[8, 58] and this protocol is detailed in Chapter 4 of this book as well
as in [43, 58]. Figure 1a, b shows an example of smFISH for the
MDN1 mRNA in wild-type cells, for side-by-side comparison with
the live imaging results shown in Fig. 1c–f. For live imaging, the
MDN1 mRNA was tagged at the 3
0 UTR with 24xMBSV6 (Fig. 1c)
[58]. To distinguish the nucleus from the cytoplasm, the nuclear
pore protein Nup49 is endogenously fused to the red fluorescent
protein tdTomato (YET443 MATa; his3Δ1; leu2Δ0; met15Δ0;
ura3Δ0 NUP49::NUP49-tdTomato KAN- CRE recombined
MDN1:: MDN1 3UTR 24MS2V6 KAN- CRE recombined;
Ycp-Lac111 CYC1p MCP-NLS-2xyeGFP).
1. For short-term imaging and fast acquisition to follow mRNAs
with high temporal resolution, stream one single Z-plane,
50 ms exposure. Detection of single mRNAs tagged with
MCP-GFP is achieved by using 10% of 100 mW 491 laser
(~1–2 mW/cm
2 measured at the objective). To detect
Nup49-tdTomato, use 1% of 50 mW 561 laser (~0.5 mW/
cm
2 measured at the objective). Under these conditions the
mRNAs in the cytoplasm and the transcription sites in the
nucleus can be visualized for 2–3 min before significant photobleaching occurs (see Supplementary Video 1).
2. For long-term imaging, i.e., over the course of a complete cell
cycle, and to cover the whole cell width, take 15 Z-stacks every
0.5 μm every 2 min (~90 Z-stacks total). An exposure of 50 ms
for each Z-plane was used under our conditions. To visualize
MBSV6-MCP-NLS-2xyeGFP-labeled mRNAs use a 491 nm
wavelength laser. For visualization of single mRNA molecules,
set the laser to 10% power (~1–2 mW/cm
2 measured at the
objective). To detect Nup49-tdTomato, use 1% of 50 mW
561 laser (~0.5 mW/cm
2 measured at the objective). Acquire
Z-planes at different stage positions and use them to detect the
number, the position, and the brightness of mRNAs in living
cells (Fig. 1d and see Note 12).
3.1.5 Imaging Analysis
1. To improve the signal-to-noise ratio, restore the images using a
deconvolution software such as the Huygens software package.
Automatically compute the theoretical point spread function
based on your microscope settings. Restore the images using
the classic maximum likelihood estimation algorithm (i.e.,
number of iterations ¼ 99; signal/noise ratio ¼ 15).
2. To measure the number, position, and brightness of the
mRNAs in single cells, use the freely available software FISHquant running on Matlab [82]. Deconvolved images can be
analyzed with FISH-quant without further filtering. Cell outlines can be created using FISH-quant or using the freely
available software CellProfiler [83]. Recent versions of FISH134
Xavier Pichon et al.
as in [43, 58]. Figure 1a, b shows an example of smFISH for the
MDN1 mRNA in wild-type cells, for side-by-side comparison with
the live imaging results shown in Fig. 1c–f. For live imaging, the
MDN1 mRNA was tagged at the 3
0 UTR with 24xMBSV6 (Fig. 1c)
[58]. To distinguish the nucleus from the cytoplasm, the nuclear
pore protein Nup49 is endogenously fused to the red fluorescent
protein tdTomato (YET443 MATa; his3Δ1; leu2Δ0; met15Δ0;
ura3Δ0 NUP49::NUP49-tdTomato KAN- CRE recombined
MDN1:: MDN1 3UTR 24MS2V6 KAN- CRE recombined;
Ycp-Lac111 CYC1p MCP-NLS-2xyeGFP).
1. For short-term imaging and fast acquisition to follow mRNAs
with high temporal resolution, stream one single Z-plane,
50 ms exposure. Detection of single mRNAs tagged with
MCP-GFP is achieved by using 10% of 100 mW 491 laser
(~1–2 mW/cm
2 measured at the objective). To detect
Nup49-tdTomato, use 1% of 50 mW 561 laser (~0.5 mW/
cm
2 measured at the objective). Under these conditions the
mRNAs in the cytoplasm and the transcription sites in the
nucleus can be visualized for 2–3 min before significant photobleaching occurs (see Supplementary Video 1).
2. For long-term imaging, i.e., over the course of a complete cell
cycle, and to cover the whole cell width, take 15 Z-stacks every
0.5 μm every 2 min (~90 Z-stacks total). An exposure of 50 ms
for each Z-plane was used under our conditions. To visualize
MBSV6-MCP-NLS-2xyeGFP-labeled mRNAs use a 491 nm
wavelength laser. For visualization of single mRNA molecules,
set the laser to 10% power (~1–2 mW/cm
2 measured at the
objective). To detect Nup49-tdTomato, use 1% of 50 mW
561 laser (~0.5 mW/cm
2 measured at the objective). Acquire
Z-planes at different stage positions and use them to detect the
number, the position, and the brightness of mRNAs in living
cells (Fig. 1d and see Note 12).
3.1.5 Imaging Analysis
1. To improve the signal-to-noise ratio, restore the images using a
deconvolution software such as the Huygens software package.
Automatically compute the theoretical point spread function
based on your microscope settings. Restore the images using
the classic maximum likelihood estimation algorithm (i.e.,
number of iterations ¼ 99; signal/noise ratio ¼ 15).
2. To measure the number, position, and brightness of the
mRNAs in single cells, use the freely available software FISHquant running on Matlab [82]. Deconvolved images can be
analyzed with FISH-quant without further filtering. Cell outlines can be created using FISH-quant or using the freely
available software CellProfiler [83]. Recent versions of FISH134
Xavier Pichon et al.