Cryo-EM samples of IDL and IDL-antibody mixture were prepared using a Leica EM GP plunge freezer (Leica, Buffalo Grove, IL, USA) equipped with a chamber for controlling the temperature and humidity during sample preparation. Prior to specimen preparation, the IDL sample was pre-incubated at temperatures below (8 °C) and above (45 °C) the lipid phase transition temperature (from ~20 to 40 °C) in a water bath for at least 30 min. The temperature of the plunge-freezer chamber was pre-set to the pre-incubation temperature, and the relative humidity was maintained at 80%. During specimen preparation, an aliquot (~3 μL) of pre-incubated IDL or IDL-antibody mixture solution (molar ratio 1:2.3) was applied to a glow-discharged lacey carbon grid (LC200-Cu, Electron Microscopy Sciences, Hatfield, PA, USA). The grid was flash-frozen after blotting by filter paper, and finally transferred to liquid nitrogen for storage.
Lc200 cu
Manufactured by Electron Microscopy Sciences
Sourced in United States
The LC200-Cu is a compact and versatile laboratory instrument designed for X-ray diffraction analysis. It features a high-intensity copper X-ray source that provides precise and reliable data for a wide range of materials. The LC200-Cu is a compact and versatile laboratory instrument designed for X-ray diffraction analysis.
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Lab products found in correlation
Tecnai g2 spirit tem, by Thermo Fisher Scientific (1 mentions)
Cf200 cu ul, by Electron Microscopy Sciences (1 mentions)
Talos tem, by Thermo Fisher Scientific (1 mentions)
Fei vitrobot mark 3, by Thermo Fisher Scientific (1 mentions)
Titan themis microscope, by Thermo Fisher Scientific (1 mentions)
Whatman no 1 filter paper, by Cytiva (1 mentions)
Whatman, by Cytiva (1 mentions)
Vitrobot, by Thermo Fisher Scientific (1 mentions)
Vivaspin protein concentrator spin columns, by Sartorius (1 mentions)
9 protocols using lc200 cu
1
Cryo-EM Preparation of IDL Samples
2
Cryo-EM Preparation of IDL Samples
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Cryo-EM samples of IDL and IDL-antibody mixture were prepared using a Leica EM GP plunge freezer (Leica, Buffalo Grove, IL, USA) equipped with a chamber for controlling the temperature and humidity during sample preparation. Prior to specimen preparation, the IDL sample was pre-incubated at temperatures below (8 °C) and above (45 °C) the lipid phase transition temperature (from ~20 to 40 °C) in a water bath for at least 30 min. The temperature of the plunge-freezer chamber was pre-set to the pre-incubation temperature, and the relative humidity was maintained at 80%. During specimen preparation, an aliquot (~3 μL) of pre-incubated IDL or IDL-antibody mixture solution (molar ratio 1:2.3) was applied to a glow-discharged lacey carbon grid (LC200-Cu, Electron Microscopy Sciences, Hatfield, PA, USA). The grid was flash-frozen after blotting by filter paper, and finally transferred to liquid nitrogen for storage.
3
Cryo-TEM and Negative Stain Imaging Protocols
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Cryo Transmission Electron Microscopy (TEM) samples were flash frozen onto lacey carbon film grids (LC200-CU, Electron Microscopy Sciences, Hatflield, PA, USA) and imaged on a FEI Talos TEM (FEI, Hillsboro, OR, USA) at an acceleration voltage of 200kV. Negative-stained samples were deposited on carbon coated square grids (CF200-Cu-UL, Electron Microscopy Sciences), dried, and stained with 2% uranyl formate, and imaged on a FEI Tecnai G2 Spirit TEM at an acceleration voltage of 120 kV.
4
Cryogenic Imaging of Protein Micelles
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Cryogenic
transmission electron microscopy was performed using samples with
a concentration of 0.5 or 5 mg/mL. Vitrified films were prepared using
a computer-controlled vitrification robot (FEI Vitrobot Mark III,
FEI Company) at 22 °C, and at a relative humidity of 100%. In
the preparation chamber of the “Vitrobot”, 3 μL
sample was applied on a Lacey film (LC200-CU, Electron Microscopy
Sciences). These films were surface plasma treated just prior to use,
with a Cressington 208 carbon coater operating at 5 mA for 40 s. Excess
sample was removed by blotting using filter paper for 3 s at −3
mm, and the thin film thus formed was plunged (acceleration about
3 g) into liquid ethane just above its freezing point. Vitrified films
were transferred into the vacuum of a CryoTITAN equipped with a field
emission gun that was operated at 300 kV, a post-column Gatan energy
filter, and a 2048 × 2048 Gatan CCD camera. Micrographs were
taken at low dose conditions, starting at a magnification of 6500
times with a defocus setting of 40 μm, and at a magnification
of 24 000 times with defocus settings of 10 and 15 μm.
