The AO tab contains the Analogue Output settings which drive the scanning mirrors. The settings tell the scan engine the maximum voltage to output to the mirrors to achieve the maximum field of view and the X and Y offsets to centre the field. Values are saved per-objective as they will differ with different magnifications.
|A||x.volt.offset||Voltage offset from 0V midpoint required to centre the scanned field in the X Axis|
|B||x.invert.image||Inverts the scan along the x-axis|
|C||x.voltage||Uneditible field displaying the voltage currently being applied to the X Galvo to achieve the desired field of view (takes current zoom level and fill fraction into account)|
|D||y.volt.offset||Voltage offset from 0V midpoint required to centre the scanned field in the Y Axis|
|E||y.invert.image||Inverts the scan along the y-axis|
|F||y.voltage||Uneditble field displaying the voltage currently being applied to the Y Galvo to achieve the desired field of view (Takes current zoom level into account)|
|G||voltage.for.max.field.of.view||Output voltage required to achieve a maximum field of view with the current objective. Maximum value is 3V.|
|H||max.field.of.view||Maximum field of view of current objective. Can be measured by using the XY stage or a calibrated slide and entered here. This value is then used to calculate the FOV displayed on the 2D frame interface at different zoom levels.|
Once an objective is calibrated these values will most likely stay fixed. Please follow our guide on calibrating new objectives for detailed instructions.
Configuring New Objectives
With SciScan it is easy to add and calibrate additional objectives. The only additional components required are a fixed fluorescence sample (such as a pollen grain slide) and a sample that will give a flat fluorescence response (such as fluorescein). The first step is to add an objective to the drop down list in the General Settings tab. This is achieved by editing the .ini file
Adding Objectives to the .ini File
The .ini file stores the calibration for different objectives:
obj1.name = "40x Olympus"
obj1.max.field.of.view = 0.000300
obj1.voltage.for.max.field.of.view = 1.600000
obj2.name = "60x Olympus"
obj2.max.field.of.view = 0.000280
obj2.voltage.for.max.field.of.view = 1.700000
active.objective = 0.000000
In the above example, two objectives are configured, a 40x and a 60x lens. To add an additional objective to the list simply duplicate the three repeated lines of code and change the objective number to the next consecutive digit. For example to add a 20x Leica objective to the list the following would need to be added to the objectives section:
obj3.name = "20x Leica"
obj3.max.field.of.view = 0.00700
obj3.voltage.for.max.field.of.view = 3.000000
Only the first line of the three is important for now, do not worry about what values are entered for .max.field.of.view or voltage.for.max.field.of.view as these will be determined through imaging, for now enter values similar to the other objectives.
Run the software and navigate to the General Settings Tab where the new objective should appear in the drop-down menu. Make sure you select your new objective from the drop-down before continuing.
Setting Maximum FOV and XY Offset
For this step, begin imaging a sample that will give you an even fluorescence response, such as Fluorescein.
The next step is to ensure that the scanned FOV is centred for the new objective and is as large as possible. To achieve this change the voltage.for.max.field.of.view to 5V as shown above. This will widen the scan angles of the mirrors to their maximum and over-scan the back of the objective.
Please note: 5V exceeds the normally recommended maximum of 3V. Please scan at a slower frame rate than normal while these large voltages are applied.
You should be able to clearly see the objective’s entire FOV surrounded by black.
From here you need to centre the objective’s FOV in the middle of the scanned area. Adjust the x.volt.offset and the y.volt.offset to shift the fluorescing area in X and Y until it is centred.
Once centred slowly reduce the voltage.for.max.field.of.view until none of the black area is visible and you have a nice even fluorescent field.
The final step is to measure and set the size of the FOV and requires you to swap to a sample with distinguishable structures, such as a fixed tissue slide or a pollen grain slide.
Pick a structure on your slide and move it to the far left or right edge of your field. Zero your XY position and move the same structure to the opposite side of the field. If done correctly, the X rel.pos should indicate the width of your field. Enter this value in microns into the max.field.of.view field and it will be used to calculate your FOV at various zoom levels.
Now every time your objective is selected, it should call the correct voltage parameters to give you the maximum and correct field of view.