PALM
®
MicroBeam User Manual - 0202 Z
21
How to Prepare the PALM
®
MicroBeam for the Application
Adjust Laser Focus
The smallest laser focus diameter is obtained when
using objectives of a high numerical aperture.
When using objectives with a lower numerical
aperture, the diameter of the laser focus increases
correspondingly.
When working with objectives providing a short
working distance, the object slides must be thin-
ner.
6.5 Adjust Laser Focus
As the laser itself is not visible, only the laser effect
(ablation) can be used for control.
To visualize the changes of the laser focus posi-
tion, you must cut (ablate) an object, whereby the
laser is permanently pulsating and the object is
being moved, simultaneously. We recommend to
use an LPC object slide (membrane-coated) for
this purpose.
Simultaneous stage movement will create a cut-
ting line within the sample. This line will be fuzzy
or fairly thick due to yet insufficient laser focus
setting.
A star-like, three-beam glass ablation around the
laser focus indicates a good focussing of the glass
surface.
With a higher energy setting cutting is possible not
only within the laser focus but also above or below.
If the laser focus is just inside the glass, smaller or
bigger pieces of glass will be blown out.
Figure 18
Zig-zag line cut with the laser with different energy
and focus settings
Find the correct focal plane in the following ways
(please refer to Part III, PALM® RoboSoftware
Manual for using the graphic tools, the automated
laser functions and how to adjust energy and focus
settings for the laser):
• use a plain membrane-slide without tissue
specimen
• draw a zig-zag line with the freehand graphic
tool spanning at least the monitor screen
• start automatic cutting with the Cut function
• reduce the laser energy with the "Page down"
key at the keyboard or with the slider for laser
energy at the monitor screen until only a faint
cut can be seen
• move the laser focus up and down with the
keys "home" and "end" at the keyboard or the
corresponding slider for laser focus at the mon-
itor screen to locate smallest cutting size
• reduce the laser energy and repeat the above
described procedure a second or third time,
until the smallest available focal spot is found.
The perfect laser focus setting is achieved, when
with a minimum of laser energy a fine cutting line
is visible, which immediately disappears as soon as
the laser focus is slightly shifted up or down.
The cutting edges have to show identical symme-
try, when the object is moved into x- or into y-
direction. With increasing energy the membrane
margins should still appear symmetrically.
Notice
For objectives with aperture diaphragm, adjust
the diaphragm to the thickness of the object
slide or the Petri dish used to ensure an opti-
mum presentation (focusing, contrast).
Adjust the laser focus to the object setting,
accordingly.
With high numerical aperture, use only the
Petri dishes and object slides that are defined
in this User Manual.
Notice
Make sure that the membrane is always
focused within the microscope focus, otherwise
the laser focus setting might be incorrect.
Notice
Preferably use a LD-40x objective.
Make sure that the membrane in this area is
tightly mounted onto the glass slide. Look for
areas where "Newton's rings" are visible (rain-
bow-colored striae): "Newton's rings" indicate
that the membrane is not perfectly mounted.
Notice
For each type of sample the laser focus adjust-
ment has to be performed individually. The
individually determined values can be stored in
a separate file.