For fluorescence recovery after photobleaching (FRAP) experiments, DIV14 neurons were transfected with Venus-mGluR5 and pSM155-mCherry or mirShank::mCherry, and imaged on a spinning disk confocal system (described above). FRAP experiments were performed using the ILas2 system (Roper Scientific). Individual spines were photobleached with a targeted 491 nm laser and imaged every 5 seconds for fluorescence recovery for a period of 5 minutes.
Ilas2 system
Manufactured by Teledyne
Sourced in France
The ILas2 system is a high-performance, multi-functional laser system designed for a variety of laboratory applications. It features a stable and reliable laser source with adjustable power output and wavelength options. The system offers flexibility in its configuration and can be customized to meet the specific needs of the user's research or experimental requirements.
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Lab products found in correlation
Axio observer inverted microscope, by Zeiss (1 mentions)
Evolve emccd camera, by Teledyne (1 mentions)
Plan apochromat, by Yokogawa (1 mentions)
Plan apochromat objective, by Yokogawa (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Ixon3 897 emccd camera, by Oxford Instruments (1 mentions)
Metamorph 7, by Molecular Devices (1 mentions)
Ix81 spinning disk confocal microscope, by Yokogawa (1 mentions)
5 protocols using ilas2 system
1
Fluorescence Recovery Dynamics in Neurons
2
Quantitative Fluorescence Recovery Assay
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For quantitative fluorescence recovery after photobleaching (FRAP) experiments, neurons were imaged on the spinning disk microscope described above. FRAP experiments were performed using the ILas2 system (Roper Scientific). Regions of primary dendrite were photobleached with high laser power (10 times at 20% laser power with a Vortran Stradus 405 nm (100 mW) laser) and imaged every 30 s for fluorescence recovery for a period of 10 min. For FRAP analysis, the mean intensity of the bleached area was corrected for background values, as well as the bleaching that occurred during image acquisition. Data were normalized to control fluorescence averaged over 1–3 initial frames before bleaching and stated as 100% intensity. Average curves were obtained and represented.
Time-lapse of nocodazole treated RFP-APC2 expressing neurons were analyzed in the same manner: background values were subtracted and data were corrected for bleaching.
Time-lapse of nocodazole treated RFP-APC2 expressing neurons were analyzed in the same manner: background values were subtracted and data were corrected for bleaching.
3
Live Imaging of Nbs and Pupae
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Live imaging of Nbs and pupae was performed at room temperature with either an Axio-observer inverted microscope (Carl Zeiss) equipped with a ×100 oil Plan-Apochromat objective lens (N/A 1.4), a spinning disk (CSUX-A1, Yokogawa), and an EMCCD Evolve camera (Princeton Instrument, Roper Scientific) or a Ti-DH inverted microscope (Nikon) equipped with a ×100 oil Plan-Apochromat objective lens (N/A 1.45), a spinning disk (CSU-W1-T1, Yokogawa), and a sCMOS camera (Roper Scientific). Images were acquired with Metamorph software (Molecular Device). Twelve to 20 Z of 0.5 µm steps were acquired every 20 s except in Fig. 3a–f, where one z plane was acquired every second (a-e) or every 5 sec (f). Fixed preparations and live embryos were observed using a DMI6000 inverted microscope equipped with a Plan-Apochromat ×63 objective (N/A 1.4) and a Leica TCS SP8 laser confocal imaging system. To induce the photoconversion of Sqh::Dendra2, cells were irradiated with a 405 nm laser (2.5% power, 1000 repetition) on a user-defined region for about 6–8 s using an Ilas2 system (Roper Scientific). Images in all figures are maximum projections except in Fig. 3a, b, d, e, f and DIC images.
4
Photoactivation of T. gondii in HFF cells
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For photoactivation studies, T. gondii tachyzoites were transiently transfected with ptubGAP40mEos2-sagCAT, inoculated into HFF cell cultures grown on glass-bottomed dishes as described above, and incubated for 12–16 h at 37°C. Samples were then transferred to an OKOLab stage-top environmental chamber (Warner Instruments, Hamden, CT) that was equipped with a digital temperature and humidity control unit as well as a manual gas controller unit and was equilibrated ∼2 h before data acquisition. Photoactivation was performed using an Olympus IX-81 spinning-disk confocal microscope equipped with an UPlanSApo 100× oil-immersion objective (NA1.4), CSU-10 scanner (Yokogawa) and iXon3 897 EMCCD camera (Andor Technology) and the iLas2 system (Roper Scientific, Paris, France) that employs a 50 mW diode-pumped crystal laser (CrystaLaser model DL405-050-O, wavelength of 405 nm).
Owing to the spontaneous photobleaching properties of mEos2, single images were acquired (no stacks) at selected time-points only using MetaMorph 7.7 (Molecular Devices). Data was collected at 488 and 561 nm (5–25% laser power; emission filters 525/50 nm for GFP and 617/73 nm for TRITC). Some images were contrast enhanced for figure presentation but all quantitative measurements were performed using unprocessed data.
Owing to the spontaneous photobleaching properties of mEos2, single images were acquired (no stacks) at selected time-points only using MetaMorph 7.7 (Molecular Devices). Data was collected at 488 and 561 nm (5–25% laser power; emission filters 525/50 nm for GFP and 617/73 nm for TRITC). Some images were contrast enhanced for figure presentation but all quantitative measurements were performed using unprocessed data.
5
Axonal Fluorescence Recovery Kinetics
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The experiments were performed using the ILas2 system (Roper Scientific). A region at the proximal axon was bleached with high laser power and fluorescence recovery was observed for a period of 10min. For FRAP analysis, the mean intensity of the bleached area was corrected with background values, as well as the bleaching that occurred during image acquisition. Data were normalized with control fluorescence averaged over 5 initial frames before bleaching and stated as 100% intensity. Average curves were obtained and represented.
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