Transient state microscopy probes patterns of altered oxygen consumption in cancer cells

Abstract

Altered cellular metabolism plays an important role in many diseases, not least in many forms of cancer, where cellular metabolic pathways requiring lower oxygen consumption are often favored (the so-called Warburg effect). In this work, we have applied fluorescence-based transient state imaging and have exploited the environment sensitivity of long-lived dark states of fluorophores, in particular triplet state decay rates, to image the oxygen consumption of living cells. Our measurements can resolve differences in oxygen concentrations between different regions of individual cells, between different cell types, and also based on what metabolic pathways the cells use. In MCF-7 breast cancer cells, higher oxygen consumption can be detected when they rely on glutamine instead of glucose as their main metabolite, predominantly undergoing oxidative phosphorylation rather than glycolysis. By use of the high triplet yield dye Eosin Y the irradiance requirements during the measurements can be kept low. This reduces the instrumentation requirements, and harmful biological effects from high excitation doses can be avoided. Taken together, our imaging approach is widely applicable and capable of detecting subtle changes in oxygen consumption in live cells, stemming from the Warburg effect or reflecting other differences in the cellular metabolism. This may lead to new diagnostic means as well as advance our understanding of the interplay between cellular metabolism and major disease categories, such as cancer.

Transient state imaging of live cells using single plane illumination and arbitrary duty cycle excitation pulse trains

“…The chosen k isc value showed the best overall agreement between experimental data and the fits. Moreover, epi-fluorescence wide-field TRAST measurements on CEDA-SE stained MCF-7 cells supported this assumption [23]. The whole decay of the normalized mean fluorescence due to build-up of the R state could not be followed in the TRAST curves within the maximally applicable pulse widths, because of pronounced photobleaching.…”

“…So far, TRAST imaging has been performed in regular and laser scanning confocal [20,21], widefield [22,23] and total internal reflection [24] mode. TRAST measurements require that a significant population difference in the monitored transient states is generated upon variation of the excitation pulse train characteristics.…”

“…Instead, the k t rates can be expected to be proportional to the local concentration of molecular oxygen, which is a predominant triplet state quencher [17]. Although a direct translation of measured k t into absolute local concentrations of oxygen is difficult, recent TRAST data have also shown that for cultured cells grown under different partial oxygen pressures the average k t rates in the cells depend linearly on this partial oxygen pressure [23]. In contrast, the k ox -images ( Figure 4C) revealed patterns much more influenced by noise.…”

Determination of molecular stoichiometry without reference samples by analyzing fluorescence blinking with and without excitation synchronization

“…While the pulse duration dependence of   ) (w F norm (the so-called TRAST-curves) can be used to extract transient state rate parameters from fluorescent molecules in solution 14,[19][20][21] as well as in live cells [22][23][24] using confocal 14,19,21 , total internal reflection 20 , wide-field 22,24 and single-plane illumination 23 imaging techniques, we are in this study mainly focusing on the relative changes of the dark states in the confocal detection volume of our FCS setup. The sum of the transient state relaxation amplitudes A 1 and A 2 are obtained by fitting…”

Stimuli-responsive protection of optically excited triplet ensembles against deactivation by molecular oxygen

“…physiologically relevant O 2 concentrations (ranging from nM-200 µM) deserve special attention. 4 For example, one can mention the transient state imaging technique, 5 single-nano-particles with a ratiometric O 2 -response 6 or phosphorescent metalloporphyrins encapsulated in hydrophobic dendrimers. 7,8 The main experimental drawback of these sensing techniques is that phosphorescence emission is an integral parameter, which depends simultaneously on the local temperature and local oxygen contamination.…”