The structure of the composite еlectrospun polyacrylonitrile-based CNF was investigated by the methods of scanning electron microscopy (SEM) using a FEI Scios microscope (FEI, Hillsboro, OR, USA), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HR TEM), scanning transmission electron microscopy with a high-angle annular dark-field detector (HAADF STEM) and energy-dispersive X-ray spectroscopy (EDX) elemental mapping using a Thermo Fisher Scientific Osiris (Waltham, MA, USA) equipped with a high-angle annular dark field (HAADF) detector and Super-X EDX detection system based on Silicon Drift Detector (SDD) technology. Electron microscope images were analyzed using Digital Micrograph (GMS 3, Gatan, Pleasanton, CA, USA), TIA (TIA 16, Siemens AG, Munich, Germany), Esprit (Esprit 2, Bruker, Billerica, MA, USA) and JEMS software (P. Stadelmann JEMS—EMS Java version 2004 EPFL, Lausanne, Switzerland). For electron microscopy studies, the samples of CNF were well-dispersed in acetone to separate the fibers using an ultrasonic bath for 20–30 min. Then, the obtained suspensions were introduced onto copper lacey carbon grids.
Digital micrograph gms 3
Manufactured by Ametek
Sourced in United States
Digital Micrograph (GMS 3) is a comprehensive software platform for electron microscopy image analysis and visualization. It provides a range of tools and functionalities for processing, analyzing, and interpreting data from various electron microscopy techniques.
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Lab products found in correlation
Scios microscope, by Thermo Fisher Scientific (2 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (2 mentions)
Osiris, by Thermo Fisher Scientific (1 mentions)
Jem 2200fs electron microscope, by JEOL (1 mentions)
Gatan oneview, by Ametek (1 mentions)
Tecnai osiris, by Thermo Fisher Scientific (1 mentions)
Titan 80 300 microscope, by Thermo Fisher Scientific (1 mentions)
Titan krios g2, by Thermo Fisher Scientific (1 mentions)
K2 camera, by Ametek (1 mentions)
Titan 80 300, by Thermo Fisher Scientific (1 mentions)
Us4000 ccd camera, by Ametek (1 mentions)
Oneview camera, by Ametek (1 mentions)
10nm gold particles, by Aurion (1 mentions)
Aztectem, by Oxford Instruments (1 mentions)
5 protocols using digital micrograph gms 3
1
Structural Analysis of PAN-based CNF
2
Transmission Electron Microscopy of Samples
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TEM images were taken
with a JEOL JEM-2200FS electron microscope
(JEOL, Freising, Germany) equipped with a cold field emission electron
gun. The microscope was operated at an acceleration voltage of 200
kV. For standard room-temperature TEM, carbon-coated copper grids
(ECF200-Cu, 200 mesh, Science Services, Munich, Germany) were used.
Next, 3 μL of a diluted sample solution was dropped on the copper
grid, and after 1 min, the residual water was blotted off with a filter
paper.
All images were recorded digitally by a bottom-mounted
camera (Gatan OneView, Gatan, Pleasanton) and processed with a digital
imaging processing system (Digital Micrograph GMS 3, Gatan, Pleasanton).
To achieve good statistics, several positions on each grid were imaged.
For the analysis of the TEM images, free software ImageJ55 (link) was used.
For cryo-TEM, the samples were
vitrified on TEM holey carbon grids
(Qantifoil R2/1, 200 mesh, Plano GmbH, Wetzlar, Germany) using a Leica
blotting and plunging device (Leica EM GP, Leica Mikrosysteme Vertrieb
GmbH, Wetzlar, Germany). The grids were plunged into liquid ethane
cooled with liquid nitrogen to achieve sufficiently fast cooling.
Subsequently, the grids were transferred to a cryo transfer and tomography
holder (Fischione Model 2550, E.A. Fischione Instruments, Pittsburgh).
with a JEOL JEM-2200FS electron microscope
(JEOL, Freising, Germany) equipped with a cold field emission electron
gun. The microscope was operated at an acceleration voltage of 200
kV. For standard room-temperature TEM, carbon-coated copper grids
(ECF200-Cu, 200 mesh, Science Services, Munich, Germany) were used.
Next, 3 μL of a diluted sample solution was dropped on the copper
grid, and after 1 min, the residual water was blotted off with a filter
paper.
All images were recorded digitally by a bottom-mounted
camera (Gatan OneView, Gatan, Pleasanton) and processed with a digital
imaging processing system (Digital Micrograph GMS 3, Gatan, Pleasanton).
To achieve good statistics, several positions on each grid were imaged.
For the analysis of the TEM images, free software ImageJ55 (link) was used.
For cryo-TEM, the samples were
vitrified on TEM holey carbon grids
(Qantifoil R2/1, 200 mesh, Plano GmbH, Wetzlar, Germany) using a Leica
blotting and plunging device (Leica EM GP, Leica Mikrosysteme Vertrieb
GmbH, Wetzlar, Germany). The grids were plunged into liquid ethane
cooled with liquid nitrogen to achieve sufficiently fast cooling.
Subsequently, the grids were transferred to a cryo transfer and tomography
holder (Fischione Model 2550, E.A. Fischione Instruments, Pittsburgh).
