Electron spin resonance microscopic imaging of oxygen concentration in cancer spheroids

Oxygen (O2) plays a central role in most living organisms. The concentration of O2 is important in physiology and pathology. Despite the importance of accurate knowledge of the O2 levels, there is very limited capability to measure with high spatial resolution its distribution in millimeter-scale live biological samples. Many of the current oximetric methods, such as oxygen microelectrodes and fluorescence lifetime imaging, are compromised by O2 consumption, sample destruction, invasiveness, and difficulty to calibrate. Here, we present a new method, based on the use of the pulsed electron spin resonance (ESR) microimaging technique to obtain a 3D mapping of oxygen concentration in millimeter-scale biological samples. ESR imaging requires the incorporation of a suitable stable and inert paramagnetic spin probe into the desirable object. In this work, we use microcrystals of a paramagnetic spin probe in a new crystallographic packing form (denoted tg-LiNc-BuO). These paramagnetic species interact with paramagnetic oxygen molecules, causing a spectral line broadening that is linearly proportional to the oxygen concentration. Typical ESR results include 4D spatial-spectral images that give an indication about the oxygen concentration in different regions of the sample. This new oximetry microimaging method addresses all the problems mentioned above. It is noninvasive, sensitive to physiological oxygen levels, and easy to calibrate. Furthermore, in principle, it can be used for repetitive measurements without causing cell damage. The tissue model used in this research is spheroids of Human Colorectal carcinoma cell line (HCT-116) with a typical diameter of ∼600μm. Most studies of the microenvironmental O2 conditions inside such viable spheroids carried out in the past used microelectrodes, which require an invasive puncturing of the spheroid and are also not applicable to 3D O2 imaging. High resolution 3D oxygen maps could make it possible to evaluate the relationship between morphological and physiological alterations in the spheroids, which would help understand the oxygen metabolism in solid tumors and its correlation with the susceptibility of tumors to various oncologic treatments.