3.3 Determining
the Parameters
for the FRAP
Experiments
1. Decide on the shape, number, and size of the areas to be
bleached (Regions Of Interest, ROI, see Note 4). Circular
areas of 0.5–1 μm diameter are applicable in most cases, and
we use 2–3 circular areas of 1 μm diameter ensuring that only
one event occurs for a cell and that no adjacent cells are
bleached. Important: The same size bleach spot within a sample
and between genotypes must be used during an experiment
[18] (see Note 5).
2. Perform test bleaching to achieve an appropriate level of
bleaching (see Note 6) within a minimal time exposure (see
Note 7). Start by bleaching the selected region for 10 ms
with 100% 488 nm laser. If the bleaching is too strong (see
Note 6), reduce the laser intensity and bleaching time. If the
fluorescence intensity in a selected ROI immediately after
bleaching is above 40% of initial pre-bleach intensity, increase
the bleaching time up to 20 ms or/and use a 405 nm laser
instead.
3. Record a freerun time series (no time interval between frames)
of the fluorescence recovery in a z-stack spanning the structure
of interest, e.g., adherens junction, for 2–5 min starting by
using the lowest power, which yields sufficient clearly visible
signal, during this phase (we use 1% intensity of 488 nm laser).
This will enable to determine the imaging parameters which
allow recording the recovery as fast as possible (see Note 8)
while minimizing any additional photobleaching (see Note 9).
4. Calculate acquisitional photobleaching by comparing the fluorescence intensity of the control (unbleached) ROI at the end
of the time series to its pre-bleach value; and the initial recovery
(between the first and second time points after bleaching, see
Subheading 3.5). Use these values to adjust the imaging parameters. If the acquisitional bleaching is more than 15%, reduce
the laser power or increase the scanning speed (see Note 9). If
the initial recovery is greater than 5%, reduce intervals between
time points by reducing image resolution, applying digital
zoom, or/and increasing scanning speed (see Note 8). If the
initial recovery is below 1%, increase the interval between time
points.
5. Repeat steps 3 and 4 until desired levels of acquisitional
bleaching and initial recovery are achieved, while maintaining sufficient spatial resolution (ROI below 10 pixel in diameter are likely to introduce large noise due to even mild
movements in XY plane). For E-cad-GFP, we use 6Â
0.38 μm sections which span the entire depth of adherens
junction. Each section is 320 Â 320 pixel, with a spatial resolution of 0.093 μm/pixel, and is taken every 20 s. We use a 63Â
magnification lens with a numerical aperture (NA) of 1.4, 1–2%
laser power, 2 μs/pixel dwell with amplification of the Hv/gain
of the PMT for optimal image acquisition.
150
Joshua Greig and Natalia A. Bulgakova
the Parameters
for the FRAP
Experiments
1. Decide on the shape, number, and size of the areas to be
bleached (Regions Of Interest, ROI, see Note 4). Circular
areas of 0.5–1 μm diameter are applicable in most cases, and
we use 2–3 circular areas of 1 μm diameter ensuring that only
one event occurs for a cell and that no adjacent cells are
bleached. Important: The same size bleach spot within a sample
and between genotypes must be used during an experiment
[18] (see Note 5).
2. Perform test bleaching to achieve an appropriate level of
bleaching (see Note 6) within a minimal time exposure (see
Note 7). Start by bleaching the selected region for 10 ms
with 100% 488 nm laser. If the bleaching is too strong (see
Note 6), reduce the laser intensity and bleaching time. If the
fluorescence intensity in a selected ROI immediately after
bleaching is above 40% of initial pre-bleach intensity, increase
the bleaching time up to 20 ms or/and use a 405 nm laser
instead.
3. Record a freerun time series (no time interval between frames)
of the fluorescence recovery in a z-stack spanning the structure
of interest, e.g., adherens junction, for 2–5 min starting by
using the lowest power, which yields sufficient clearly visible
signal, during this phase (we use 1% intensity of 488 nm laser).
This will enable to determine the imaging parameters which
allow recording the recovery as fast as possible (see Note 8)
while minimizing any additional photobleaching (see Note 9).
4. Calculate acquisitional photobleaching by comparing the fluorescence intensity of the control (unbleached) ROI at the end
of the time series to its pre-bleach value; and the initial recovery
(between the first and second time points after bleaching, see
Subheading 3.5). Use these values to adjust the imaging parameters. If the acquisitional bleaching is more than 15%, reduce
the laser power or increase the scanning speed (see Note 9). If
the initial recovery is greater than 5%, reduce intervals between
time points by reducing image resolution, applying digital
zoom, or/and increasing scanning speed (see Note 8). If the
initial recovery is below 1%, increase the interval between time
points.
5. Repeat steps 3 and 4 until desired levels of acquisitional
bleaching and initial recovery are achieved, while maintaining sufficient spatial resolution (ROI below 10 pixel in diameter are likely to introduce large noise due to even mild
movements in XY plane). For E-cad-GFP, we use 6Â
0.38 μm sections which span the entire depth of adherens
junction. Each section is 320 Â 320 pixel, with a spatial resolution of 0.093 μm/pixel, and is taken every 20 s. We use a 63Â
magnification lens with a numerical aperture (NA) of 1.4, 1–2%
laser power, 2 μs/pixel dwell with amplification of the Hv/gain
of the PMT for optimal image acquisition.
150
Joshua Greig and Natalia A. Bulgakova