3. Remove the excess of liquid with a tissue and air bubbles by
gently tapping the coverslip with tweezers.
4. Seal the coverslip to the slide with rubber cement or nail polish
(see Note 67).
3.4 Methods
for Microscopy
Acquisition and Data
Analysis
Here we chose to use a spinning disk microscope to perform
observation of DNA-FISH labeled nuclei in 3D. Prior to imaging
it is assumed that the required laser lines are aligned to the sample
using the appropriate dichroic and emission filters. Note that other
microscopes (epifluorescence, confocal, high-throughput imaging
setup. . .) may be appropriate as long as the setup is capable to
acquire z-stacks.
3.4.1 Spinning Disk
Image Stacks Acquisitions
Laser intensities and camera integration times were optimized for
each channel and each sample. 3D acquisitions were performed in
51 focal planes at 200 nm steps. For each condition, a minimum of
150 fields were acquired to obtain a minimum of 1500 nuclei.
3.4.2 Image Analysis
The DNA-FISH labeled loci of each nucleus appearing on the
multichannel image stacks were detected and their position
reported using a custom-made script running on ImageJ (https://
imagej.nih.gov/ij/; Macro language), enabling automated treatment of the data. Note that commercial scripts are also available, in
particular with high-throughput microscopes.
1. Perform nuclei segmentation by doing a Z-projection of the
image stack (Hoechst staining) followed by (a) a Smooth filter
of the resulting image (b) a binarization by an automatic intensity threshold (Li methods), (c) a Fill Holes function and (d) an
Adjustable Watershed function (tolerance ¼ 3). We extracted
the nuclei morphology parameters (radii length, area, roundness. . .) with the Analyze Particles function (size ¼ 120-Infinity, Circularity ¼ 0.65–1.00).
2. Determination of the nuclei Masks (in 3D) by applying to each
stack of the Hoechst channel a smoothing filter, followed by
binarization applying an automatic intensity threshold
(Li method) and a Fill Holes function. We then extracted the
volume of the nuclei with the 3D object counter plugin [17].
3. Determination of coordinates of the DNA-FISH labeled loci.
In each individual nucleus (Region of interest determined step
1) we apply on every DNA-FISH channel staining (a) a Smooth
filter (b) the Enhance Contrast function (saturated ¼ 0.0015)
(c) the Subtract Background function (rolling ¼ 6) and (d) a
binarization applying an automatic intensity threshold
(RenyiEntropy method). The coordinates of each locus were
recorded using the 3D Object Counter plugins.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
375
gently tapping the coverslip with tweezers.
4. Seal the coverslip to the slide with rubber cement or nail polish
(see Note 67).
3.4 Methods
for Microscopy
Acquisition and Data
Analysis
Here we chose to use a spinning disk microscope to perform
observation of DNA-FISH labeled nuclei in 3D. Prior to imaging
it is assumed that the required laser lines are aligned to the sample
using the appropriate dichroic and emission filters. Note that other
microscopes (epifluorescence, confocal, high-throughput imaging
setup. . .) may be appropriate as long as the setup is capable to
acquire z-stacks.
3.4.1 Spinning Disk
Image Stacks Acquisitions
Laser intensities and camera integration times were optimized for
each channel and each sample. 3D acquisitions were performed in
51 focal planes at 200 nm steps. For each condition, a minimum of
150 fields were acquired to obtain a minimum of 1500 nuclei.
3.4.2 Image Analysis
The DNA-FISH labeled loci of each nucleus appearing on the
multichannel image stacks were detected and their position
reported using a custom-made script running on ImageJ (https://
imagej.nih.gov/ij/; Macro language), enabling automated treatment of the data. Note that commercial scripts are also available, in
particular with high-throughput microscopes.
1. Perform nuclei segmentation by doing a Z-projection of the
image stack (Hoechst staining) followed by (a) a Smooth filter
of the resulting image (b) a binarization by an automatic intensity threshold (Li methods), (c) a Fill Holes function and (d) an
Adjustable Watershed function (tolerance ¼ 3). We extracted
the nuclei morphology parameters (radii length, area, roundness. . .) with the Analyze Particles function (size ¼ 120-Infinity, Circularity ¼ 0.65–1.00).
2. Determination of the nuclei Masks (in 3D) by applying to each
stack of the Hoechst channel a smoothing filter, followed by
binarization applying an automatic intensity threshold
(Li method) and a Fill Holes function. We then extracted the
volume of the nuclei with the 3D object counter plugin [17].
3. Determination of coordinates of the DNA-FISH labeled loci.
In each individual nucleus (Region of interest determined step
1) we apply on every DNA-FISH channel staining (a) a Smooth
filter (b) the Enhance Contrast function (saturated ¼ 0.0015)
(c) the Subtract Background function (rolling ¼ 6) and (d) a
binarization applying an automatic intensity threshold
(RenyiEntropy method). The coordinates of each locus were
recorded using the 3D Object Counter plugins.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
375