This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.
This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe’s lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 μm is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.
This protocol shows how ESR micro-imaging can be applied to map oxygen concentration near live small samples. A spatial resolution of ~30 x 30 x 100 μm is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve. In the near future we plan on further improving this methodology to provide live sample images with a resolution of a few microns, providing contrast parameters such as super oxide concentration, acidity (pH), probe diffusion coefficient and, of course, oxygen concentration. These capabilities are complementary to the current optical-based methodologies both in terms of contrast type and also of samples characteristics (e.g non-transparent thick samples and, in some cases, intracellular vs. extracellular measurements).
The authors have nothing to disclose.
This work was partially supported by grant no. 213/09 from the Israeli Science Foundation, grant no. 2005258 from the BSF foundation, grant no. 201665 from the European Research Council (ERC), and by the Russell Berrie Nanotechnology Institute at the Technion. We acknowledge the help of Prof. Noam Adir and Faris Salame from the Schulich Faculty of Chemistry at the Technion regarding the supply and handling of the cyanobacteria. The help and the support of Svetlana Yoffis from the Technion Micro-Nano Fabrication Unit is greatly appreciated.
Material Name | Type | Company | Catalogue Number | Comment |
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Centrifuge | Kendro | Heraus, 75003235 | ||
Perdeuterated triarylmethyl (trityl) radical | Synthesized at Novosibirsk using the method described in reference 6. | |||
BG-11 buffer | For instruction preparation, see Scheme 1 and references 8, 9. | |||
Syringe | Hamilton | Microliter 7000.5 | ||
Ultraviolet Curing | Norland Products, Inc. | NOA63, or NOA61. |