All imaging took place in Leibovitz’s L-15 supplemented with 10 µg/mL cycloheximide (Sigma-Aldrich, C4859) and cells were pulsed with biotin as described above. Imaging was performed on a Leica SP8 SMD system at 37 °C, equipped with an HC PL APO CS2 63x/1.20 Water objective. Fluorophores were excited with a pulsed white-light laser, operating at 80 MHz. mCitrine was excited at 514 nm, two separate HyD detectors were used to collect photons, set at 521–565 nm and 613–668 nm, respectively. Photons were collected for 1 min and lifetime histograms of the donor fluorophore were fitted with monoexponential decay functions convoluted with the microscope instrument response function in Leica LAS X.
Sp8 smd system
Manufactured by Leica
The SP8 SMD system is a confocal microscope designed for high-resolution imaging. It features advanced scanning technology and a modular design to accommodate a variety of sample types and imaging techniques. The core function of the SP8 SMD system is to provide researchers with a versatile and powerful tool for visualizing and analyzing their samples at the cellular and subcellular level.
Automatically generated - may contain errors
2 protocols using sp8 smd system
1
Fluorescence Lifetime Imaging of Biotinylated Cells
2
Time-Correlated Single-Photon Counting FLIM Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Time-correlated single-photon counting FLIM
imaging was performed on a Leica SP8 SMD system at 37 °C, equipped
with a HC PL APO CS2 63×/1.20 Water objective. pHLuorin2 was
excited at 488 nm with a pulsed white light laser, operating at 80
MHz. Photons were collected for 1 min or 30 s for time-lapse experiments
with a HyD detector set at 502–530 nm, and lifetime histograms
of the donor fluorophore were fitted with a monoexponential decay
function convoluted with the microscope instrument response function
in Leica LAS X. For reconstructing the images, tiff files with τ
values were generated using FLIMFit77 (link) and
2 × 2 spatial binning and then convoluted with the fluorescence
intensities using a custom-written ImageJ Macro. Ratiometric pH measurements
were done similarly to the FLIM measurements, but the imaging was
performed on a Leica SP8 SMD system at 37 °C, equipped with a
HC PL APO CS2 63×/1.20 Water objective or a Zeiss LSM 800 system
at 37 °C, equipped with a Plan Apochromat 1.4× Oil objective.
RpHLuorin2 was excited at 405 and 488 nm sequentially, images were
acquired with an emission wavelength bandwidth (495–560 nm)
that included an emission wavelength of 508 nm.
See theSupporting Information for experimental details.
imaging was performed on a Leica SP8 SMD system at 37 °C, equipped
with a HC PL APO CS2 63×/1.20 Water objective. pHLuorin2 was
excited at 488 nm with a pulsed white light laser, operating at 80
MHz. Photons were collected for 1 min or 30 s for time-lapse experiments
with a HyD detector set at 502–530 nm, and lifetime histograms
of the donor fluorophore were fitted with a monoexponential decay
function convoluted with the microscope instrument response function
in Leica LAS X. For reconstructing the images, tiff files with τ
values were generated using FLIMFit77 (link) and
2 × 2 spatial binning and then convoluted with the fluorescence
intensities using a custom-written ImageJ Macro. Ratiometric pH measurements
were done similarly to the FLIM measurements, but the imaging was
performed on a Leica SP8 SMD system at 37 °C, equipped with a
HC PL APO CS2 63×/1.20 Water objective or a Zeiss LSM 800 system
at 37 °C, equipped with a Plan Apochromat 1.4× Oil objective.
RpHLuorin2 was excited at 405 and 488 nm sequentially, images were
acquired with an emission wavelength bandwidth (495–560 nm)
that included an emission wavelength of 508 nm.
See the
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