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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. You can stop the scan by clicking RepeatStop. Clicking the Focus x2 or Focus x4 button will also initiates a Live View, but these initiate a live scan. These are high-speed scans will that have a lower resolution . You but 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.
- 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 change the LUT to whatever colour you want.
To stop the Live View click the RepeatStop button in the Image Acquisition Control windowBefore starting the image there are a few more parameters you can adjust in the Acquisition Setting panel:
In Mode you can pick between the unidirectional (Oneway) or bidirectional (Roundtrip) scan mode. Roundtrip on this microscope tends to display unwanted phase shifts in the images. It is recommended to use the Oneway rather than the Roundtrip scan mode on this machine. If you select one of the ROIs to the right of the scan mode you can acquire images with special formats such as Lines, Points or Rectangles. The pixel dwell time can be adjusted using the slider and dragging it to the desired scan speed. Decreasing the pixel dwell time allows faster scanning at the expense of signal to noise ratio.
In the Size section the frame format (number of pixels that will be imaged) can be changed by adjusting the slider position.
In the Area section the field that is being imaged can be changed or rotated around. You can also zoom using the slider on the right to look at a smaller field of view.