2.2 Grid Surface
Treatment
1. Several surface treatment systems are adequate [19]. We
describe use of a PELCO easiGlow, glow discharge, and plasma
generation system capable of 30 mA plasma currents, with
vacuum control and containing a platform with a recess suited
for 25 Â 75 mm slide. This equipment also requires a rotary
vacuum pump and gas and vent inlets.
2. Clean a standard glass micro-slide, 75 Â 25 mm, 1 mm thick
onto which grids can be placed for glow discharging. Other
grid holders can be used instead.
2.3 Cryo Storage and
Cryo-Transfer Systems
1. Several sample vitrification tools are available to perform rapid
sample cooling following the Dubochet method
[20, 21]. Here, we describe use of a Vitrobot Mark IV cryosample plunger capable of automated sample vitrification into
liquid ethane.
2. While a number of sample transfer holders are available, we
describe use of the Gatan 626 cryo-transfer holder with a high
tilt range and low sample drift.
3. The Gatan cryo-transfer holder pairs with Gatan dry pumping
station model 655 and Gatan cryo-transfer station.
2.4 Microscope
Components
1. Microscope types include Tecnai series FEI microscopes such as
the F20 TEM (200 keV) [13], F30 TEM (300 keV) [22], FEI
Talos Arctica S/TEM (200 keV) [4] or Titan (300 keV), or
JEOL JEM-3200, JEM-2200/2100 or cryo ARM series
[23]. Here, we describe use of a Tecnai F30 TEM (300 keV)
in microprobe mode.
2. Electron source: Field-emission gun operating with accelerating voltages in the 100–300 keV range (see Note 1).
3. Selected area aperture: Aperture diameters in the 50–150 μm
range are recommended. Depending on lens system, aperture
sizes can correspond to ~0.5–4.0 μm in diameter at the image
plane.
4. Sample stage: Stage accuracy tilt range and tilt speed should be
carefully calibrated to maintain eucentricity at the sample.
Typical stage parameters include a tilt range of Æ70
and tilt
speed of 0.1–10
/s.
2.5 Cameras and
Computing
1. Several detectors are suitable for data collection including some
that have been developed specifically for MicroED. We focus
on use of the TVIPS TemCam-XF416: a 16-megapixel CMOS
sensor with integrated electronics delivering 16-Bit images at
up to 48 frames/s with correlated double sampling [22] (see
Note 2).
MicroED of Small Macromolecules
331
Treatment
1. Several surface treatment systems are adequate [19]. We
describe use of a PELCO easiGlow, glow discharge, and plasma
generation system capable of 30 mA plasma currents, with
vacuum control and containing a platform with a recess suited
for 25 Â 75 mm slide. This equipment also requires a rotary
vacuum pump and gas and vent inlets.
2. Clean a standard glass micro-slide, 75 Â 25 mm, 1 mm thick
onto which grids can be placed for glow discharging. Other
grid holders can be used instead.
2.3 Cryo Storage and
Cryo-Transfer Systems
1. Several sample vitrification tools are available to perform rapid
sample cooling following the Dubochet method
[20, 21]. Here, we describe use of a Vitrobot Mark IV cryosample plunger capable of automated sample vitrification into
liquid ethane.
2. While a number of sample transfer holders are available, we
describe use of the Gatan 626 cryo-transfer holder with a high
tilt range and low sample drift.
3. The Gatan cryo-transfer holder pairs with Gatan dry pumping
station model 655 and Gatan cryo-transfer station.
2.4 Microscope
Components
1. Microscope types include Tecnai series FEI microscopes such as
the F20 TEM (200 keV) [13], F30 TEM (300 keV) [22], FEI
Talos Arctica S/TEM (200 keV) [4] or Titan (300 keV), or
JEOL JEM-3200, JEM-2200/2100 or cryo ARM series
[23]. Here, we describe use of a Tecnai F30 TEM (300 keV)
in microprobe mode.
2. Electron source: Field-emission gun operating with accelerating voltages in the 100–300 keV range (see Note 1).
3. Selected area aperture: Aperture diameters in the 50–150 μm
range are recommended. Depending on lens system, aperture
sizes can correspond to ~0.5–4.0 μm in diameter at the image
plane.
4. Sample stage: Stage accuracy tilt range and tilt speed should be
carefully calibrated to maintain eucentricity at the sample.
Typical stage parameters include a tilt range of Æ70
and tilt
speed of 0.1–10
/s.
2.5 Cameras and
Computing
1. Several detectors are suitable for data collection including some
that have been developed specifically for MicroED. We focus
on use of the TVIPS TemCam-XF416: a 16-megapixel CMOS
sensor with integrated electronics delivering 16-Bit images at
up to 48 frames/s with correlated double sampling [22] (see
Note 2).
MicroED of Small Macromolecules
331