16
Make any final adjustments to both the 45
o
and 0
o
screws to ensure optimal alignment of the laser line
with the target. Once completed, remove the camera from the microscope stage and the alignment
target from the nosepiece.
4.2 Laser setup considerations
Before setting up the laser to allow autofocus function, consider the nature of your microscope
system. The PF209 setup slide used for this procedure has a number of different options. Please
note that Line mode systems (PF850M) have a slightly different procedure than spot mode systems.
4.2.1 Biological samples
Use the coverslip sample, which most closely mimics permanent slides or petri dish samples.
Transmitted light only (diascopic) microscope systems can only be setup using the coverslip
sample. Alternatively, the mirror sample can be used if sufficient ambient light is available to
illuminate the sample. If using a reflected light technique (e.g. fluorescence) for the majority
of imaging, the mirror sample can be used. The sample provided on the PF209 also may not
fluoresce at the required wavelength.
4.2.2 Samples requiring reflected light
Use the mirror sample, which is the simplest reflective sample. This sample closely mimics
wafers. When using biological samples on microscopes without diascopic illumination, Prior
Scientific recommends this sample for ease of use. Please note that large increases in laser
power may be required when setting up the samples you intend to image.
4.3 Setting the sensor (pinhole) centre
Please refer to section 4.3.1 for slice mode systems (PF850) and section 4.3.2 for line mode
systems (PF850M).
4.3.1 Sensor centre setup for slice mode systems
In the PureFocus850 GUI, go to the offset submenu and click ‘Go to Factory Home’. The offset
lens will move. Go to the laser submenu and increase the value to 100. Click set.
Go to the mode submenu and ensure slice mode is selected.
Set the PureFocus controller to MAN FOC mode.
Move an objective into the light path. Prior Scientific recommends a 10x objective for this
procedure due to the working distance relative to higher magnifications, but any objective of
higher or lower magnification can be used.
Place the PF209 onto the stage and focus on either the coverslip sample or the mirror sample.
Use the digipot, in MAN FOC mode, to achieve focus.
Go to the setup submenu, which will open a graph that indicates the laser intensity over the
PureFocus850 sensor. Adjust focus until a peak appears in the signal. Make this peak as sharp
as possible. It is optimal for the peak to fill almost the entire y-axis of the graph, which can