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Pre-scan acquisition settings

*Consult the labelled diagram of the A1Plus Compact GUI (page 10) if you're not sure

where any of the buttons mentioned below are

1. Before you can scan with the lasers you have to remove the laser interlock by

pressing the red "Remove Interlock button".

2. The scan direction buttons indicate unidirectional and bidirectional scanning. When

bidirectional mode is selected the image acquisition frame rate doubles. Only

consider bidirectional mode when carrying out a live cell experiment.

3. Select an image size (e.g. 512x512 pixels in xy). The size affects the xy image

sampling with larger sizes resulting in smaller um/pixel values and thus better

spatial resolution but the scan time is also increased. The most commonly used

sizes are 512 or 1024.

4. Now choose a scan speed, start by selecting the lowest number when the control

by "Pixel dwell" option is enabled. Higher values represent a longer pixel dwell and

hence a slower scan. You may need to use a slower scan speed if your signal is

low but this could

increase the

photobleaching rate.

5. Ensure that the "Normal" and "Ch Series" buttons are enabled (green).

Channel Series enables sequential scanning mode which means only one

laser is scanning at any particular time. If Channel Series is turned off then

all lasers scan simultaneously which can lead to fluorescence crosstalk

where emission from one fluorophore is detected by more than one detector.

Press the "Optimal Pinhole" button, this sets the detection pinhole to a diameter that

gives the best compromise between light throughput, noise and resolution. Closing

the pinhole results in a thinner optical

section and better lateral and axial

resolution due to rejection of out of focus

light.