Monitoring Metabolic Responses of Single Mitochondria within Poly(dimethylsiloxane) Wells: Study of Their Endogenous Reduced Nicotinamide Adenine Dinucleotide Evolution Emmanuel Suraniti, † Venkata Suresh Vajrala, † Bertrand Goudeau, † Serge P. Bottari, ‡ Michel Rigoulet, § Anne Devin, § Neso Sojic,* ,† and Stephane Arbault* ,† † University Bordeaux, ISM, UMR5255, F-33400 Talence, France, and CNRS, ISM, UMR5255, F-33400 Talence, France ‡ Laboratory of Fundamental and Applied Bioenergetics, University Joseph Fourier-Grenoble, INSERM U1055, 2280 rue de la Piscine, 38400 Saint Martin d’Hè res, France § University Bordeaux Segalen, CNRS, IBGC UMR5095, 1 rue Camille Saint Saë ns, 33077 Bordeaux, France * S Supporting Information ABSTRACT: It is now demonstrated that mitochondria individually function differently because of specific energetic needs in cell compartments but also because of the genetic heterogeneity within the mitochondrial pool-network of a cell. Consequently, understanding mitochondrial functioning at the single organelle level is of high interest for biomedical research, therefore being a target for analyticians. In this context, we developed easy-to-build platforms of milli- to microwells for fluorescence microscopy of single isolated mitochondria. Poly(dimethylsiloxane) (PDMS) was determined to be an excellent material for mitochondrial deposition and observation of their NADH content. Because of NADH autofluorescence, the metabolic status of each mitochondrion was analyzed following addition of a respiratory substrate (stage 2), ethanol herein, and a respiratory inhibitor (stage 3), Antimycin A. Mean levels of mitochondrial NADH were increased by 32% and 62% under stages 2 and 3, respectively. Statistical studies of NADH value distributions evidenced different types of responses, at least three, to ethanol and Antimycin A within the mitochondrial population. In addition, we showed that mitochondrial ability to generate high levels of NADH, that is its metabolic performance, is not correlated either to the initial energetic state or to the respective size of each mitochondrion. M itochondria are major organelles of aerobic cells because they constitute the most prominent source of ATP and they are involved in multiple anabolic and catabolic pathways. 1,2 In addition, mitochondria regulate a continuum of cellular functions, spanning from physiological metabolism to stress responses and cell necrosis. 3-5 Mitochondrial genetic defects or function alterations underpin a large number of human diseases, including premature aging, neurodegenerative disorders, cardiovascular disorders, and cancer. 6-9 Consequently, the monitoring of the mitochondrial metabolic status in physiological or pathological situations is of major interest. Methods dedicated to mitochondrial metabolic studies are usually performed with large populations of isolated organelles. 7 In particular, the electrochemical methods including oxygen measurements with the Clark electrode necessitate large quantities of mitochondria. This allowed defining standard energetic states observed as variations of the mean oxygen consumption by a population of typically millions of mitochondria (milligram amounts of proteins). 10 This allowed also for modern instruments to study the effects of a variety of pharmaceutical agents based on parallelization of measurements within microtiter plates. However, it is now demonstrated that mitochondria constituting the cell’ s mitochondrial network are genetically (heteroplasmy) and metabolically heterogeneous. 5,11,12 As a consequence, refined metabolic studies targeting mitochondria at the individual level should provide further insights into patho-physiological pathways. 13 Fluorescence microscopy has up to now been the gold standard method for single mitochondria studies, particularly because dozens of fluorescence dyes and sensors are commercially available to stain or detect many metabolic activities. Fluorescence microscopy, especially in confocal mode, was used successfully for studies of metabolic waves involving mitochondria in single cells, 14 including myocytes wherein the mitochondrial network is organized along the cytoskeleton. 14-17 In addition, the mitochondrial network appeared as an excellent model for the application to subcellular structures of super-resolution fluorescence micros- Received: February 15, 2013 Accepted: April 19, 2013 Published: April 19, 2013 Article pubs.acs.org/ac © 2013 American Chemical Society 5146 dx.doi.org/10.1021/ac400494e | Anal. Chem. 2013, 85, 5146-5152