LEFT TOOL AREA AND HARDWARE CONTROL TOOLS

LEFT TOOL AREA AND HARDWARE CONTROL TOOLS
LSM 700
5.2.18
LEFT TOOL AREA AND HARDWARE CONTROL TOOLS
Acquisition Tab
Carl Zeiss
Image Optimization
This section describes an example how to acquire
an image, using an excitation wavelength of
488nm and a fluorescence emission range above
510 nm. We presume that no predefined
configuration is available for this imaging task and
that Smart Setup is not used to do this task.
The description applies to the use of any
microscope.
5.2.18.1
Requirements
x Click on the Locate tab on the Left Tool Area
and set the microscope according to your
needs.
x Position the specimen under the objective and
focus onto the desired imaging plane.
x Click Acquisition tab in the Left Tool Area
toolbar.
Click on Show all tools to display the tools in the
tool group Setup Manager.
x Open the Light Path tool. Choose Channel
mode for the image acquisition (Fig. 98).
Fig. 98
Light Path tool
Fig. 99
Laser line and laser power selection
Fig. 100
Channel activation and color
selection
x Click on the laser icon, select the 488 line, and
set the transmission slider to about 2 %. Make
sure other lasers are not checked (Fig. 99).
Optimize the laser intensity subsequently via
the transmission slider when imaging.
x Set the VSD to the position 500 nm and
activate PMT 2 via the check box. You may
want to assign a different color to the channel
which will be taken over for the initial display of
the image. Click the LUT part of the list to open
the color table and select the desired color
(Fig. 100).
x Click on the Dye pull down list and select the
desired dye (e.g. FITC). This will display the
emission spectrum of the desired dye in the
upper part of the light path.
x If required, deactivate all the other channels
(Ch1, transmission, R1-2) via the check boxes.
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LEFT TOOL AREA AND HARDWARE CONTROL TOOLS
Acquisition Tab
Carl Zeiss
5.2.18.2
LSM 700
Defining the Scan Parameters
x Open the Acquisition Mode tool (Fig. 101).
x Select Frame mode and a Frame Size of
512 x 512 to start with.
x The Optimal button automatically adjusts the
frame size to match the magnification and
resolution of the objective. Thus, images are
acquired without collecting redundant image
information.
Fig. 101
Acquisition Mode tool
x The Scan Speed is set to 9 per default. A lower
speed produces better images as the light
collection per pixel is higher. Use a speed from
7 to 9 to begin with.
x Start with the following settings on the Pixel Depth, Scan Direction & Scan Average panel:
Data depth:
8 bits
Average:
Number: 1 (no averaging)
The default settings for Acquisition Mode are suitable for good image quality and do not need to
be changed for initial scanning.
5.2.18.3
Defining the Detection
Parameters
x Activate the Channels tool (Fig. 102).
x Set the Pinhole to two Airy Units using the
slider. The value for the pinhole is displayed in
the tooltip.
The pinhole diameter should be so small that
there is still enough variation for the setting
of the Gain (Master) and that sufficient
image information is still available. 1 Airy is a
good value to enable a confocal fluorescence
XY-image to be obtained.
Fig. 102
Channels tool
A small pinhole diameter will increase the
depth of focus, but reduce the light intensity
received by the PMT photomultiplier (for
reflection mode confocal images start with a
pinhole value of 0.3 Airy Units).
x Use the Set Exposure button.
The system determines the settings for the
Gain (Master) and Digital Offset to match
the scan speed, pinhole size and laser power.
Finally, the system builds up the image.
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LEFT TOOL AREA AND HARDWARE CONTROL TOOLS
Acquisition Tab
LSM 700
5.2.18.4
Carl Zeiss
How to Optimize Image Quality
As a rule, the first scanned image is not ideal, since
the settings of the laser and therefore also the
photomultiplier might not match the light output.
More often than not, the screen image is dull and
needs subsequent optimization.
x Use the lookup table Range Indicator (see
section Dimensions). This display mode shows
overexposed regions in red and underexposed
regions in blue. Some red and some blue pixels
should be visible to ensure the signal is spread
over the dynamic range of the detector and no
image information is lost.
x Start a Continuous scan and quickly adjust the
laser power, Gain (Master) and Digital Offset
to set the image into the dynamic range.
x Try to lower the impact of laser light onto the
sample. Reduce the laser power and adjust the
gain accordingly.
Fig. 103
Image displaying bad adjustment of
gain and offset (blue regions are
underexposed, red regions are
overexposed)
Fig. 104
Image displaying good adjustment
of gain and offset
x To adjust the black level (background), use the
Digital Offset slider so that areas without
picture content just show a trace of blue.
x If necessary, re-amplify brightness with the
Digital Gain slider.
Do not change the Digital Gain setting
unless the settings made so far are
insufficient for image optimization.
x Stop scanning.
x Save the imaging configuration again as this
time it will also save the correct settings for the
detector, the laser, and the pinhole.
The signal-to-noise ratio can be substantially
improved by reducing the scan speed to an
acceptable level and averaging over several scans
(i.e. with an average Number greater than 1 for
the Mean average Method in the Scan Control
window).
x Use the Speed slider in the Acquisition Mode
tool set the slowest acceptable scan speed.
x In the Number text box of the Averaging
panel enter the number of images to be
averaged.
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Carl Zeiss
LEFT TOOL AREA AND HARDWARE CONTROL TOOLS
Acquisition Tab
LSM 700
Image optimization can be achieved faster if you select a smaller frame, since less data have to be
processed.
The greater the number of averages selected using Mean as average Method, the better the
image quality will be; the scan time is prolonged accordingly.
A similar procedure applies for image optimization if more than one channel is used and simultaneous
image acquisition is performed.
Make sure the suitable lasers are switched on and the channels are activated.
Pay attention to address the right channel when Pinhole size, Gain (Master), and Digital Offset are
controlled as these are set individually for each channel.
Use the split view (see section Split View) to see the effect of the controls onto each channel separately.
If the acquisition is performed sequentially in with multiple tracks it is faster to activate only one track at a
time for image optimization.
.
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