LS Microscope Startup Manual 2018.7.25
17
K.
Setting Up Autofocus (
LS720 only
)
1.
Use Manual Image and Manual XYZ to find a focused live image in a desired XY
position as described in Section II.F. above. In Manual XYZ, click the Autofocus Setup
button to open its dialog box.
2.
Transfer the manually determined Focus Z level in Manual XYZ to the Center position
field of Autofocus Setup. The Focus range will be set to a default value of 200 um.
Change this to the recommended Range for each objective magnification according
to the following table:
3.
The Max focus time will be set to the default of 15 sec. This can be reduced as
performance of the autofocus is confirmed. The Max focus time should be at least
that required for successful autofocus but optimally only a few seconds longer.
Autofocus of 3 seconds should be possible in flat labware with 2D samples.
4.
Click OK and autofocus will occur. The Focus Z level in Manual XYZ will now be
updated to the new autofocused Z level. The actual time taken to reach autofocus, i.e., the Autofocus
time (sec), will also be shown.
5.
If autofocus did not result in a sharp live image, make sure the Center position transferred was correct.
In general, if autofocus consistently uses the full Max focus time, the range is too great for the time
entered or the time is insufficient. It may be necessary to decrease the Focus range and/or increase
the Max focus time.
6.
Upon successful autofocus, move to other locations distant from the initial location. Click Select
Labware in Manual XYZ and the appropriate Labware Map will open (see Section II.E.3. above). Select
a location and repeat Steps 1-4 above.
7.
Continue to move to other locations far away from the initial ones and repeat autofocus. It may be
necessary to re-enter the Center position and/or adjust the range. To determine the final parameters,
review the upper and lower Z levels obtained and use the approximate mean for the Center position.
For the range, determine the spread between the highest and lowest Z values and add the suggested
buffer listed in the following table:
L.
Writing and Using Protocols
Magnification
Range (um)
Magnification
Range (um)
4x
350
40x
45
10x
200
60x
30
20x
100
100x
20
Magnification
Buffer (um)
Magnification
Buffer (um)
4x
400
40x
50
10x
200
60x
36
20x
120
100x
24