The sizes of the observed micelles were measured manually using Fiji
software.
transmission electron microscopy was performed using samples with
a concentration of 0.5 or 5 mg/mL. Vitrified films were prepared using
a computer-controlled vitrification robot (FEI Vitrobot Mark III,
FEI Company) at 22 °C, and at a relative humidity of 100%. In
the preparation chamber of the “Vitrobot”, 3 μL
sample was applied on a Lacey film (LC200-CU, Electron Microscopy
Sciences). These films were surface plasma treated just prior to use,
with a Cressington 208 carbon coater operating at 5 mA for 40 s. Excess
sample was removed by blotting using filter paper for 3 s at −3
mm, and the thin film thus formed was plunged (acceleration about
3 g) into liquid ethane just above its freezing point. Vitrified films
were transferred into the vacuum of a CryoTITAN equipped with a field
emission gun that was operated at 300 kV, a post-column Gatan energy
filter, and a 2048 × 2048 Gatan CCD camera. Micrographs were
taken at low dose conditions, starting at a magnification of 6500
times with a defocus setting of 40 μm, and at a magnification
of 24 000 times with defocus settings of 10 and 15 μm.
The sizes of the observed micelles were measured manually using Fiji
software.
5
Characterization of SLNC Structures and Particles
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A Phenom
XL G2 SEM was used for imaging of SLNC structures and sprayed particle
morphology. Top-down SEM views were produced using as-processed, uncoated
samples. Cross-sectional samples of SLNCs were produced through freeze-fracturing.
50 mm × 5 mm strips were cut from melt-infiltrated coupons and
submerged in liquid nitrogen for 20 min. Samples were then broken
into two parts using tweezers to snap the sample, mounted to aluminum
stubs, and sputter coated with gold to prevent charging. A Hitachi
SU8010 SEM was used to collect images of individual particles for
particle size analysis. Particles were dispersed under sonication
at 10mg/L in water or CHCl3, for rGO and rGO-dd particles,
respectively, and deposited on a lacey carbon-coated copper transmission
electron microscopy (TEM) grid for imaging (Electron Microscopy Services,
LC200-Cu).
XL G2 SEM was used for imaging of SLNC structures and sprayed particle
morphology. Top-down SEM views were produced using as-processed, uncoated
samples. Cross-sectional samples of SLNCs were produced through freeze-fracturing.
50 mm × 5 mm strips were cut from melt-infiltrated coupons and
submerged in liquid nitrogen for 20 min. Samples were then broken
into two parts using tweezers to snap the sample, mounted to aluminum
stubs, and sputter coated with gold to prevent charging. A Hitachi
SU8010 SEM was used to collect images of individual particles for
particle size analysis. Particles were dispersed under sonication
at 10mg/L in water or CHCl3, for rGO and rGO-dd particles,
respectively, and deposited on a lacey carbon-coated copper transmission
electron microscopy (TEM) grid for imaging (Electron Microscopy Services,
LC200-Cu).
6
Characterization of Hb@AuNCs by DLS, Zeta, and STEM
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The hydrodynamic size of the Hb@AuNCs was evaluated by dynamic light scattering (DLS) (Zetasizer Nanoseries nano-ZS, Malvern, UK). The zeta (ζ)-potentials were assessed also using the Zetasizer. The morphology of the Hb@AuNCs was evaluated by scanning transmission electron microscopy (STEM), using a Titan Themis microscope (Thermo Fisher Scientific) operating at 200 kV with a probe of 20 mrad convergence angle and 50 pA current. The microscope was equipped with a high-brightness Schottky emitter (X-FEG), a probe aberration correction system (CEOS DCOR) and a Super-X energy dispersive X-ray (EDX) detector. Annular dark-field (ADF) STEM images were acquired with a collection angle of 31–192 mrad and EDX spectrum images were acquired with a probe current of 250 pA. Colored elemental maps were processed using Velox software, after removal of the background in the spectra (net count maps). The Cliff-Lorimer method were applied for EDX quantification, using the Brown-Powell ionization cross-sections. The samples were prepared by powder dispersion on lacey carbon films supported by copper meshed TEM grids (LC200-Cu, Electron Microscopy Sciences, PA, US).
