Alignment of the detectors

Alignment of the detectors for cbf and calcium measurements
The position of the analysis area is critical for the formation of a clear signal representing ciliary activity. The cyclic motion of cilia is drawn in profile below. The amount of variation in position, and therefore range in image intensity, increases towards the ciliary tip.
		Cells viewed from the side showing the profile of ciliary movement.
Consequently, small areas of interest (RED squares) are selected towards the ciliary tip to measure beat frequency. The detector area is designed to be small so that it detects only a few cilia. This greatly reduces the complicating effects that metachronal activity of closely packed cilia can have for the intensity waveform.
		Cells viewed from above to show relative placement of detector areas.
Changes in [Ca²⁺]_ioccur within the cell and are most likely to effect the cilia, at least initially, through their bases. As a result, the area of interest for calcium measurements (GREEN squares) are aligned with the base of the cilia. A slightly larger area is used to decrease the signal to noise ratio.

Focus Level: The ciliary and calcium events occur at different planes. The cilia are above the cell surface while the calcium changes occur within the cell. This difference is accommodated by having the CCD (cilia) and SIT (calcium) camera focused at a the slight different planes (see Simultaneous imaging).	Optics: The ciliary activity is best observed with phase-contrast optics as this enhances the variation in image intensity as the cilia move.