PerkinElmer IVIS Lumina III Series, Hardware Manual

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IVIS ® Lumina XRMS Series III Hardware Manual Chapter 8 | Fluorescence Module
64
Acquiring Fluorescent Images
1.
If it is not already on, start the acquisition computer and Living Image
®
software.
The control panel appears ( Figure 8.10 ).
2.
Click Initialize IVIS system.
After initialization, the Temperature box in the center of the panel should be green, indicating
that the CCD camera is adequately cooled. (Allow 10 to 15 minutes for the camera to reach the
proper temperature.) The Temperature box changes from red to green when the CCD camera
has reached the proper operating temperature.
3.
Place the sample to be imaged in the center of the stage in the imaging chamber and close the
door.
4.
Select the Fluorescent check box.
5.
Make a selection from the Field of View drop-down menu on the left side of the Control Panel.
6.
Enter the approximate (0.5 cm) Subject Height (height) in the lower left entry box (or focus
manually).
7.
Select the Emission Filter and Excitation Filter. If the Filter Lock box is checked, select the
excitation or emission filter of interest. Select only one, as the other filter will be selected
automatically.
8.
Select High or Low from the Fluor Lamp Level drop-down menu. High is the recommended
setting
9.
Set the Exposure Time, Binning, and F/Stop.
Fluorescence is generally brighter than bioluminescence, so the exposure time is shorter and F/
Stop higher (smaller lens opening). Typical fluorescence image camera settings might be 1 sec
exposure time, Binning = Medium, and F/Stop = 2.
10.
Click Acquire.
After the exposure is completed, the overlay image is displayed.
11.
To save the data, select Living Image
➞
Save Living Image Data from the main menu bar.
This completes the data acquisition. To obtain additional images, repeat the process, beginning
with
step 3 .
The image window that displays the fluorescent image includes annotations specific to
fluorescence (including emission filter, excitation filter, and fluorescence level) as well as
standard annotations such as exposure time, F/Stop, FOV, and acquisition date/time.
8.6 Troubleshooting
Hardware Problems
If you have difficulty during fluorescent imaging, it may be due to the lack of excitation light. Loss
of excitation light can result from either a burned out quartz tungsten halogen lamb or a blown line
fuse. The following procedure describes a troubleshooting process for determining the problem.
Figure 8.3 on page 59 shows the fluorescence light source module.
1.
Verify that the fiber optic cables are not loosened or disconnected from their proper connectors.
2.
Adjust "Fluor Light Level" (excitation lamp) to a value of high in the IVIS Acquisition Control
Panel.
NOTE:
During the fluorescent image acquisition, the Acquire button becomes a Stop button,
which can be used to terminate the exposure if necessary.