IVIS Fluorescent Imaging Guide - Optical Imaging …oicf.bsd.uchicago.edu/docs/IVIS200 Fluorescence Imaging...IVIS® Fluorescent Imaging Quick Start Guide 3 Correcting For Tissue Autofluorescence

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IVIS® Fluorescent Imaging Quick Start Guide


1 Acquiring an Image

First take an image of your subject to determine the best settings for your images.

■ Start the Living Image® 2.5 software (double click the icon on the desktop).

In the IVIS System Control panel that appears, do the following:

1. Click Initialize IVIS system.

2. Choose the Fluorescent option.

3. Select a field of view (FOV).

4. Enter the exposure time (usually 1-10 seconds).

5. Select a binning level (a lower level gives better spatial resolution).

6. Select an f/stop setting. A higher f/stop gives better depth of field (sharper image). Set the f/stop to higher values for fluorescent imaging (f/stop of 2 or 4 is recommended).

7. Select the Excitation filter (for example, Cy5.5 is selected in Figure 1.1.If the Filter Lock option is checked, the software automatically sets the emission filter.

8. Click Acquire.

Figure 1.1 IVIS System Control: Steps to acquire a fluorescent image.

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Adjust the settings to obtain a signal between 1,000 and 65,000 counts. If the fluorescent signal of interest is hidden by instrument autofluorescence, proceed to “Subtracting Instrument Autofluorescence”. If the signal is hidden by animal tissue fluorescence, proceed to “Correcting For Tissue Autofluorescence”.

Subtracting Instrument Autofluorescence


2 Subtracting Instrument Autofluorescence

The IVIS® Imaging Systems use fused silica optics, low-autofluorescent materials, and high quality filters to minimize autofluorescence and background caused by instrumentation. However, a residual background may be detected during fluorescent imaging. This background is often higher near the edges and corners of the CCD, resulting in a ring-shaped pattern (Figure 2.1). If the ring-shaped pattern is significant, it can be subtracted out of the image using the following procedure.

1. Remove the fluorescent subject(s) from the imaging chamber.

2. Select Living Image ➞Fluorescent Background ➞Measure and Replace Fluorescent Background on the menu bar (Figure 2.2).

Figure 2.1 IVIS fluorescent images.Left: Instrument autofluorescence can result in a ring-shaped background. Right: Image after the instrument autofluorescence is subtracted.

Note: If an instrument background has been taken, the Sub Fluor Bkg check box appears in the IVIS System Control panel. If this box is checked, the instrument background is subtracted from the original image.

!IMPORTANT Confirm that all of the imaging parameters in the IVIS System Control

panel are set to the same values used to acquire the image. When generating an image series or imaging a group of animals, perform instrument subtraction first and only once. For further details, see the Living Image® 2.50 Software User Manual.



3 Correcting For Tissue Autofluorescence

High levels of tissue autofluorescence limit the sensitivity of detection of exogenous fluorophores. The IVIS® Imaging Systems implement a subtraction method using background excitation filters to minimize this effect.

Two images are required for this procedure: one taken with the primary excitation filter and one taken with the corresponding background excitation filter. Both images use the same emission filter.

1. Place the subject(s) in the imaging chamber.

2. In the IVIS System Control Panel (Figure 1.1), click Select sequential mode.

— The Sequential Setup dialog box appears (Figure 3.2).

Figure 2.2 Living Image menu bar

Figure 3.1 Image corrected for tissue autofluorescence (right)

Correcting For Tissue Autofluorescence


3. Set the imaging parameters for the primary excitation filter image (exposure time, binning, f/stop, fluor lamp level, and FOV) and click Set.

4. Set the excitation filter to the corresponding background filter and click Set.

For example, if the primary excitation filter is Cy5.5, select Cy5.5Bkg for this step.

5. Click Acquire Sequence.

— A sequence panel that contains both images appears (Figure 3.3).

Figure 3.2 Sequential Setup dialog box

Figure 3.3 Sequence panel

Select efficiency for quantifying fluorescent measurements.

Click Display to open both images.



6. Inspect both the primary excitation filter image and the background excitation filter image to determine if instrument background needs to be subtracted.

If the instrument background is significant in either image, perform instrument background subtraction on both images (see “Subtracting Instrument Autofluorescence,” page 2). If the instrument background is not significant, continue to step 7.

7. Determine the scale factor (k) that is used to subtract the primary excitation filter image from the background excitation filter image.

The scale factor is the ratio of the autofluorescent signal measured using the background excitation filter to that of the primary excitation filter in a region on the animal with no fluorophore present.

To determine the scale factor:

a. In the sequence panel, select efficiency for the image units (Figure 3.3).

It is recommended that you quantify all fluorescent measurements in units of efficiency.

b. Click Display on the Sequence panel.

— Both images open.

c. Choose Apply to Sequence on the individual image panel (Figure 3.4).

d. Create an ROI:

1. Click Create on the image panel.

2. Adjust the ROI size and location so that it does not include the fluorophore signal. For example, the ROI was positioned on the scruff of the animal in (Figure 3.1).

NOTE If the primary excitation filter image and the background excitation filter image were taken separately (not using the sequential mode), save and close the images. Then use the Living Image Browser window to load the images as a group and display the images in a sequence panel.

Figure 3.4 Individual image panel

Choose Apply to Sequence.

Click to create an ROI.



e. Click Measure in the image panel. In the Measurements table that appears, click Configure (Figure 3.5).

f. In the Configure Measurements dialog box, select Efficiency from the Measurement Type drop-down list (Figure 3.5).

g. To compute the scale factor k, use a calculator to divide the average ROI value from the primary excitation filter image by the average ROI value from the background excitation filter image.

8. Select LI Tools ➞Image Math for... on the Living Image menu bar.

— The Image Math window opens (Figure 3.6).

NOTE Since Apply to Sequence is selected in the individual image panel, the same ROI is created in both images. Adjustments to ROI size or location are applied to both ROIs.

Figure 3.5 Image panel (top), Measurements table (middle), and Configure Measurements dialog box (bottom)

Individual image panel

Measurements table

Configure Measurements dialog box

Select Efficiency from the Measurement Type drop-down list.



9. Select A-B*k (subtraction mode) from the Results = drop-down list.

“A” represents the image taken with the primary excitation filter. “B” represent the image taken with the background excitation filter.

10. Check to make sure the correct image number is highlighted for “A” and “B” in the Image Math window.

11. To display the corrected image, click Display Result for Measuring.

Figure 3.6 Image Math window

Select the subtraction mode from the Result drop-down list.

Enter the value for k that you calculated in step g.



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IVIS Fluorescent Imaging Guide - Optical Imaging …oicf.bsd.uchicago.edu/docs/IVIS200 Fluorescence Imaging...IVIS® Fluorescent Imaging Quick Start Guide 3 Correcting For Tissue Autofluorescence