...
- The easiest way to create suitable laser and detector settings in the FV10-ASW software is by using the Dye List. This can be accessed by clicking the on the Dye List icon located on the right hand side of the Image Acquisition Control panel.
In the Dye List menu (shown below) drag the dyes you would like to use into the Selected Dyes box or double-click to select them. If you want to remove any dyes you can drag them out of the list individually or click clear all to remove all dyes and start over. Click Apply and the lightpath for the selected dye combination will be configured automatically (includes activation of lasers and selection of detectors and filters)
Note You cannot select more than three dyes at a time using the Dye List and certain dye combinations are not possible because the dichroic mirror combinations do not allow them. If you would like to image such a forbidden dye combination this can be achieved by using the Virtual Channels tool. How to set this up is described in the section USING VIRTUAL CHANNELS TO ACQUIRE MULTICHANNEL CONFOCAL IMAGES.
- The combination of detectors activated in the Image Acquisition Control window (CHS1, CHS2, CH3, etc.) depends on the dyes you chose. The lasers you need for fluorescence excitation will be automatically ticked in the Acquisition Setting menu. Click the XY Repeat button to open a Live View window to preview your specimen. Clicking the Focus x2 or Focus x4 button also initiates a Live View, but these high-speed scans will have a lower resolution. You can use them to focus easily on your specimen and adjust laser power, gain etc.
- The Live View window below shows a saturated fluorescent root section. You will need to adjust the acquisition parameters until there is no more saturation in your images or if your sample is very dim so that you detect sufficient fluorescence signal