11. Using the foot pedal, cycle through the following steps: Raise
sample into the blotting chamber, raise the ethane cup to the
bottom of the blotting chamber, and lower the tweezers into
the sample addition/hand blotting position (typically used for
adding sample to the grid).
12. Insert long tweezers holding the folded blotting paper, from
the side opposite the cells. Gently place the blotting paper flush
against the back of the grid, being careful not to bend the grid.
13. Blot for ~2 s, making sure the media has blotted into the paper
(see Note 10 and Fig. 3b, c).
14. Using the foot pedal, plunge the grid into the liquid ethane.
15. Transfer grid from liquid ethane to the liquid nitrogen quickly,
but smoothly. Place grid inside of a grid box for storage under
liquid nitrogen until ready for imaging.
3.6 Imaging
This section describes the use of Thermo Fisher’s “Tomography”
software for the batch collection of tilt-series on a Titan Krios cryoTEM equipped with a Gatan Imaging Filter and K2 direct electron
detector. There are many other configurations of microscope hardware and software that can achieve similar results, and many of the
same principles discussed here (especially in the detailed notes) will
apply to any tomography data collection session. This protocol
assumes the following: (a) The sample has been loaded into the
Thermo Fisher “Autogrid” assembly and transferred under LN into
the Krios, (b) the sample has been loaded on the stage, (c) the
microscope is properly aligned (including the GIF), and (d) the
“Tomography” software is properly calibrated for batch data collection. For a detailed video protocol of all these steps, please
consult the curriculum online at Thermo Fisher Scientific’s
CryoEM University [8], which was a joint effort with Caltech
professor Grant Jensen. See Note 11.
3.6.1 Set Imaging Modes
In order to collect a batch of multiple tilt-series using Thermo
Fisher’s “Tomography” software package, the user is required to
define eight different optical modes in the “Preparation” tab. Once
established, the setting can be saved and recalled in future sessions.
See Table 1 for an example of these settings. The magnification for
“Tracking,” “Exposure,” “Focus,” and “Zero Loss” should be the
same value and are typically the only modes that are adjusted
between imaging sessions. These optical setting should be established over a broken hole in the carbon.
To set the exposure mode for imaging with the K2 in counted
mode, find a region of representative ice and center the stage over a
cellular feature similar to ones you will collect data from during
your session (in this case, a neurite). Change to the exposure mode
and use Gatan’s Digital micrograph software to start the camera’s
32
Ryan K. Hylton et al.
sample into the blotting chamber, raise the ethane cup to the
bottom of the blotting chamber, and lower the tweezers into
the sample addition/hand blotting position (typically used for
adding sample to the grid).
12. Insert long tweezers holding the folded blotting paper, from
the side opposite the cells. Gently place the blotting paper flush
against the back of the grid, being careful not to bend the grid.
13. Blot for ~2 s, making sure the media has blotted into the paper
(see Note 10 and Fig. 3b, c).
14. Using the foot pedal, plunge the grid into the liquid ethane.
15. Transfer grid from liquid ethane to the liquid nitrogen quickly,
but smoothly. Place grid inside of a grid box for storage under
liquid nitrogen until ready for imaging.
3.6 Imaging
This section describes the use of Thermo Fisher’s “Tomography”
software for the batch collection of tilt-series on a Titan Krios cryoTEM equipped with a Gatan Imaging Filter and K2 direct electron
detector. There are many other configurations of microscope hardware and software that can achieve similar results, and many of the
same principles discussed here (especially in the detailed notes) will
apply to any tomography data collection session. This protocol
assumes the following: (a) The sample has been loaded into the
Thermo Fisher “Autogrid” assembly and transferred under LN into
the Krios, (b) the sample has been loaded on the stage, (c) the
microscope is properly aligned (including the GIF), and (d) the
“Tomography” software is properly calibrated for batch data collection. For a detailed video protocol of all these steps, please
consult the curriculum online at Thermo Fisher Scientific’s
CryoEM University [8], which was a joint effort with Caltech
professor Grant Jensen. See Note 11.
3.6.1 Set Imaging Modes
In order to collect a batch of multiple tilt-series using Thermo
Fisher’s “Tomography” software package, the user is required to
define eight different optical modes in the “Preparation” tab. Once
established, the setting can be saved and recalled in future sessions.
See Table 1 for an example of these settings. The magnification for
“Tracking,” “Exposure,” “Focus,” and “Zero Loss” should be the
same value and are typically the only modes that are adjusted
between imaging sessions. These optical setting should be established over a broken hole in the carbon.
To set the exposure mode for imaging with the K2 in counted
mode, find a region of representative ice and center the stage over a
cellular feature similar to ones you will collect data from during
your session (in this case, a neurite). Change to the exposure mode
and use Gatan’s Digital micrograph software to start the camera’s
32
Ryan K. Hylton et al.