Any advice on EGFP and luciferase interference for IVIS measurements ?

Dear Colleague,

When conducting in vivo imaging studies using the IVIS (In Vivo Imaging System) technology, simultaneous measurement of Enhanced Green Fluorescent Protein (EGFP) and luciferase can present challenges due to potential spectral overlap and interference. However, with careful planning and optimization, reliable and distinct measurements of both reporters can be achieved. Here are some strategies to minimize interference and maximize the fidelity of your IVIS measurements:

Optimize Reporter Gene Selection: Choose luciferase and fluorescent protein variants with distinct spectral properties to minimize overlap. For example, use firefly luciferase with EGFP, as they have sufficiently separated emission peaks (approximately 560 nm for firefly luciferase and 509 nm for EGFP), which helps in reducing spectral crosstalk.

Use Spectral Unmixing: Modern IVIS systems are equipped with spectral unmixing capabilities that can differentiate between signals from closely overlapping spectra. This feature can be particularly useful when imaging multiple reporters. Ensure to include controls for each reporter alone to set up the spectral unmixing correctly.

Optimize Filter Selection: Carefully select the appropriate filters for both excitation and emission to match the peak wavelengths of EGFP and the chosen luciferase. This optimization helps in enhancing signal specificity and sensitivity for each reporter.

Temporal Separation of Measurements: Since luciferase activity requires the addition of a substrate (e.g., luciferin) and produces a transient signal, you can temporally separate the measurements of luciferase and EGFP. Measure the luciferase signal shortly after substrate injection, and then measure the EGFP signal once the luciferase signal has decayed. This approach relies on the different kinetics of the two reporters.

Spatial Resolution Enhancement: Employ imaging modalities or settings that allow for high spatial resolution, aiding in the distinction between signals when reporters are localized to different tissues or cells within the same organism.

Quantitative Analysis Adjustments: Apply software tools and quantitative analysis techniques provided with the IVIS system to adjust for background fluorescence and to calibrate the measurements based on known standards. This can help in accurately quantifying the signals from each reporter.

Experimental Controls: Include appropriate controls in your experimental design, such as animals or samples expressing only one reporter at a time. These controls are invaluable for identifying and correcting for any potential interference between the reporters.

Literature and Manufacturer Guidelines: Review relevant literature for tips and protocols optimized for co-reporter studies. Additionally, consult the manufacturer's guidelines and technical support for advice specific to your IVIS model and software version.

By incorporating these strategies, you can effectively reduce interference between EGFP and luciferase signals in your IVIS imaging experiments, enabling accurate and reliable in vivo visualization and quantification of gene expression.

Yours sincerely,

This protocol list might provide further insights to address this issue.

Cédric Chaveroux

Ok thanks a lot for the reply

Albert Rizvanov

Detection of luminescence and fluorescence occur under different conditions. During luminescence acquisition there is no illumination with excitation light, so there should be no signal from FLUORESCENT proteins. I would suggest to use red fluorescent proteins since red light has better tissue penetration.

And again, when you are detecting fluorescent proteins, you don't inject luc substrate, so there should be no interference.

Benjamin Thoha Thomas

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