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- 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 below shows a green and red fluorescenct section of plant root. The signal in both channels is saturated, meaning that the light is too bright and the detector is measuring maximum signal in large parts of the image. It would be impossible to quantify fluorescence within the saturated regions because the intensity value is off the scale. In such a case it is necessary to adjust the imaging settings to reduce the intensity to a measurable level.
- Before adjusting the settings it can be useful to change the image colour look-up table (LUT) to Hi-Lo. This is a greyscale LUT with the brightest and dimmest pixel values (usually 255 and 0) coloured red and blue, respectively. 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 Hi-Lo on the right. There is a choice of other LUTs if you want to change the appearance of your image later on.
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 Increasing the laser power (2 below) will increase the fluorescence emitted by your specimen but will also increase the bleach rate and phototoxicity. Also, the intensity will no longer increase if all the fluorophores in your specimen are already in the excited state. The detector voltage HV (3 below) increases or decreases the brightness by changing the amplification of the signal. This won't result in bleaching but it does amplify the photon noise to the same degree as the signal and also adds some multiplicative noise. Gain is a 'digital gain' that simply applies a multiplication factor to the digital value of all the pixels. Offset changes the black level of the image. Increasing the offset will make the background darker by setting more pixel values at the lower end of the scale to zero. Make these adjustments for all channels that you would like to image. Then , then change the LUT back to the colour you would like to view your channel inwhatever colour you want.
To stop the Live View click the RepeatStop button in the Image Acquisition Control window.