3
Nanocomposite Structural Analysis by Electron Microscopy
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The solution with CNF was applied onto standard copper grids with a holed amorphous carbon film and subsequently dried under normal conditions. The structure of nanocomposites was investigated by the methods of scanning electron microscopy (SEM) using an FEI Scios microscope (FEI, Hillsboro, OR, USA), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), scanning transmission electron microscopy with a high-angle annular dark-field detector (HAADF STEM), electron diffraction, energy-dispersive X-ray analysis (EDX), and elemental mapping using a FEI Titan 80–300 microscope and a FEI Tecnai Osiris (FEI, Hillsboro, OR, USA) with accelerating voltage of 200 kV equipped with a special SuperX EDS system including four silicon detectors for rapid obtaining of chemical distribution maps. Electron microscope images were analyzed using Digital Micrograph (GMS 3, Gatan, Pleasanton, CA, USA), Esprit (Esprit 2, Bruker, Billerica, MA, USA), TIA (TIA 16, Siemens AG, Munich, Germany) and JEMS software (P. Stadelmann JEMS—EMS Java version 2004 EPFL, Lausanne, Switzerland).
4
Cryogenic and Room-temperature TEM Imaging of Virus-like Nanoparticles
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For cryogenic transmission electron microscopy (cryo-TEM), GVNP samples were vitrified by ultra-rapid freezing. Typically, 3 μl of the sample was put on a pre-glow-discharged copper grid coated with a holy carbon layer and blotted from both sides. The blotted grid was plunge-frozen in liquid ethane using Vitrobot Mark IV (ThermoFisher Scientific) and transferred into liquid nitrogen. Samples imaging was performed on Titan Krios G2 (Thermo Fisher Scientific) equipped with a K2 camera (Gatan) placed at the end of the energy filter (Gatan) and operated at 300 kV. Typically, electron doses of ∼50 electrons/ Å2 were used.
For room-temperature (RT-)TEM, 4 μl of the purified GVNPs sample was drop casted on pre-glow-discharged carbon film copper grids (Electron Microscopy Sciences) and incubated for 1 min for adsorption. After that, the sample was removed with filter paper. Negative staining was performed by adding 4 µl of 2% uranyl acetate for 1 min. Afterward, the uranyl acetate stain was removed with filter paper, and the grids were air-dried. Images were taken in different magnifications with a US4000 CCD camera (Gatan, Inc., USA) or OneView camera (Gatan, Inc., USA) on a Titan 80–300 (Thermo Fisher Scientific). The lengths and diameters of 25 GVNPs were measured manually in DigitalMicrograph GMS3 (Gatan, Inc., USA) with a selected library.
For room-temperature (RT-)TEM, 4 μl of the purified GVNPs sample was drop casted on pre-glow-discharged carbon film copper grids (Electron Microscopy Sciences) and incubated for 1 min for adsorption. After that, the sample was removed with filter paper. Negative staining was performed by adding 4 µl of 2% uranyl acetate for 1 min. Afterward, the uranyl acetate stain was removed with filter paper, and the grids were air-dried. Images were taken in different magnifications with a US4000 CCD camera (Gatan, Inc., USA) or OneView camera (Gatan, Inc., USA) on a Titan 80–300 (Thermo Fisher Scientific). The lengths and diameters of 25 GVNPs were measured manually in DigitalMicrograph GMS3 (Gatan, Inc., USA) with a selected library.
5
Immunogold Labeling of Exosomes for TEM Analysis
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Transmission
electron microscopy (TEM) characterization was performed using a JEOL
JEM-2100F field emission S/TEM equipped with an Oxford X-MaxN 80 mm2
SDD detector for elemental analysis. TEM images were acquired at a
200 kV accelerating voltage and 116 μA emission current. DigitalMicrograph
GMS3 (Gatan) and Aztec TEM (Oxford Instruments) software packages
were used for the TEM data and energy dispersive X-ray (EDX) spectral
analysis and interpretation, respectively. Briefly, for the immunogold
labeling with antibodies, GPC-1 antibodies (pa5–51290, Thermo
Scientific) were attached on 10 nm gold particles (AURION) according
to the manufacturer’s instructions at room temperature. Healthy
plasma and PDAC plasma sample pools were prepared. 200 μL from
each of 5 healthy or 5 PDAC plasma samples was taken to form a healthy
plasma or PDAC plasma sample pool. Exosomes were isolated as previously
described. Then isolated exosome samples were conjugated with the
gold-particles attached GPC-1 antibodies at the appropriate dilution
overnight at 4 °C. Samples for TEM characterization were drop-cast
from dilute aqueous suspensions onto amorphous carbon coated (200
nm) Cu grids (Agar Scientific) and dried naturally overnight.
electron microscopy (TEM) characterization was performed using a JEOL
JEM-2100F field emission S/TEM equipped with an Oxford X-MaxN 80 mm2
SDD detector for elemental analysis. TEM images were acquired at a
200 kV accelerating voltage and 116 μA emission current. DigitalMicrograph
GMS3 (Gatan) and Aztec TEM (Oxford Instruments) software packages
were used for the TEM data and energy dispersive X-ray (EDX) spectral
analysis and interpretation, respectively. Briefly, for the immunogold
labeling with antibodies, GPC-1 antibodies (pa5–51290, Thermo
Scientific) were attached on 10 nm gold particles (AURION) according
to the manufacturer’s instructions at room temperature. Healthy
plasma and PDAC plasma sample pools were prepared. 200 μL from
each of 5 healthy or 5 PDAC plasma samples was taken to form a healthy
plasma or PDAC plasma sample pool. Exosomes were isolated as previously
described. Then isolated exosome samples were conjugated with the
gold-particles attached GPC-1 antibodies at the appropriate dilution
overnight at 4 °C. Samples for TEM characterization were drop-cast
from dilute aqueous suspensions onto amorphous carbon coated (200
nm) Cu grids (Agar Scientific) and dried naturally overnight.
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