9. Background strains that are ADE2+, thus not producing the
red pigment accumulating in mutant cells, show reduced cellular background during live imaging.
10. For short-term imaging, not requiring strict temperature control, other types of glass-bottom dishes can be used, i.e.,
MatTek.
11. High-quality and reproducible imaging is achieved only if the
cells are imaged while growing exponentially.
12. The parameters for imaging single mRNAs in living cells (laser
power, exposure time) can be modified to increase the fluorescence intensity of single mRNAs. Choose parameters that will
keep the fluorescence intensity signal in the dynamic range of
the camera while minimizing photo-bleaching of the sample.
13. At this point active lentivirus is being produced. Strict adherence to biosafety class II is necessary. All materials in contact
with virus-containing liquid must be bleached prior to
disposal.
14. Viruses may be stored at 4
C for short periods (hours to days).
For long-term storage, aliquots should be frozen at À80
C.
15. Polybrene increases the efficiency of retrovirus-mediated gene
transfer; the conditions should be optimized for each cell type.
16. Expression levels of the MCP-FP transgene should show a
strong spot for the transcription site, smaller dots throughout
the nucleus (single-molecule mRNA), and a low nuclear background of free unbound MCP-FP. If needed, the best pool can
be re-sorted by FACS to obtain a more homogenous population. Positive cells can be FACS-sorted upon a single-cell clonal
dilution to isolate pure clones. Select the most suitable
sub-cloned cells that display the optimal signal-to-noise ratio
for the live-cell imaging acquisition.
17. We recommend a stable integration of the MCP-GFP with viral
infection followed by sorting a pool of low-expressing
MCP-FP cells. This gives much better results than a crude
transient transfection. Optimization of the MCP-GFP expression further improves the signal-to-noise ratio of singlemolecule detection and enhances the quality of the data
recorded.
18. We recommend stacks of 11 planes with a Z-spacing of 0.6 μm.
This size allows accurate quantification of single mRNA molecules when using 100Â, N.A. 1.4 objectives.
19. These short and long movies are important if one wants to
capture the entire promoter dynamics (see ref. 60), and they are
required since at high temporal resolution, bleaching limits the
acquisitions to about 30 min.
Imaging Single mRNAs in Living Eukaryotic Cells
139
red pigment accumulating in mutant cells, show reduced cellular background during live imaging.
10. For short-term imaging, not requiring strict temperature control, other types of glass-bottom dishes can be used, i.e.,
MatTek.
11. High-quality and reproducible imaging is achieved only if the
cells are imaged while growing exponentially.
12. The parameters for imaging single mRNAs in living cells (laser
power, exposure time) can be modified to increase the fluorescence intensity of single mRNAs. Choose parameters that will
keep the fluorescence intensity signal in the dynamic range of
the camera while minimizing photo-bleaching of the sample.
13. At this point active lentivirus is being produced. Strict adherence to biosafety class II is necessary. All materials in contact
with virus-containing liquid must be bleached prior to
disposal.
14. Viruses may be stored at 4
C for short periods (hours to days).
For long-term storage, aliquots should be frozen at À80
C.
15. Polybrene increases the efficiency of retrovirus-mediated gene
transfer; the conditions should be optimized for each cell type.
16. Expression levels of the MCP-FP transgene should show a
strong spot for the transcription site, smaller dots throughout
the nucleus (single-molecule mRNA), and a low nuclear background of free unbound MCP-FP. If needed, the best pool can
be re-sorted by FACS to obtain a more homogenous population. Positive cells can be FACS-sorted upon a single-cell clonal
dilution to isolate pure clones. Select the most suitable
sub-cloned cells that display the optimal signal-to-noise ratio
for the live-cell imaging acquisition.
17. We recommend a stable integration of the MCP-GFP with viral
infection followed by sorting a pool of low-expressing
MCP-FP cells. This gives much better results than a crude
transient transfection. Optimization of the MCP-GFP expression further improves the signal-to-noise ratio of singlemolecule detection and enhances the quality of the data
recorded.
18. We recommend stacks of 11 planes with a Z-spacing of 0.6 μm.
This size allows accurate quantification of single mRNA molecules when using 100Â, N.A. 1.4 objectives.
19. These short and long movies are important if one wants to
capture the entire promoter dynamics (see ref. 60), and they are
required since at high temporal resolution, bleaching limits the
acquisitions to about 30 min.
Imaging Single mRNAs in Living Eukaryotic Cells
139