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  1. The FV10-ASW 4.2 confocal image acquisition software is very modular and the main panels are:

    1. Acquisition Setting: allows you to set up the parameters for the confocal image acquisition
    2. Image Acquisition Control: here you can set up the lightpath for your experiment and start an image preview or the image acquisition
    3. Live View: shows as live preview of your sample
    4. Data Manager: displays properties of selected image files
    5. Explorer: here you can access saved files and load acquisition parameters of previous experiments

  2. First you need to set up the light path including lasers and detectors using the Dye List menu. This can be accessed by clicking the on the Dye List icon located on the right hand side of the Image Acquisition Control panel.


     
  3. 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)



  4. Please bear in mind that you cannot select more than three dyes at a time and that certain dye combinations are not available such as green, red and far red. If you would still like to image this 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.

  5. If you would like to check that the light path is configured correctly or want to manually adjust it you can do so by clicking the Light Path & Dyes icon. 



  6. When you have finished with the lightpath configuration the dyes you have selected will be shown in the Image Acquisition Control window and the lasers you are using for fluorescence excitation will be 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.



  7. 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.



  8. Click the LUT (Look Up Table) button in the Live View window to open the LUT panel. Select the channel for which you would like to change the LUT, i.e. CH1, then select a LUT on the right. To check for saturation click Hi-Lo. Saturated areas in the image will be shown in red and non-saturated areas in white (see step 9). The image Offset (minimal signal intensity detected by the sensor) can be adjusted by changing the Offset value in the Image Acquisition Control window. Adjust the value until no or only few blue pixels are displayed.



  9. There are several ways to adjust the intensity of your images. Panel 1 below shows a saturated fluorescence image of a root section at the top and an adjusted image at the bottom. You can adjust the laser power (2), gain or detector voltage HV (3, lower it if saturation, increase if signal too low) for each channel (dye). Be careful not to increase the laser power too much, as this may lead to photobleaching or photodamage of your sample. Make sure to do these adjustments for all channels that you would like to image. Then change the LUT back to the colour you would like to view your channel in.



  10. To stop the Live View click the RepeatStop button in the Image Acquisition Control window.






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