Time-lapse fluorescence as an imaging modality for effective surgical tumor removal

In the surgical treatment of cancer, it is important to distinguish tumors from healthy tissue. Fluorescent markers can help do this by enhancing the contrast of tumors during surgery. Some markers show a phenomenon called “delayed fluorescence” (DF), which is based on the detection of “hypoxia” (or low oxygen concentration), a condition often seen in tumors.

Real-time hypoxia imaging can provide high contrast between tumors and healthy cells. This can allow surgeons to remove a tumor effectively. However, real-time imaging of hypoxia for surgical monitoring has not yet been achieved.

In a new study published in Journal of Biomedical Optics (JBO), researchers proposed an optical imaging system that enables real-time imaging of tissue oxygen concentrations in tumors with chronic or transient hypoxia. The team achieved this by using an endogenous molecule called protoporphyrin IX (PpIX), which exhibits DF in the red to near-infrared region.

“It is a truly unique reporter of the local partial pressure of oxygen in tissues. PpIX is endogenously synthesized by mitochondria in most tissues, and the special emission property of DF is directly related to the low concentration of oxygen in the microenvironment,” explains Brian Pogue, Chair of the Department of Medical Physics at the University of Wisconsin-Madison. is an adjunct professor of engineering at Dartmouth College and senior author of the study. “Healthy cells show little to no DF because it is quenched in the presence of molecular oxygen.”

The technical challenge in the detection of DF due to its low intensity; background noise makes detection difficult without a single photon detector.

The team overcame this problem with a highly sensitive, time-controlled imaging system that allows signal detection only in a defined time window. This significantly reduces background noise and allows direct display of wide-field partial pressure of oxygen (pO₂) varies with the received DF signal. The result is real-time metabolic information, a useful map for surgical guidance.

Lead author Arthur Pettuso, a doctoral candidate in engineering at Dartmouth College, explains that “acquiring both instantaneous and delayed fluorescence in a rapid sequential cycle allowed us to image oxygen levels in a manner independent of PpIX concentration’.

Petusseau’s team demonstrated the effectiveness of their technique using mouse models of pancreatic cancer that exhibited hypoxic tumor growth. The DF signal obtained from the cancer cells was more than five times stronger than that from the surrounding healthy, oxygenated tissue. Signal contrast was further enhanced by tissue palpation prior to imaging to further enhance transient hypoxia.

According to Frédéric LeBlond, a professor of engineering physics at the Polytechnic of Montreal and associate editor of JBO, “The results reported by Pettuso’s team suggest hypoxia imaging as an effective approach to tumor identification in cancer treatment. The detection of PpIX DF uses a known clinical dye and an already validated human marker, with great potential for surgical monitoring and more.”

Petusseau notes that the image of pO₂ in tissues may also allow control of tissue metabolism. This, in turn, will help us better understand biochemistry oxygen supply and consumption.