Nikon A1r - 2: acquisition

Right-click on of the default OC (Resonant or Galvano) and duplicate the OC. The scanner can be changed also later in the settings.

Galvano scanner is more flexible in terms of image format

Resonant scanner is very fast, suitable ,e.g., live cell imaging

Rename your OC and click OK

The naming depends on you. You can choose some general name or more specific that will link this setting to this specific experiment.

Drag and drop the OC to the sub-list My Experiments.

Click on the Dye Assistent

Choose in the setting mode "Auto"

You can choose the number of channels you need

And choose from the menu dye

If you can't find your dye, choose the one with a similar excitation and emission spectrum.

Confirm with OK

In the A1plus Compact GUI Pad you can now see selected channels

Click on the wavelength to activate laser and change the laser power.

Rather start with low laser power

Adjust a voltage on detector

Avoid adjusting the Offset setting because it artificially alters the baseline signal of the detector.

Start Scan to create an live image

You can see the preview image in A1plus Scan Area window as well as in the Frozen pop-up window

Continue setting up the other channel

If you do not see any signal, you may be slightly out of focus or you need to scale the histogram.

Activate the navigation with mouse, so you can use mouse to move in XY and mouse wheel in Z.

Click on the Autoscale button to enhance the contrast

If you start to see something in the backgroung you can move up or down with an objective to focus on the cell

Do not forget to change the speed on the joystick to one arrow as your sample must me close, if you used the initial navigation in WF mode.

(Optional) Open the pinhole to have a thicker focal plane.

Turn ON the saturation control

(Pixel that has intensity higher then 4094 = 12bit image, will be displayed in chosen color)

You can split the view of the channels

Or see the split view and overlay.

Or keep an overlay and toggle between displaying single channels.

In the A1plus Compact GUI pad you can control more settings of the scannnig

You can set up the sequential scan.

You can explore sequential scan for multiple channels in the drop down menu.

Scanning speed - either controlled by Frame/sec or Pixel Dwell time

The dimensions of the image in pixels.

You can use an line averaging of the signal

And line accumulations (=sum of the signal)

There is no universal settings that can be used for all samples and users. You need to optimize these setting for your sample, based on the signal and resolution you need to achieve.