18. Cryo-tomograms normally suffer from low signal-to-noise
ratio and thus further processing to enhance contrast is
required. There are several procedures for this, and the best
option needs to be determined empirically for each sample. We
commonly use the smooth filter option in the IMOD clip
program or the “Nonlinear Anisotropic Diffusion” filter.
3.5 Segmentation
Model Generation
Segmentation models are a great way to extract essential information from tomograms of the crowded cell interior and an excellent
way to visualize its complex three-dimensionality. Segmentation
model generation can be performed with the IMOD software
package and a detailed tutorial can be found on the IMOD website
(https://bio3d.colorado.edu/imod/doc/3dmodguide.html). In
brief, models in IMOD are structured in three levels:
1. “Object” (e.g. all microtubules in a tomogram).
2. “Contours” (e.g. an individual microtubule).
3. “Points” (points that are placed to trace an individual microtubule; see below). In other words, an “Object” is made of
“Contours” that consist of “Points”. Furthermore, segmentation models can also be used to pursue subtomogram averaging, for example, using the IMOD software PEET (http://
bio3d.colorado.edu/PEET/). Here, we will briefly explain
the generation of segmentation models on three different cellular features. These are:
(a) Filaments, e.g. microtubules.
(b) Membrane vesicles.
(c) Small globular densities, e.g. ribosomes (Fig. 3b, c).
3.5.1 Generate
Segmentation Model
1. Open tomogram in 3dmod.
2. In the “Information window” choose “Model” under mode.
3. Make a new object or use the current one if not in use already.
4. Example 1: Segmenting filaments.
(a) Set “Object type” to “Open”.
(b) Open the “Slicer window” by using the hot key “\”.
(c) Alter rotation angles and “View axis position” so that the
filament of interest appears as straight as possible.
(d) Start at one end of filament and place points along the
center of the filament axis until the other end of the
filament is reach. Adjust tomogram z-slice as necessary.
(e) Make sure that all points selected in (step d) are contained
in the same contour and appear in a linear order.
(f) To trace a second filament, generate a new contour by
using the hot key “n” and repeat the steps c–e.
Cryo-ET of Cellular Structures
15
ratio and thus further processing to enhance contrast is
required. There are several procedures for this, and the best
option needs to be determined empirically for each sample. We
commonly use the smooth filter option in the IMOD clip
program or the “Nonlinear Anisotropic Diffusion” filter.
3.5 Segmentation
Model Generation
Segmentation models are a great way to extract essential information from tomograms of the crowded cell interior and an excellent
way to visualize its complex three-dimensionality. Segmentation
model generation can be performed with the IMOD software
package and a detailed tutorial can be found on the IMOD website
(https://bio3d.colorado.edu/imod/doc/3dmodguide.html). In
brief, models in IMOD are structured in three levels:
1. “Object” (e.g. all microtubules in a tomogram).
2. “Contours” (e.g. an individual microtubule).
3. “Points” (points that are placed to trace an individual microtubule; see below). In other words, an “Object” is made of
“Contours” that consist of “Points”. Furthermore, segmentation models can also be used to pursue subtomogram averaging, for example, using the IMOD software PEET (http://
bio3d.colorado.edu/PEET/). Here, we will briefly explain
the generation of segmentation models on three different cellular features. These are:
(a) Filaments, e.g. microtubules.
(b) Membrane vesicles.
(c) Small globular densities, e.g. ribosomes (Fig. 3b, c).
3.5.1 Generate
Segmentation Model
1. Open tomogram in 3dmod.
2. In the “Information window” choose “Model” under mode.
3. Make a new object or use the current one if not in use already.
4. Example 1: Segmenting filaments.
(a) Set “Object type” to “Open”.
(b) Open the “Slicer window” by using the hot key “\”.
(c) Alter rotation angles and “View axis position” so that the
filament of interest appears as straight as possible.
(d) Start at one end of filament and place points along the
center of the filament axis until the other end of the
filament is reach. Adjust tomogram z-slice as necessary.
(e) Make sure that all points selected in (step d) are contained
in the same contour and appear in a linear order.
(f) To trace a second filament, generate a new contour by
using the hot key “n” and repeat the steps c–e.
Cryo-ET of Cellular Structures
15