Cryo-TEM samples were prepared from resuspended or extracted solutions onto Quantifoil R2/2 (Electron Microscopy Sciences) grids. Grids were glow discharged for 70 s to increase hydrophilicity prior to sample loading. Vitrification was carried out by an Automatic Plunge Freezer ME GP2 (Leica Microsystems) with 3 μL of sample. Grid preparation was performed at 95% humidity and the grids were blotted for 3 s prior to plunging into liquid propane. Cryo-TEM samples were then placed on a Gatan Cryo-TEM holder and imaged on a JEOL 2100F TEM using a Schottky type field emission gun operating at 200 keV. Images were recorded using DigitalMicrograph (Gatan) software with a Gatan OneView CMOS camera at 4k × 4k resolution.
Automatic plunge freezer me gp2
Manufactured by Leica
The Automatic Plunge Freezer ME GP2 is a laboratory equipment designed for rapid freezing of samples. It features an automated plunging mechanism that submerges samples into a cryogenic liquid, enabling fast and uniform freezing. The core function of this device is to provide a controlled and reproducible freezing process for various research and analytical applications.
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3 protocols using automatic plunge freezer me gp2
1
Cryo-TEM Imaging of Biological Samples
2
Cryo-TEM Sample Preparation Protocol
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Cryo-TEM samples were prepared using a Quantifoil
R2/2 Holey Carbon Films from Electron Microscopy Sciences or 400 Mesh
Carbon grids from TedPella. Prior to sample application, glow discharge
was applied to the grids for 70 s. Reaction solutions at various time
points were centrifuged for ∼ 2 s, and 3 μL of each sample
was taken from the reaction solutions and underwent vitrification
using an Automatic Plunge Freezer ME GP2 (Leica Microsystems). Vitrification
was performed at a ∼95% humidity with a blot time of 4 s, and
samples were plunged into liquid propane. Samples were then analyzed
using a JOEL-2100 TEM with a Schottky field-type emission gun set
to 200 kV. Images were obtained using Serial EM software or a Gatan
OneView Camera.
R2/2 Holey Carbon Films from Electron Microscopy Sciences or 400 Mesh
Carbon grids from TedPella. Prior to sample application, glow discharge
was applied to the grids for 70 s. Reaction solutions at various time
points were centrifuged for ∼ 2 s, and 3 μL of each sample
was taken from the reaction solutions and underwent vitrification
using an Automatic Plunge Freezer ME GP2 (Leica Microsystems). Vitrification
was performed at a ∼95% humidity with a blot time of 4 s, and
samples were plunged into liquid propane. Samples were then analyzed
using a JOEL-2100 TEM with a Schottky field-type emission gun set
to 200 kV. Images were obtained using Serial EM software or a Gatan
OneView Camera.
3
Cryo-TEM Analysis of Polymeric Micelles
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Cryo-TEM
samples were prepared on QUANTIFOIL R2/2 (Electron Microscopy Sciences)
grids. Grids were glow-discharged for 70 s to increase hydrophilicity
prior to sample loading. Vitrification was carried out using an Automatic
Plunge Freezer ME GP2 (Leica Microsystems) with 3 μL of the
sample. Grid preparation was performed at 95% humidity, and the grids
were blotted for 3 s prior to plunging into liquid propane. Cryo-TEM
samples were then placed on a Gatan cryo-TEM holder and imaged on
a JEOL 2100F TEM using a Schottky-type field emission gun operating
at 200 keV. Images were recorded using SerialEM software in the low-dose
mode with a Gatan OneView CMOS camera at a 4k × 4k resolution.
Prior to vitrification, samples of PCL30-b-PEG45 (50 mg/mL) were heated up to 70 °C and cooled
with an average cooling rate of 20 °C/min. Following cooling,
samples were immediately diluted to 10 mg/mL and vitrified. Dilution
was necessary in order to image the sample using cryo-TEM. Selected
images have been analyzed to determine the size of the spherical micelles
and the cross-sectional diameter of the cylindrical micelles. The
analysis (seeFigure S4d,e ) was performed
on 100 particles per sample; the data in the main text are presented
as the average ± standard deviation.
samples were prepared on QUANTIFOIL R2/2 (Electron Microscopy Sciences)
grids. Grids were glow-discharged for 70 s to increase hydrophilicity
prior to sample loading. Vitrification was carried out using an Automatic
Plunge Freezer ME GP2 (Leica Microsystems) with 3 μL of the
sample. Grid preparation was performed at 95% humidity, and the grids
were blotted for 3 s prior to plunging into liquid propane. Cryo-TEM
samples were then placed on a Gatan cryo-TEM holder and imaged on
a JEOL 2100F TEM using a Schottky-type field emission gun operating
at 200 keV. Images were recorded using SerialEM software in the low-dose
mode with a Gatan OneView CMOS camera at a 4k × 4k resolution.
Prior to vitrification, samples of PCL30-b-PEG45 (50 mg/mL) were heated up to 70 °C and cooled
with an average cooling rate of 20 °C/min. Following cooling,
samples were immediately diluted to 10 mg/mL and vitrified. Dilution
was necessary in order to image the sample using cryo-TEM. Selected
images have been analyzed to determine the size of the spherical micelles
and the cross-sectional diameter of the cylindrical micelles. The
analysis (see
on 100 particles per sample; the data in the main text are presented
as the average ± standard deviation.
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