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Bright field and fluorescent images of the beads and cells were then acquired using an Axiovert 200M microscope (Zeiss, Minneapolis, MN) in the multiple channel mode that was equipped with a Photometric CoolSNAP HQ CCD camera (Photometrics, Tucson, AZ).
Once a cell containing bound beads was aligned in the focal plane a time series of bright field and fluorescent images were acquired in the multiple channel mode of the Axiovert 200M microscope.
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For each spectrum, 10 20 continuum scans were averaged in multiple channel analyzer mode.
For each spectrum, 50 continuum scans were averaged in multiple channel analyzer (MCA) mode.
For each spectrum, 9 to 150 continuum scans were averaged in multiple channel analyzer (MCA) mode.
Data were acquired over a period of 2 20 min, in multiple-channel acquisition mode and with a dwell time for each ion species of 50 ms.
For the multiple-channel model, BTs can send and receive data with multiple nodes simultaneously in infrastructure mode.
Essentially the algorithm solves a nonlinear, multiple channel dynamic system.
We used the pre-bleach channel mode image and the final post-bleach channel mode image (after 19 bleach exposures) for standard acceptor photobleaching FRET studies.
The final channel mode image set captured after the last bleach exposure corresponded to the post-bleach channel mode image set (Step 4).
In Step 2, we switched to channel mode and collected a pre-bleach channel mode image set using CFP excitation/emission and YFP excitation/emission settings.
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