Confocal spectroscopy for cell 3D microthermometry

One in two people will be diagnosed with cancer during their lifetime, presenting a significant challenge to the UK goal of "staying healthy for longer". For some cancers, therapeutic innovations have increased survival, but for other cancers, such as brain cancer, outcomes have changed little in 20 years. A topic of increasing interest in the cancer research community is the critical role of metabolism in cancer cell behaviour. The Warburg effect, a metabolic switch from oxidative to glycolytic metabolism in cancer cells, has been documented for over decades, but much remains unknown about the nature and significance of cancer cell metabolism. The intrinsic pyrogenic substances secreted by tumour cells induce distinct hyperthermia in the temperature range of 37 to 42 C. Simultaneously, different parts of the cell can be at different temperatures, with mitochondria more than 10 C above basal temperature.
We need to investigate fundamental unknowns about cancer cell metabolism, its role in cancer growth and the potential of targeting more metabolically active regions within cancer for therapy. Significantly, there is increasing awareness that this needs to be done in the context of intact cancer tissue, where the cancer cell interactions with the cellular microenvironment can be observed. Cancer cell-microenvironment interactions influence cancer cell biology and are not effectively modelled using in vitro cancer cell cultures. Crucially, then, cancer cell metabolism must be interrogated in tissue slice culture, and ultimately in rodent models, for which we need innovative technologies as proposed here.
For this, it is necessary to have an imaging technique capable of working in three dimensions in thick tissue, and able to provide the temperature distributions in the cancer environment. This can be achieved by using luminescent nanoparticles as probes. Such nanoparticles can be 500 times smaller than a red blood cell, and when they are excited with light of a wavelength ("colour"), they will re-emit light in a different wavelength. The analysis of this re-emitted light can provide information about the temperature of its environment. Importantly, certain wavelengths can propagate longer in tissue without being attenuated, which can be used for obtaining information from inner areas. This will enable the 3D reconstruction of the map of temperatures.

We need to investigate cancer cell metabolism in order to understand its role in cancer growth and the potential of targeting more metabolically active regions within cancer for therapy. This can be done by studying the temperature distributions in affected tissue.
This project will develop an instrument and new technology capable of mapping the temperature of in vitro 3D cell cultures and human tissue slices with micrometre resolution in 3D.
The technique relies on the use of anti-Stokes reporters (lanthanide-based upconversion nanoparticles). These reporters are studied for bioimaging, but they also offer opportunities for thermal imaging, which is the target of this project. They present many advantages: (i) The anti-Stokes emission avoids tissue self-fluorescence (ii) The method allows auto-calibration because it will monitor the ratio of two different emissions coming from the same position, (vi) The use of lanthanides prevents photo-bleaching, the main problem of dyes. (v) Because some lanthanides provide ladder-like energy levels, the efficiency of the luminescence is very high, allowing lower power levels and simpler lasers and avoiding tissue heating.
A fundamental aspect of this research is to study real tissue (in 3D) rather than just 2D isolated cells. For this, the penetration depth has to be maximized. This will be achieved by: (i) The use of a confocal arrangement which allows the 3D temperature imaging of the volume by scanning plane by plane, by filtering the optical signal with a pinhole. (ii) The use of NIR excitation (in the 3rd biological window) enables deep penetration (>0.1 mm), because the attenuation coefficient in tissue is 3x smaller than for visible light, and the Rayleigh scattering is reduced since it is inversely proportional to the fourth power of wavelength, allowing better focusing on selected planes.