Question:  what is the sharpness of edge of bleaching field in X, Y and Z axes when bleaching with Zeiss Duo?

To address this, we used green fluorescent Plexiglas distributed by Chroma Technology as a sample because we assumed it is fluorescent, has a refractive index close enough to aqueous material to be relevant (1.5) and effectively does not have diffusion of the fluorescence.

We used the 100X N.A. 1.4 objective with Zeiss oil (IR 1.518) without a coverslip.  We focused approximately 40 um up into the Plexiglas and bleached 100 to 200 iterations at 100% intensity with the 489 nm laser.  This was followed by a Z series at 0.5 um steps with laser at approximately 4% intensity.

Results follow.

Z axis resampling shows that bleaching in cell cultures is a column of bleaching through the entire depth of the cells.

Illustration of sharpness of boundary of bleach (without quantification of steepness of gradient).

 

Z axis reslice with intensity plot profiles.
Left: XY scan at plan of bleach.
Center:  YZ reslice along magenta line at left with lines drawn on to estimate cone of bleaching (red) and profile for intensity measurement (magenta).
Right:  Intensity plots.

YZ fft filtered to pass features less than 64 pixels in diameter.

Gaussian blurred 16 radius.  Intensity plot is right magenta line.

 

IFRAP may have higher Z axis resolution due to the cone of bleaching although the XY edges of the bleaching region are not so sharp.

Z-axis-IFRAP.jpg (101674 bytes)

 

We have not looked at whether the angle of the Z axis being skewed is due to our sample's placement on the stage or other alignment considerations.  More for a later date...

 

Michael Cammer cammer@aecom.yu.edu 20071209