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Pinhole size was set such that the optical slice was 1 µm for all channels.
To determine this scattering effect, a calibration of the optical slice sizes is necessary depending on the pinhole size.
The optical slice was set to 1 μM (FWHM).
Figure 1 figure supplement 2D has 3 mistakes: 1) The optical slice depth 400nm is an overestimation.
The optical slice described previously in transparent media should then be modified according to this scattering effect.
Three or two channel recordings were performed consecutively in Z-series with gaps of 250 nm between the optical slices.
In Figure 4a, we show the evolution of fluorescence recovery along one of the optical slices.
Indeed, as it was shown, the optical slices represent the collected signal from the surface down to optical depths (z) that are collected by each pinhole size.
In our system the typical spectral acquisition time for single optical slice is in the range of minutes.
Bottom, the same optical slice imaged for Yan-YFP fluorescence.
We therefore sampled from similar areas of cell membrane wherever the two-photon optical slice passed through the cells.
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