11
5. The Offset is also found in the PMT settings menu and controls the image black
level. As an example setting an offset of zero means that pixel intensity values
between 1-4095 have a grey scale value and will be visible in the image. If you
set the offset to -10% this means that any pixel with an intensity lower than 409.5
(4095 x 0.10) will be set to black (zero). You can use this to reduce background
fluorescence but be aware that you can cancel out specific signal if not set
correctly.
6.
You can determine if the signal fills the dynamic range of the detectors by using
the saturation indicator which is found in the toolbar to the left of the images.
Press the button once and the indicator will be activated. Now any pixels which
have an intensity of 4095 (max) will be coloured blue.
7.
The aim is to set each channel image so that the pixel intensities are just under
saturation level (4095). It’s not a concern if there are a few blue pixels. Press
the saturation indicator button again twice to see the channel images in their
original colours.
Capturing a Multi-‐Wavelength 2D Image (Optical Slice)
1. If you have followed the sections above you just need to
decide if the image is to be captured simultaneously or
sequentially.
2. Press the Capture Image button at the bottom right of the left monitor and a
single scan of each channel will be acquired.
Saving Images
1. Captured images will be added to the experiment. You can
access the experiment images by selecting the Experiments
tab in the top left corner of the left monitor.
2. Images are given a default name “Image” followed by an
incremental number unless you uncheck the “Default image
basename” option.
3. You can rename an image by right clicking on it then select rename.
4. Images in the experiment are stored temporarily so you must ensure you save
the experiment to disk at regular intervals and at the end of the session using the
Save All option at the bottom of the experiment window.
5. Images are stored as Leica LIF format by default which can be opened with
FIJI’s Bio-Formats plugin.
Summary of Contents for SP5 confocal
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