7
Cryo-TEM Imaging of Vitrified BTA Samples
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Imaging was performed on samples with a BTA concentration
of 250
μM incubated overnight with 20 mg/mL BSA in PBS. Vitrified films
were prepared in a “Vitrobot” instrument (PC controlled
vitrification robot, patent applied, Frederik et al. 2002, patent
licensed to FEI, Vitrobot Mark III) at 22 °C and at a relative
humidity of 100%. In the preparation chamber of the “Vitrobot”,
3 μL samples were applied on Lacey grids (LC200-Cu, Electron
Microscopy Sciences), which were surface plasma treated just prior
to use (Cressington 208 carbon coater operating at 5 mA for 40 s).
Excess sample was removed by blotting using filter paper for 4 s at
−3 mm, and the thin film thus formed was plunged (acceleration
about 3 g) into liquid ethane just above its freezing point. Vitrified
films were transferred into the vacuum of a CryoTITAN equipped with
a field emission gun that was operated at 300 kV, a postcolumn Gatan
energy filter, and a 2048 × 2048 Gatan CCD camera. Virtrified
films were observed in the CryoTITAN microscope at temperatures below
−170 °C. Micrographs were taken at low dose conditions,
starting at a magnification of 6500 with a defocus setting of −40
μm and at a magnification of 24000 with a defocus setting of
−10 μm.
of 250
μM incubated overnight with 20 mg/mL BSA in PBS. Vitrified films
were prepared in a “Vitrobot” instrument (PC controlled
vitrification robot, patent applied, Frederik et al. 2002, patent
licensed to FEI, Vitrobot Mark III) at 22 °C and at a relative
humidity of 100%. In the preparation chamber of the “Vitrobot”,
3 μL samples were applied on Lacey grids (LC200-Cu, Electron
Microscopy Sciences), which were surface plasma treated just prior
to use (Cressington 208 carbon coater operating at 5 mA for 40 s).
Excess sample was removed by blotting using filter paper for 4 s at
−3 mm, and the thin film thus formed was plunged (acceleration
about 3 g) into liquid ethane just above its freezing point. Vitrified
films were transferred into the vacuum of a CryoTITAN equipped with
a field emission gun that was operated at 300 kV, a postcolumn Gatan
energy filter, and a 2048 × 2048 Gatan CCD camera. Virtrified
films were observed in the CryoTITAN microscope at temperatures below
−170 °C. Micrographs were taken at low dose conditions,
starting at a magnification of 6500 with a defocus setting of −40
μm and at a magnification of 24000 with a defocus setting of
−10 μm.
8
Cryo-TEM Analysis of Extracellular Vesicles
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Cryo-TEM measurements were carried out on a Tecnai G2Sphera 20 electron microscope (Thermo Fisher, Waltham, MA, USA) equipped with a Gatan 626 cryo-specimen holder and an LaB6 gun. The samples were prepared by plunge-freezing [26 (link)]. Briefly, 3 μL of the sample, either isolated lEVs or plasma supernatant depleted of lEVs, was applied to a copper grid covered with a perforated carbon film forming woven-mesh-like openings of different sizes and shapes (the lacey carbon grids #LC-200 Cu, Electron Microscopy Sciences, Hatfield, PA, USA), which was then glow discharged for 40 s with a 5 mA current prior to specimen application. Most of the sample was removed by blotting (Whatman no. 1 filter paper) for approximately 1 s, and the grid was immediately plunged into liquid ethane held at −183 °C. The grid was transferred without rewarming into the microscope. Images were recorded at an accelerating voltage of 120 kV and with magnifications ranging from 6000× to 29,000× using a GatanUltraScan 1000 slow-scan CCD camera. All cryo-TEM pictures were carefully inspected for possible artefacts such as radiation damage and ice crystals, and high-quality images were CTF-corrected and band-pass filtered in order to suppress both ice thickness variations and noise to below a 1 nm detail size.
9
Characterization of mRNA Lipid Nanoparticles
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The mRNA LNPs at pH 7.4 were first concentrated to a total lipid mass concentration of ~10 mg/mL using Vivaspin Protein Concentrator Spin Columns with a MWCO of 100,000 Da (Sartorius, Cat# VS0141). 5 μL of the concentrated LNP sample was deposited onto copper grids coated by a lacey carbon film (Electron Microscopy Services, Cat# LC200-CU) that had been treated with glow discharge. The sample was then vitrified by plunging into liquid ethane operated on a Vitrobot (Thermo Fisher Scientific). The Vitrobot parameters were as follows: blot time = 3 seconds, blot force = 0, offset = 0, wait time = 3 seconds, relax time = 0, and humidity = 100%. After vitrification, the grids were kept under liquid nitrogen and were transferred to a F200C Talos transmission electron microscope (Thermo Fisher Scientific) operated at an acceleration voltage of 200 kV. Images were captured using a Ceta camera equipped on the Talos instrument.
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