Hypoxia imaging for surgical guidance has never been possible, yet it is well known that most tumors have microregional chronic and/or cycling hypoxia present as well as chaotic blood flow. The ability to image oxygen partial pressure (pO₂) is therefore a unique control of tissue metabolism and can be used in a range of disease applications to understand the complex biochemistry of oxygen supply and consumption.

Delayed fluorescence (DF) from the endogenous molecule protoporphyrin IX (PpIX) has been shown to be a truly unique reporter of the local oxygen partial pressure in tissue. PpIX is endogenously synthesized by mitochondria in most tissues and the particular property of DF emission is directly related to low microenvironmental oxygen concentration. Here it is shown that protoporphyrin IX (PpIX) has a unique emission in hypoxic tumor tissue regions, that is measured as a delayed fluorescence (DF) signal in the red to near-infrared spectrum.

A time-gated imaging system was used for PpIX DF for wide field direct mapping of pO₂ changes. Acquiring both prompt and delayed fluorescence in a rapid sequential cycle allowed for imaging oxygenation in a way that was insensitive to the PpIX concentration. By choosing adequate parameters, the video rate acquisition of pO₂ images could be achieved, providing real-time tissue metabolic information.

In this report, we show the first demonstration of imaging hypoxia signals from PpIX in a pancreatic cancer model, exhibiting >5X contrast relative to surrounding normal oxygenated tissues. Additionally, tissue palpation amplifies the signal and provides intuitive temporal contrast based upon neoangiogenic blood flow differences.