Field Illumination

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Homogeneous field acquisition.
Zooming helps decreasing intensity range between corner (min) and center (max) of the image.

Consequences when measuring intensities in samples

Acquired on LSM710: 3 channels, 1AU pinhole for each channel, pixel size 210nm/px and zoom 0.5.
First the cell in the corner was acquired, then the cell in the center, to show the trend goes beyond photobleaching.

Values on the right represent rounded average intensity for the same cell at the bottom right of the field of view (top row) at the center of the field of view (bottom row)
The loss of intensity is represented by dimming the color of the boxes around the measurements.

Loss

Compensating Uneven Illumination

One way to compensate is to estimate the effect of the illumination using homogeneous samples. Chroma slides, like in the experiment on top, are simple. Other methods involving saturated dye solutions yield better results, especially if the dyes are the same fluorophores as the ones used in imaging.

See the associated article
M. A. MODEL, J. L. BLANK, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope
Journal of Microscopy, Vol. 229, Pt 1 2008, pp. 12–16

Applying FlatField illumination correction at the BIOP

We developed an ImageJ Groovy script to process this data, which can be downloaded below. It works on tiles LSM, CZI and LIF files.
rBIOPFFSTITCH