3.4 Imaging
Fluorescent Targets
in the Titan Krios Using
Cryo-CLEM
1. Use SerialEM to collect a 200Â magnification montage for
each EM grid loaded into the Titan Krios (see Note 17).
2. Find a cell of interest imaged previously by cryo-LM using the
letters, symbols, or numbers of the grid support.
3. Use the whole-grid 200Â montage to generate a polygon
montage of the thin cellular peripheral area of interest and
surrounding area. It is advisable to capture fluorescent microspheres in the surrounding area in this montage for registration
of the EM and LM images (see Note 18).
4. Using your polygonal montage, evaluate if the ice is too thick
for tilt-series collection by evaluating the hole that is closest to
the fluorescent puncta using the cryo-LM aligned phase contrast/fluorescence images for orientation.
5. If the ice is thin enough, overlay the fluorescence images with
the polygonal montage images using the fluorescent microspheres seen in both the LM and EM modalities. Any image
processing software can be used to roughly align the images.
6. Next, localize more precisely the location of the fluorescent
puncta in the thin edge.
7. Collect a tilt-series in this location.
4 Data Processing and Tomographic Reconstruction
4.1 Conventional
Tilt-Series
We reconstruct tomograms from raw tilt-series using the IMOD
software [41, 43, 44]. We refer the reader to the useful IMDO
tutorial
(http://bio3d.colorado.edu/imod/doc/tomoguide.
html). We also refer the reader to our recently published chapter
[32] where a similar procedure is described for bacterial secretion
systems.
The tilt-series is opened (“Build Tomogram”) using the IMOD
program Etomo’s graphical user interface (GUI). Fill the diameter
of the gold fiducials and specify the tilt-series axis type. Click “Scan
header” to scan the pixel size and image rotation. Then, click
“Create Computer Scripts”. Following this, several steps will
appear on the GUI that need to be performed. These steps include:
4.1.1 Pre-processing
During this step, pixels with very high or low values are removed
using “Ccderaser” to circumvent the generation of artifacts. Use
value 10 for “Peak criterion” and 8 for “Difference criterion”.
Generate a stack by clicking “Create Fixed Stack”, then check the
quality of the stack by clicking “View Fixed Stack” and remove any
bad images from the stack then click “Use Fixed Stack”.
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Mohammed Kaplan et al.
Fluorescent Targets
in the Titan Krios Using
Cryo-CLEM
1. Use SerialEM to collect a 200Â magnification montage for
each EM grid loaded into the Titan Krios (see Note 17).
2. Find a cell of interest imaged previously by cryo-LM using the
letters, symbols, or numbers of the grid support.
3. Use the whole-grid 200Â montage to generate a polygon
montage of the thin cellular peripheral area of interest and
surrounding area. It is advisable to capture fluorescent microspheres in the surrounding area in this montage for registration
of the EM and LM images (see Note 18).
4. Using your polygonal montage, evaluate if the ice is too thick
for tilt-series collection by evaluating the hole that is closest to
the fluorescent puncta using the cryo-LM aligned phase contrast/fluorescence images for orientation.
5. If the ice is thin enough, overlay the fluorescence images with
the polygonal montage images using the fluorescent microspheres seen in both the LM and EM modalities. Any image
processing software can be used to roughly align the images.
6. Next, localize more precisely the location of the fluorescent
puncta in the thin edge.
7. Collect a tilt-series in this location.
4 Data Processing and Tomographic Reconstruction
4.1 Conventional
Tilt-Series
We reconstruct tomograms from raw tilt-series using the IMOD
software [41, 43, 44]. We refer the reader to the useful IMDO
tutorial
(http://bio3d.colorado.edu/imod/doc/tomoguide.
html). We also refer the reader to our recently published chapter
[32] where a similar procedure is described for bacterial secretion
systems.
The tilt-series is opened (“Build Tomogram”) using the IMOD
program Etomo’s graphical user interface (GUI). Fill the diameter
of the gold fiducials and specify the tilt-series axis type. Click “Scan
header” to scan the pixel size and image rotation. Then, click
“Create Computer Scripts”. Following this, several steps will
appear on the GUI that need to be performed. These steps include:
4.1.1 Pre-processing
During this step, pixels with very high or low values are removed
using “Ccderaser” to circumvent the generation of artifacts. Use
value 10 for “Peak criterion” and 8 for “Difference criterion”.
Generate a stack by clicking “Create Fixed Stack”, then check the
quality of the stack by clicking “View Fixed Stack” and remove any
bad images from the stack then click “Use Fixed Stack”.
102
Mohammed Kaplan et al.