized by bringing the Ag-coated CaF 2 windows in close proximity and by carefully controlling their parallelism with the kinematic mirror holders. Subsequently, the cavity was filled by adding a few droplets of an appropriate solution onto the edges of the CaF 2 windows using a micropipette. The solution flowed into the cavity by capillary action and remained stable between the mirrors during the entire experiment.
Open microcavity preparation 54, 55 The Ag films, typically 10 nm thick (unless otherwise explicitly stated), were deposited on a CaF 2 substrate by a high vacuum sputter coater (Leica EM MED 020 coating system). The thickness of the metal film was controlled using a quartz microbalance film thickness monitoring system (EM QSG100) as a reference during sputtering. The microcavity was realized by bringing the Ag-coated CaF 2 windows in close proximity and by carefully controlling their parallelism with the kinematic mirror holders. Subsequently, the cavity was filled by adding a few droplets of an appropriate solution onto the edges of the CaF 2 windows using a micropipette. The solution flowed into the cavity by capillary action and remained stable between the mirrors during the entire experiment.
3
Visualizing Osteoclast-Calcium Phosphate Interactions
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To visualize osteoclasts and their interactions with the calcium phosphate coating, SEM analysis was performed. After 7 days of culture, three specimens from each group were fixed in 4% paraformaldehydephosphate buffer solution for 15 minutes, followed by 2.5% glutaraldehyde-PB solution for 20 minutes.
The samples were then post-fixed with 1% osmium tetroxide (OsO4)-PB solution for 60 minutes. The samples were dehydrated in ascending concentrations of ethanol (30%, 50%, 70%, 95% and 100%) for 10 minutes each, followed by CO2 critical point drying. To study detailed interaction between osteoclasts and AiM-CaP coatings, cross-sections through the cells and the underneath CaP coating were prepared and imaged by FIB-SEM (Helio NanoLab 650 Focused Ion Beam SEM). SEM observation was performed as previously described. After SEM observation, the samples were coated with 20nm of iridium (Leica EM MED020 Coating System). In order to obtain a smoother cross-section, a thin layer of platinum (Pt)
was deposited on the sample surface in order to reduce the curtaining effect. An accelerating voltage of 2 kV and high pressure were used for imaging (BSE mode).
The samples were then post-fixed with 1% osmium tetroxide (OsO4)-PB solution for 60 minutes. The samples were dehydrated in ascending concentrations of ethanol (30%, 50%, 70%, 95% and 100%) for 10 minutes each, followed by CO2 critical point drying. To study detailed interaction between osteoclasts and AiM-CaP coatings, cross-sections through the cells and the underneath CaP coating were prepared and imaged by FIB-SEM (Helio NanoLab 650 Focused Ion Beam SEM). SEM observation was performed as previously described. After SEM observation, the samples were coated with 20nm of iridium (Leica EM MED020 Coating System). In order to obtain a smoother cross-section, a thin layer of platinum (Pt)
was deposited on the sample surface in order to reduce the curtaining effect. An accelerating voltage of 2 kV and high pressure were used for imaging (BSE mode).
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