Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
LSM 880 Left Tool Area and Hardware Control Tools ZEISS
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The pinhole is adjusted using a dye solution. The general approach is to move the pinhole cyclically in x
and y until the intensity maximum is found. For each excitation wavelength a suitable dye must be used.
We recommend:
−
Rhodamine 6 Green (Rh6G) or Alexa 488 for 458, 488 and 514 nm,
−
Tetra-Methyl-Rhodamine (TMR) or Alexa 546 for 543 nm and
−
Cy 5 or Alexa 633 for 633 nm.
It is also recommended to work with a relatively concentrated solution (10
-6
mol/l) and low laser power to
achieve smooth intensity curves.
•
Press the
Coarse
button of x and y to perform a coarse adjustment in x and y. The pinhole will travel
over the maximum range for each axis. Perform a fine adjustment in x and y by pressing the Fine
button. The pinhole will travel only a limited distance around the peak found in the coarse alignment.
If the pinholes are adjusted for the first time, the coarse adjustment must be performed first and
then the fine adjustment, each time for x and y. For subsequent readjustments, the fine adjustment
is normally sufficient.
•
Click on the
X
and on
Y
auto adjust buttons to perform a coarse / fine alignment of the pinhole in x
and y, respectively. In either case, the Pinhole Adjustment window will appear (Fig. 270). In the
Count Rate/Position diagram, the crosses correspond to the measured intensity (count rate). The line
corresponds to the intensity curve fitted to the data points to find the optimum pinhole position. The
pinhole will be automatically positioned to the maximum. If a maximum will not be found in the fine
alignment, the system will show a warning message. In this case, a coarse alignment must be
performed before the fine adjustment.