3i SDC - Z series, Multiposition & Definite Focus

In most cases you will use the ASI Adept Piezo for Z series, so this should always be selected by default in the Z Stage area of the Focus window.

Single Position: Use Top and Bottom

1. Click the Z tab in the Focus window
2. Use the arrows in the Z Stage area to move the piezo to where you want to start the Z series
3. Click Set Bottom
4. Move the piezo up to where you want the Z series to end
5. Click Set Top
6. Specify the Step Size in the Capture Information area of the Z tab
7. In the Capture window, check the 3D box
8. Select the ‘Use top and bottom positions’ radio button in the 3D Capture area
9. The number of planes and Step Size will automatically be copied over from the Focus window

Note that that Top and Bottom are absolute positions of the piezo, so this approach will only be useful for z series at a single position and cannot be used for multi-position capture.

Single Position: Use Reference

Use this if you have a plane of interest that you want to use as a reference position in the stack, or if you need to use the Definite Focus (DF) to keep the specimen in focus

1. Click the Z tab in the Focus window
2. Use the arrows in the Z Stage area to set a Top and Bottom
3. Use the arrows again to move the focus to your plane of interest, then click Set next to Reference: in the Z tab
4. Specify Step Size in the Capture Information area of the Z tab
5. In the Capture window, check the 3D box
6. Select the ‘Use reference position’ radio button

Note that you can also set the Reference position before the Top and Bottom positions.

Single Position: Range around current/reference

If you check the ‘range around’ box in the Capture window it will use the center or (probably more usefully) the reference plane as the centre of a symmetrical Z series. The range will be taken from the value in the Range (um) field. Note that you can type a value into that field if you don’t want to use the Top and Bottom positions.

Method 1: Using the Definite Focus at a single position using the Reference

To use the DF at a single position in this way you must have a Reference position specified, so set up your Z series as for ‘Single Position: Use Reference’ or ‘Range around reference’.

1. In the Focus window, click the Focus tab and select ‘Use Definite Focus to focus when autofocus is enabled’
2. Select the ‘Use individual offsets for multipoint capture’. This isn’t essential for single position capture but it is the most useful option for multipoint, so it’s worth setting it by default
3. In the Capture window, click the Advanced button to open the Capture Preferences window
4. In Capture Preferences, click the Focus tab and enable the autofocus by checking the box that says ‘Autofocus during time-lapse/multipoint captures’
5. Back in the Focus window, go to the Reference point by clicking Go in the Z tab. Note that if the focus has drifted, you can reposition the Reference point by moving the microscope focus knob and clicking Set
6. In the Focus tab, click the ‘Set Parameters’ button. This calculates the DF offset for the one reference position
7. You can now start the acquisition and provided there is enough time between captures the DF will refocus on the Reference point before each Z series

Method 2: Using the Definite Focus at a single position using an XY point

This method uses the same approach as would be used for multi-position acquisition but only marks a single point.

1. In the Z Stage section of the Focus window, make sure the piezo is at 0.0
2. Focus on a plane of interest in your specimen using the microscope focus drive
3. Click on the XY tab of the Focus window
4. Click Set Point
5. The point will have four numbers: X stage position, Y stage position, microscope focus position and the piezo position, which should be 0.0. Note that this position takes the place of the reference point in Method 1.
6. In the Z tab, set a Top and Bottom for your Z series using the piezo
7. Set the Step Size in the Capture Information area
8. Click the Focus tab and select ‘Use Definite Focus to focus when autofocus is enabled’
9. Select the ‘Use individual offsets for multipoint capture’. This isn’t essential for single position capture but it is the most useful option for multipoint, so it’s worth setting it by default

10. In the Capture window, click the Advanced button to open the Capture Preferences window

11. In Capture Preferences, click the Focus tab and enable the autofocus by checking the box that says ‘Autofocus during time-lapse/multipoint captures’

12. In the XY tab, return to the plane of interest by double clicking the marked point or by selecting it and pressing Visit Point. This will make sure you’re ‘zeroed’ before starting the acquisition

13. Back in the Focus window, select the Focus tab and click the ‘Compute All Offsets’ button. This calculates the DF offset for all XY points. The offset should be visible next to the point in the XY tab (it will be in this sort of form: 0x919ffffffc901000010)

14. Make sure you are at your plane of interest (see 12 above) and in the Capture window, select the Current Location button in the Multiple XY Location Capture area. You need to do this for the next step

15. Select ‘Use top and bottom positions’ in the 3D Capture area to import the range, Offset* and Step Size from the Focus window.  Reselect use reference position. 

16. Select Multipoint List in the Multiple XY Location Capture area. You have to do this when using this method, even if you only have one point

*Note that the Offset (um) in the 3D Capture area is not the same as the DF offset. It is the distance from the zero/reference position of the piezo to the bottom of the Z series. This value, together with the range, defines the lower and upper limit of the Z series.

Multi-position Note:

The procedure above will also work for multi-position acquisition. You would simply set more XY points at stage 4 (piezo should be 0.0 for each). When you click Compute All Offsets at stage 13 it will calculate the offsets for all the points.