3.6 Single-Molecule
Detection
To detect single mRNAs, we recommend the use of the freely
available software FISH-quant [31]. Here we summarize the standard procedure using FISH-quant to measure the number of
mRNAs in the cytoplasm and nascent transcripts at transcription
sites. smFISH-IF was performed for the CLN2 mRNA and for
tubulin proteins (Fig. 3). The details for the mRNA quantification
steps are exemplified in Fig. 6.
1. Define the outline of all the cells included in the analysis using
either the Cy3 or the Cy5 channel. At this stage also select the
transcription sites.
2. Subtract the background using the 3D_LoG option (Size 5;
Standard deviation 1).
3. Pre-detect the mRNAs for one specific cell for which you
defined the outline before (see step 1), and where you can easily
count the mRNAs. Adjust the parameters to make sure that the
program detects the number of mRNAs that you expect. Apply
the settings to the entire image.
4. Fit the spots in the cytoplasm to a three-dimensional
(3D) Gaussian to determine the coordinates of the mRNAs.
5. Check if the selected setting recognized most mRNAs (>95%).
The intensity and width of the 3D Gaussian curve can be
adjusted to exclude nonspecific signal. If the selected settings
are satisfactory, you can save the detection settings and
detected spots (Fig. 6b).
6. If many images are analyzed, create the outlines for all your
images first. Then open in Tools> Batch processing. Load your
previously saved settings for all your outlines. Process all your
images in batch.
7. Use the option “average of thresholded spots” to obtain the
average intensity of all the mRNAs and use it to determine the
intensity of each transcription site.
8. To generate the settings for the transcription site selection
open Tools> TxSite quantification. Load the image of the
“average of thresholded spots” and quantify the TxSite for all
sites. Save the setting that can be now loaded in the Batch
processing mode for automatic transcription site quantification
for all images.
9. Save the results. From the menu select: Batch > Save > Summary
nascent and mature mRNAs, which gives the number of spots
in each cell. From the menu select: Batch > Save > Summary of
All Thresholded spots, which give the information about the x,
y,z position and intensity of each spot. Because these files are .
txt files, the information can be easily copied to other analysis
Detection of mRNA and Proteins in Single Cells by smFISH-IF
63
Detection
To detect single mRNAs, we recommend the use of the freely
available software FISH-quant [31]. Here we summarize the standard procedure using FISH-quant to measure the number of
mRNAs in the cytoplasm and nascent transcripts at transcription
sites. smFISH-IF was performed for the CLN2 mRNA and for
tubulin proteins (Fig. 3). The details for the mRNA quantification
steps are exemplified in Fig. 6.
1. Define the outline of all the cells included in the analysis using
either the Cy3 or the Cy5 channel. At this stage also select the
transcription sites.
2. Subtract the background using the 3D_LoG option (Size 5;
Standard deviation 1).
3. Pre-detect the mRNAs for one specific cell for which you
defined the outline before (see step 1), and where you can easily
count the mRNAs. Adjust the parameters to make sure that the
program detects the number of mRNAs that you expect. Apply
the settings to the entire image.
4. Fit the spots in the cytoplasm to a three-dimensional
(3D) Gaussian to determine the coordinates of the mRNAs.
5. Check if the selected setting recognized most mRNAs (>95%).
The intensity and width of the 3D Gaussian curve can be
adjusted to exclude nonspecific signal. If the selected settings
are satisfactory, you can save the detection settings and
detected spots (Fig. 6b).
6. If many images are analyzed, create the outlines for all your
images first. Then open in Tools> Batch processing. Load your
previously saved settings for all your outlines. Process all your
images in batch.
7. Use the option “average of thresholded spots” to obtain the
average intensity of all the mRNAs and use it to determine the
intensity of each transcription site.
8. To generate the settings for the transcription site selection
open Tools> TxSite quantification. Load the image of the
“average of thresholded spots” and quantify the TxSite for all
sites. Save the setting that can be now loaded in the Batch
processing mode for automatic transcription site quantification
for all images.
9. Save the results. From the menu select: Batch > Save > Summary
nascent and mature mRNAs, which gives the number of spots
in each cell. From the menu select: Batch > Save > Summary of
All Thresholded spots, which give the information about the x,
y,z position and intensity of each spot. Because these files are .
txt files, the information can be easily copied to other analysis
Detection of mRNA and Proteins in Single Cells by smFISH-IF
63