Fluorescence molecular imaging is the visualization of cellular and biological function in vivo to gain deeper insights into disease processes and treatment effects. Designing a more effective study requires careful consideration but can ultimately yield better and more meaningful results. The following steps will allow you to gain unique and meaningful biological information from your fluorescent imaging studies.

1. Set clear goals

   To start, be sure to define your goals clearly in order to identify which approach will maximize your research. For example, is your primary goal to explore early disease, or late disease? Waiting until the end of the study to understand the limited biology available at that time can miss a lot of biology that has already happened.

2. Know your animal model

   When choosing your animal model, make sure you know its biology extremely well. The timing and/or kinetics of biology is important. If you are imaging cells that don’t arrive on the scene until a certain time, then it is important to plan your probe injection and the imaging window to coincide with when the biology is operant.

3. Identify optimal probe objectives

   What do you need your probe to be able to do? This could be binding to a specific cell surface receptor or fluoresce only in the presence of a certain biomarker. If you’re looking at working with antibodies, be cautious about using them as probes.

4. Understand probe performance in regard to your model

   Consider probe characteristics such as route of metabolism, time frame from injection to imaging, and tissue washout time. Make sure to test probes to identify best imaging strategies, timepoints, and analysis approaches.

   Depending on your imaging strategy, choosing probes to detect disease markers may yield early biological information to characterize disease onset and progression, whereas probes for detecting treatment markers may provide data predictive of non-imaging terminal readouts.

5. Choose proper imaging controls

   Appropriate study design should include both positive and negative (i.e., un-diseased) control mice injected with the probe.

6. Confirm results ex vivo

   Corroborate in vivo quantifications using in situ and/or ex vivo tissue readouts. For fluorescence microscopy, frozen sections are the recommended method over formalin fixing, as fixation can potentially degrade a fluorescent signal.

7. Validate imaging results against non-imaging gold standard metrics

   Proper imaging results will correlate or sometimes surpass gold standard readouts, such as histology.

Incorporating vital imaging practices such as depilation, consistent animal positioning, careful injection method, and proper analysis along with properly selected, validated probes is key to maximizing the results of your in vivo imaging study. To learn more about what to keep in mind when designing your study and other tips and tricks for fluorescence imaging including multiplexing, read our best practice guide: