Single Cell Viability Measured by Scanning Electrochemical Microscopy and Live/DeadTM Staining
Microscopy and Microanalysis
Metabolic activity is an unambiguous indicator of cell viability and health. A widely utilized probe molecule for cell cultures is resazurin, which is reduced to resorufin by living cells. Scanning electrochemical microscopy (SECM) can probe the metabolic activity of single cells via O 2 reduction ; respiration causes a decrease in the O 2 concentration above living cells. However, based on its standard reduction potential, oxidized ferrocenemethanol (FcCH 2 OH) may act as an alternate
... an alternate electron acceptor. By locally oxidizing FcCH 2 OH at a microelectrode, we probe metabolic activity using feedback measurements, which have higher resolution than passive measurements of O 2 diffusion profiles. If FcCH 2 OH is regenerated by the cell, the current will increase when the microelectrode is positioned above a cell. This is known as positive feedback and has been reported for HeLa cells  . However if FcCH 2 OH diffusion is blocked by the cell, the current will decrease when the microelectrode is positioned above a cell. This is known as negative feedback and has been reported for COS-7 cells . Living cells have been probed via SECM using a variety of redox mediators; the information obtained depends critically on the interaction of the probe molecule and the living cell. Cellimpermeable mediators such as Fe(CN) 6 3-/4or Ru(NH 3 ) 6 3+/2+ are always blocked by the membrane, leading to negative feedback or topography [4, 5] . On the other hand, cell-permeable mediators, most notably quinones, can participate in cellular redox processes. Here mediator concentration becomes critical, as intracellular redox processes are not detectable when the concentration of mediator in solution far exceeds the concentration of redox centers. Therefore, while intracellular redox activity is obtained at a low (40 µM) concentration of menadione or 1,2-naphthoquinone , at high (1000 µM) concentration, many redox mediators detect only topography , despite intracellular regeneration. Both menadione and 1,2-naphthoquinone are reduced by the microelectrode, meaning that the cell must re-oxidize them to obtain positive feedback. We examined adherent HeLa cells in cell culture medium containing 500 µM FcCH 2 OH. The three electrode system comprised a Pt microelectrode of about 5 µm radius, a Pt wire counter electrode, and a Ag/AgCl reference electrode. All experiments utilized the reversible, one-electron oxidation of FcCH 2 OH, which is diffusion-limited at E = 0.5 V vs. Ag/AgCl. When the potential on a Pt microelectrode is stepped to this potential, it reaches a steady, diffusion-limited current (I lim ). First the microelectrode was brought into contact with the culture medium and positioned near a group of cells. Approach curves were acquired by moving the microelectrode towards the polystyrene substrate at 1.0 µm/s; because the microelectrode was positioned away from the cells, the normalized current (I tip /I lim ) vs. distance curve matched simulated curves for approach to an insulating surface  . Approach was halted when I tip /I lim ~ 0.8, corresponding to an imaging height of 10 µm. Images were obtained at 40.8 µm/s or 61.2 µm/s.