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Distance measurement
To measure cell substrate distance, the cell is attached to a silicon chip with microscopic steps made of transparent silicon oxide. These steps modulate the distance to the silicon chip and thus the fluorescence intensity. The cell substrate distance provides a shift of the fluorescence intensity versus oxide thickness. You can simulate a FLIC distance measurement with the applet below, just move the sliders to change the main parameters.

[when the applet doesn't work correctly...]
Have a look at the systematic error and at the measurement of ghosts.

See the 4 oxide applet