7. Wash the labeled cells thrice with 0.05% TritonX-100 for
5 min.
8. Incubate cells in PBS, and then use FITC and Cy5 filter sets to
image MB and IF signals, respectively. Representative fluorescence images of centromeres are shown in Fig. 2b (see
Note 23).
3.5 Dual-Color
Single-Particle
Tracking Analysis
Dynamic properties of genomic loci can be determined by singleparticle tracking analysis as described below (see Notes 14 and 24).
1. Open time-lapse images acquired at 100 ms per frame for
600 frames in Fiji.
2. Set image interval properties. Select Image > Properties, set
Channels (c) to 1, Slices (z) to 1, Frames (t) to 600, Unit of
length to “μm,” Pixel width and Pixel height to 0.16, Voxel depth
to 0, Frame interval to 104 ms (see Note 25), and Origin to
0,0. Select Global.
3. Click Plugins > Tracking > TrackMate, check the parameters
input in step 2, and click next.
4. Select LoG detector and click next.
5. Pre-filter the spots. Set Estimated blob diameter to 0.5 μm, and
set appropriate Threshold to see most light spots. Select Do
sub-pixel localization. Click next.
6. Review the settings for the pre-filtering process in step 5, and
click next.
7. Improve spot selection by adjusting the initial threshold on the
Quality feature and click next.
8. Select HyperStack Displayer so that the spots can be manually
edited and click next.
9. Apply more filters, such as Quality, Minimal Intensity, and
Maximal intensity, as needed. Click next.
10. Select Simple LAP tracker. Click next.
11. Set parameters for Simple LAP tracker. Set Linking max distance to 0.5 μm and Gap-closing max frame gap to 4 μm. For
telomeres, Gap-closing max distance is set to 1 μm, and for
centromeres, Gap-closing max distance is set to 0.7 μm (see
Note 26). Click next.
12. Review the previous settings and click next.
13. Filter tracks by adjusting the threshold on the Number of spots
in track feature. Click next.
14. Select Display spots and set Spot display radius ratio to 1. Select
Display tracks and set Limit frame depth to 10. Click Analysis to
obtain track statistics.
Visualizing Genomic Loci by CRISPR/MB
367
5 min.
8. Incubate cells in PBS, and then use FITC and Cy5 filter sets to
image MB and IF signals, respectively. Representative fluorescence images of centromeres are shown in Fig. 2b (see
Note 23).
3.5 Dual-Color
Single-Particle
Tracking Analysis
Dynamic properties of genomic loci can be determined by singleparticle tracking analysis as described below (see Notes 14 and 24).
1. Open time-lapse images acquired at 100 ms per frame for
600 frames in Fiji.
2. Set image interval properties. Select Image > Properties, set
Channels (c) to 1, Slices (z) to 1, Frames (t) to 600, Unit of
length to “μm,” Pixel width and Pixel height to 0.16, Voxel depth
to 0, Frame interval to 104 ms (see Note 25), and Origin to
0,0. Select Global.
3. Click Plugins > Tracking > TrackMate, check the parameters
input in step 2, and click next.
4. Select LoG detector and click next.
5. Pre-filter the spots. Set Estimated blob diameter to 0.5 μm, and
set appropriate Threshold to see most light spots. Select Do
sub-pixel localization. Click next.
6. Review the settings for the pre-filtering process in step 5, and
click next.
7. Improve spot selection by adjusting the initial threshold on the
Quality feature and click next.
8. Select HyperStack Displayer so that the spots can be manually
edited and click next.
9. Apply more filters, such as Quality, Minimal Intensity, and
Maximal intensity, as needed. Click next.
10. Select Simple LAP tracker. Click next.
11. Set parameters for Simple LAP tracker. Set Linking max distance to 0.5 μm and Gap-closing max frame gap to 4 μm. For
telomeres, Gap-closing max distance is set to 1 μm, and for
centromeres, Gap-closing max distance is set to 0.7 μm (see
Note 26). Click next.
12. Review the previous settings and click next.
13. Filter tracks by adjusting the threshold on the Number of spots
in track feature. Click next.
14. Select Display spots and set Spot display radius ratio to 1. Select
Display tracks and set Limit frame depth to 10. Click Analysis to
obtain track statistics.
Visualizing Genomic Loci by CRISPR/MB
367