The AO tab is responsible for changing 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.

The X Resonant Galvo and the standard Y Galvo scale differently to one another when zooming so therefore require two different calibrations. The AO tab is responsible for changing 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.

The maximum FOV in A and B refers to the real world distances in your sample and depend on the objective used. In this example the maximum measured field was 430 micrometers in x and 300 micrometers in y; (the LabVIEW notation “u” denotes “micro-” or 10^{-6}.).

To measure the field of view use a graticule slide or a motorised XY stage with an accurate position readout.

Label | Variable Name | Function |
---|---|---|

A | max.field.of.view | Maximum field of view in Y for the given objective |

B | voltage.for.max.field.of.view | Voltage required to achieve field of view stated in max.field.of.view - Galvo mirrors are driven by a command voltage with a fixed voltage/angle relationship (typically 0.25V/optical degree). The voltage for max.field.of.view represents the maximum absolute value, ie. 1.5 V generates a command voltage ranging from -1.5V to +1.5V. Set empirically by lowering voltage until Y FOV = X FOV to give a square aspect ratio - measured using XY stage - Maximum 3V |

C | max.field.of.view.resonant | Maximum field of view in X for the given objective |

D | voltage.for.max.field.of.view.resonant | Resonant mirrors are not driven by a command voltage in the same way as the galvo mirrors. Instead, a DC voltage is used to set the scan amplitude (ZOOM) with 5 V being the maximum value accepted by the drive card. |

E | voltage.for.min.field.of.view.resonant | A theoretical voltage value that is used to account for non-linear scaling of resonant mirror angles. - To set this value, zoom to maximum zoom level (ie. smallest field of view) and adjust this value until aspect ratio appears square |

Please Note:SciScan does not limit the maximum voltage applied to either scanning mirror. Please consult component manuals for maximum acceptable input for your scanners.

## Configuring New Objectives

With SciScan it is easy to add and calibrate additional objectives. The only additional component required is a fixed fluorescence sample (such as a pollen grain slide). 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:

[objectives]

obj1.name = “40x Olympus”

obj1.max.field.of.view = 0.000300

obj1.voltage.for.max.field.of.view = 1.600000

obj1.max.field.of.view.resonant = 0.000300

obj1.voltage.for.max.field.of.view.resonant = 5.000000

obj1.voltage.for.min.field.of.view.resonant = 6.000000

obj2.name = “60x Olympus”

obj2.max.field.of.view = 0.000280

obj2.voltage.for.max.field.of.view = 1.700000

obj2.max.field.of.view.resonant = 0.000280

obj2.voltage.for.max.field.of.view.resonant = 5.000000

obj2.voltage.for.min.field.of.view.resonant = 7.000000

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 five 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 = 0.0007000

obj3.voltage.for.max.field.of.view = 1.700000

obj3.max.field.of.view.resonant = 0.00070000

obj3.voltage.for.max.field.of.view.resonant = 3.000000

obj3.voltage.for.min.field.of.view.resonant = 5.000000

Only the first line of the five is important for now, do not worry about what values are entered for the voltage values or for the field of view measurements as these will be set while imaging.

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 Voltages for New Objectives

For this step, begin imaging a sample with some structure, such as a pollen grain slide or some fixed tissue.

The next step is to ensure that the scanned FOV is as large as possible and that the combination of the X and Y mirrors produce an image with a square aspect ratio. To begin, using the controls on the Analogue Output Tab set the **voltage.for.max.field.of.view.resonant** to 5V (the maximum input for the resonant mirror).

Now, using the XY stage, measure the field of view in X by moving a structure from one side of your visible field to the other, noting position readout before and after to give you the width of your field. Enter this value into the field called **max.field.of.view.resonant.**

To ensure a square aspect ratio, the voltage used to drive the Y mirror must now be set so that it scans over the same distance as the X Mirror. Measure the field of view in Y using the same method as with the X axis and adjust the voltage in **voltage.for.max.field.of.view** until the field of view is the same size in Y as it is in X. Higher voltages will increase the range over which it scans, smaller voltages will decrease the FOV. Once you have found the correct value, enter the size of the Y field of view in the field marked **max.field.of.view.**

The resonant scanner and the traditional galvo scanner scale their amplitude differently with a change in voltage resulting in a non-square aspect ratio when zooming in. In order to get around this problem we have introduced a scale factor called **voltage.for.min.field.of.view.resonant.** To set this value properly; zoom into a small structure in your sample (around 5-8x zoom should be enough). You’ll notice that the image appears stretched in one direction. Measure the field of view in Y while zoomed in and then adjust the **voltage.for.min.field.of.view.resonant** until the field of view in X is equal to the field of view in Y. With this value set properly you should be able to zoom in and out while maintaining a square aspect ratio.

With all the voltages and FOV measurements set correctly the objective is properly configured. The values entered in the **Analogue Output Tab** will automatically update and be stored in the .ini file for the next time the objective is used.