3.9 Image
Quantification
The number of localization events for each transcript can be calculated using Volocity or any other spot detection software, for
example SpotCounter plug-in of ImageJ [13], or FISH-quant
[14]. We used Volocity for generating the figure. However, directions for using the more readily available ImageJ FIJI software are
provided below (For Volocity software parameters, see Note 1).
1. Remove noise using a noise filter. For ImageJ users, this step is
done using Plugin ! Analysis ! SpotCounter. Set the prefilter
to Gaussian1_5.
2. Set the intensity threshold and size limit to capture only the
smFISH signal dots but not the background noise. The
smFISH signal looks like punctate dots, while the background
usually has undefined shapes and mixed intensity values. In
Image J, set BoxSize to 6. Choose the Noise tolerance level
so that only the smFISH dots were detected (with the check
box for Check Settings selected).
3. Count the number of RNA spots detected.
4. At least three replicates should be used for statistical analysis.
4 Notes
1. We used Volocity for detecting smFISH signal. Any other dot
detection software and smFISH image processing software can
also be used, for example FISH-quant [14]. For Volocity users,
the pathway we created was as follows: Remove noise using
Volocity default noise filter. Create a Find Object pipeline for
the R1 channel. We set a lower intensity threshold at 2, with a
minimum object size of 0.01 μm
2 . The parameters we used are
Clip to ROIs (draw a circle at the region of interest), Separate
Touching Objects (0.05 μm
3
), Exclude Object (<0.05 μm
3 ),
and Exclude Object (>0.8 μm
3 ).
2. A good chemical fixation is critical for preserving the RNAs and
shapes of the plant cells. Some plant samples are very difficult to
fix. The ultimate standard is to have the sample sink to the
bottom of the fixative. An extended vacuum time can be
applied for hard-to-fix samples. A lightweight object could
also be used to hold the sample below the fixation solution
level to help penetration of the fixative.
3. Protease concentration and incubation times could affect the
signal/background ratio. Generally, an incubation with
50 mg/mL protease for 20 min works for most plant tissues.
If the obtained smFISH signal is weak, a higher protease concentration and longer incubation time can be used to increase
probe penetration.
4. Treatment with triethanolamine-acetic anhydride (TAE) acetylates the positively charged amino groups. We use this step to
30
Kun Huang et al.
Quantification
The number of localization events for each transcript can be calculated using Volocity or any other spot detection software, for
example SpotCounter plug-in of ImageJ [13], or FISH-quant
[14]. We used Volocity for generating the figure. However, directions for using the more readily available ImageJ FIJI software are
provided below (For Volocity software parameters, see Note 1).
1. Remove noise using a noise filter. For ImageJ users, this step is
done using Plugin ! Analysis ! SpotCounter. Set the prefilter
to Gaussian1_5.
2. Set the intensity threshold and size limit to capture only the
smFISH signal dots but not the background noise. The
smFISH signal looks like punctate dots, while the background
usually has undefined shapes and mixed intensity values. In
Image J, set BoxSize to 6. Choose the Noise tolerance level
so that only the smFISH dots were detected (with the check
box for Check Settings selected).
3. Count the number of RNA spots detected.
4. At least three replicates should be used for statistical analysis.
4 Notes
1. We used Volocity for detecting smFISH signal. Any other dot
detection software and smFISH image processing software can
also be used, for example FISH-quant [14]. For Volocity users,
the pathway we created was as follows: Remove noise using
Volocity default noise filter. Create a Find Object pipeline for
the R1 channel. We set a lower intensity threshold at 2, with a
minimum object size of 0.01 μm
2 . The parameters we used are
Clip to ROIs (draw a circle at the region of interest), Separate
Touching Objects (0.05 μm
3
), Exclude Object (<0.05 μm
3 ),
and Exclude Object (>0.8 μm
3 ).
2. A good chemical fixation is critical for preserving the RNAs and
shapes of the plant cells. Some plant samples are very difficult to
fix. The ultimate standard is to have the sample sink to the
bottom of the fixative. An extended vacuum time can be
applied for hard-to-fix samples. A lightweight object could
also be used to hold the sample below the fixation solution
level to help penetration of the fixative.
3. Protease concentration and incubation times could affect the
signal/background ratio. Generally, an incubation with
50 mg/mL protease for 20 min works for most plant tissues.
If the obtained smFISH signal is weak, a higher protease concentration and longer incubation time can be used to increase
probe penetration.
4. Treatment with triethanolamine-acetic anhydride (TAE) acetylates the positively charged amino groups. We use this step to
30
Kun Huang et al.