Geographical mapping of metabolites in biological tissue with quantitative bioluminescence and single photon imaging

Abstract

This article features a novel technique for measuring the spatial distribution of metabolites, such as ATP, glucose, and lactate, in rapidly frozen tissue. Concentration values are obtained in absolute terms and with a spatial resolution of single-cell dimension. The method is based on enzymatic reactions that link the metabolite of interest to luciferase with subsequent light emission. Using a specific array, cryosections are brought into contact with the enzymes in a well-defined, reproducible way inducing a distribution of light across the section with an intensity that is proportional to the metabolite concentration. The emitted light can be visualized through a microscope and an imaging photon counting system, and the respective image can be transferred to a computer for image analysis. Measurements in spherical cell aggregates with central necrosis demonstrate a close correlation between the distribution of ATP and of cellular viability at a microregional level. Similarly, ATP and glucose are correlated with the geometrical arrangement of more viable and more necrotic tissue regions in human melanomas xenografted in nude mice. Lactate did not show such a structure-related distribution in these tumours. Structure-related distributions of ATP, glucose, and lactate are found in cervix tumours of patients. In contrast to the heterogeneous distributions in tumours, the distribution patterns were much more homogeneous in normal tissues. Regional differences were present, but were much more gradual than in malignancies. This was illustrated for heart muscle where ATP concentrations were found that agreed with data in the literature, and that showed a decrease in periventricular areas.

Spatial Heterogeneity and Oxygen Dependence of Glucose Consumption in R3230Ac and Fibrosarcomas of the Fischer 344 Rat

“…From this, we conclude that both tumor lines depend on oxygen and the lack of it is probably mainly responsible for the formation of necrosis. Although this is true for many solid tumors, there is evidence that in some tumors, the lack of glucose may be causative for necrosis formation as well (23,28). This is further supported by the finding that a-ketoacids, such as pyruvate, have a protective effect against oxidative stress.…”

“…The microscope stage was protected from extraneous light using a custom-fit black box. The sections were brought into contact with reaction solutions for either glucose or lactate, which were prepared following earlier published protocols (23)(24)(25). Light emission from the enzymatic activity of the luciferase reporter was detected through the bottom of the coverslip.…”

Effect of aerobic exercise on tumor physiology in an animal model of human breast cancer

“…The energy status of the tumor was measured using bioluminescence for ATP as described previously (31). In brief, bioluminescence enzymatic reaction was conducted with a temperature-controlled, black box-encased fluorescence microscope using a cooled 16-bit CCD camera with photon-counting capability.…”

Mapping of ATP, Glucose, Glycogen, and Lactate Concentrations Within the Arterial Wall

“…14,17 Optimization of measurement conditions (described in the online supplement) has reduced this artifact, but true tissue gradients will be slightly underestimated. This is most clearly illustrated at tissue borders, where an infinitely steep gradient would be expected but a less steep gradient is registered (profiles in Figures 4 and 5).…”