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Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

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Comparing fluorescent contrast agents for fluorescence guided surgery using 3-D cryo-imaging.

Augustino V Scorzo¹, Caleb Y Kwon¹, Rendall R Strawbridge¹

¹Dartmouth College, Thayer School of Engineering.

Proceedings of Spie--The International Society for Optical Engineering

|October 11, 2024

Summary

Fluorescence cryo-imaging requires stable fluorescent probes. Most tested agents were stable when frozen, but FITC and its PEGylated derivatives showed significant fluorescence loss.

Keywords:

cryo-imaging fluorescence guided surgery fluorescent contrast agent oncopig phantom imaging

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Area of Science:

Biomedical Imaging
Optical Imaging
Fluorescence Imaging

Background:

Fluorescence cryo-imaging offers high-resolution 3-D biodistribution analysis.
Effective application requires fluorescent agents stable at low temperatures (-20°C or below).
Consistent fluorescence intensity under physiological and cryo-imaging conditions is ideal.

Purpose of the Study:

To assess the fluorescence intensity stability of 11 contrast agents during freezing.
To identify fluorescent probes suitable for cryo-imaging applications.

Main Methods:

Evaluating 11 fluorescent contrast agents in a tissue-simulating phantom.
Measuring mean fluorescence intensity before and after freezing.
Demonstrating cryo-imaging of a whole pig brain with a selected agent.

Main Results:

Most fluorescent contrast agents exhibited stability within approximately 25% after freezing.
FITC (fluorescein isothiocyanate) and PEGylated FITC derivatives showed a marked decrease in fluorescence intensity upon freezing.
Cryo-imaging of a whole pig brain was successfully demonstrated.

Conclusions:

Fluorescent contrast agent stability varies significantly with temperature changes.
FITC and its derivatives are not ideal for fluorescence cryo-imaging due to reduced fluorescence at low temperatures.
Careful selection of fluorescent probes is crucial for successful fluorescence cryo-imaging.