acquisition) where one Z-stack is recorded every 3 s for 30 min
and long movies where one Z-stack is recorded every 3 min for
>8 h (see Note 19).
3. Analyze the time-lapse movies of transcription sites with dedicated software tools that are available upon request
(MS2-quant, RampFinder, RampFitter, ON-quant, see ref.
60) (see Note 20).
4 Notes
1. Yeast microscopy experiments were performed on a home-built
microscope built around an IX71 stand (Olympus). For excitation, a 491 nm laser (CalypsoTM, Cobolt) and a 561 nm laser
(JiveTM, Cobolt) were combined and controlled by an
acoustic-optic tunable filter (AOTF, AOTFnC-400.650-TN,
AA Opto-electronic) before being coupled into a single-mode
optical fiber (Qioptiq). The output of the fiber was collimated
and delivered through the back port of the microscope and
reflected into an Olympus 150x 1.45 N.A. oil immersion
objective lens with a dichroic mirror (zt405/488/561rpc,
2 mm substrate, Chroma). The tube lens (180 mm focal
length) was removed from the microscope and placed outside
of the right port. A triple-band notch emission filter (zet405/
488/561 nm) was used to filter the scattered laser light. A
dichroic mirror (T560LPXR, 3 mm substrate, Chroma) was
used to split the fluorescence onto two precisely aligned
EMCCDs (Andor iXon, Model DU-897 U-CS0, pixel size
16 μm) mounted on alignment stages (x, y, z, θ-, and φangle). Emission filters FF03-525/50-25 and FF01-607/7025 (Semrock) were placed in front of green and red channel
cameras, respectively. The two cameras were triggered for
exposure with a TTL pulse generated on a DAQ board (Measurement Computing). The microscope was equipped with a
piezo stage (ASI) for fast z-stack and a Delta-T incubation
system (Bioptech) for live-cell imaging. The microscope
(AOTF, DAQ, stage, and cameras) was automated with the
software MetaMorph (Molecular Devices).
2. XL1-blue competent cells are bacteria of choice for transformation of plasmid containing MS2 repeats. Note that the
bacteria can be grown at 30
C if the plasmid is unstable.
3. Other Flp-in mammalian cell lines can be used.
4. A stable Flag-Tat-expressing cell line was created by CRISPR
genome editing using an AAVS1 repair vector [60] in HeLa
Flp-in H9 cell line (available upon request). Individual clones
were picked and analyzed by immunofluorescence with an antiImaging Single mRNAs in Living Eukaryotic Cells
137
and long movies where one Z-stack is recorded every 3 min for
>8 h (see Note 19).
3. Analyze the time-lapse movies of transcription sites with dedicated software tools that are available upon request
(MS2-quant, RampFinder, RampFitter, ON-quant, see ref.
60) (see Note 20).
4 Notes
1. Yeast microscopy experiments were performed on a home-built
microscope built around an IX71 stand (Olympus). For excitation, a 491 nm laser (CalypsoTM, Cobolt) and a 561 nm laser
(JiveTM, Cobolt) were combined and controlled by an
acoustic-optic tunable filter (AOTF, AOTFnC-400.650-TN,
AA Opto-electronic) before being coupled into a single-mode
optical fiber (Qioptiq). The output of the fiber was collimated
and delivered through the back port of the microscope and
reflected into an Olympus 150x 1.45 N.A. oil immersion
objective lens with a dichroic mirror (zt405/488/561rpc,
2 mm substrate, Chroma). The tube lens (180 mm focal
length) was removed from the microscope and placed outside
of the right port. A triple-band notch emission filter (zet405/
488/561 nm) was used to filter the scattered laser light. A
dichroic mirror (T560LPXR, 3 mm substrate, Chroma) was
used to split the fluorescence onto two precisely aligned
EMCCDs (Andor iXon, Model DU-897 U-CS0, pixel size
16 μm) mounted on alignment stages (x, y, z, θ-, and φangle). Emission filters FF03-525/50-25 and FF01-607/7025 (Semrock) were placed in front of green and red channel
cameras, respectively. The two cameras were triggered for
exposure with a TTL pulse generated on a DAQ board (Measurement Computing). The microscope was equipped with a
piezo stage (ASI) for fast z-stack and a Delta-T incubation
system (Bioptech) for live-cell imaging. The microscope
(AOTF, DAQ, stage, and cameras) was automated with the
software MetaMorph (Molecular Devices).
2. XL1-blue competent cells are bacteria of choice for transformation of plasmid containing MS2 repeats. Note that the
bacteria can be grown at 30
C if the plasmid is unstable.
3. Other Flp-in mammalian cell lines can be used.
4. A stable Flag-Tat-expressing cell line was created by CRISPR
genome editing using an AAVS1 repair vector [60] in HeLa
Flp-in H9 cell line (available upon request). Individual clones
were picked and analyzed by immunofluorescence with an antiImaging Single mRNAs in Living Eukaryotic Cells
137