The authors describe an efficient technique for the selective chemical and biological …

• Abstract
• Introduction
• Materials and Methods
• Results and Discussion
• Conclusion
• Acknowledgement
• References
• Figures

Biology Articles » Biophysics » Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes » Introduction

Introduction

- Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes

In the last decade, we have witnessed a tremendous growth in high-resolution techniques driven by the need to understand cellular biochemical and biophysical processes in ever greater detail. For example, patch clamp measurements of ion channel activities (1) have profoundly shaped our understanding of ion channel physiology and cellular communication. The use of carbon fiber ultramicroelectrodes for in vivo voltammetry have revealed exciting information on single transmitter-vesicle exocytotic release events (2, 3). Confocal microscopy and two-photon microscopy have provided striking images of the workings of cellular machinery, such as the dynamics of intracellular calcium ion and the localization of single serotonin-containing granulae in RBL cells (4, 5). The Abbe resolution limit even is bypassed in near-field spectroscopic probes, in which an optical resolution as high as 12 nm has been achieved (6). The manipulation of single organelles and even single biomolecules has been made possible by optical trapping, and this has since been applied to a wide range of interesting biological problems (7-10).

Although numerous high-resolution techniques exist to detect, image, and analyze the contents of single cells and subcellular organelles, few methods exist to control and manipulate the biochemical nature of these compartments. In this study, we demonstrate a high-resolution technique to alter the biochemical content of single cells and organelles in situ, based on permeabilization of phospholipid bilayer membranes by pulsed electric fields (electroporation). During the effective pore-open time, cell-impermeant solutes added to the extracellular medium can enter the cell interior by diffusion. In contrast to microinjection techniques for single cells and single nuclei (11), electric field-induced reagent transfer can be applied for biological containers of subfemtoliter (10¹⁵ liters) volumes and can be extremely fast and precisely timed (12, 13), which is of importance in studying fast reaction phenomena.

The membrane voltage V_m, at different loci on phospholipid bilayer spheres during exposure in a homogenous electric field of duration t, can be calculated from

[ 1 ]

where E is the electric field strength, r_c is the cell radius,  is the angle in relation to the direction of the electric field, and t is the capacitive-resistive time constant. Pore-formation will result at spherical coordinates exposed to a maximal potential shift, which is at the poles facing the electrodes (cosa = 1 for a = 0; cos a = 1 for a = p ). Generally, electric field strengths on the order of 1 to 1.5 kV/cm for durations of a few µs to a few ms are sufficient to cause transient permeabilization in 10-µm outer-diameter spherical cells (14-16). A recent study shows that isolated mitochondria, because of their correspondingly smaller size, require 7- to 10-fold higher electric field strengths to incorporate a 7.2-kilobase plasmid DNA (17). Mitochondrial outer-membrane fusion at lower electric field strengths of »2.5 kV/cm also has been observed (18).

Conventional electroporation using high-voltage pulse generators is made in a batch mode in relatively large containers, which typically permeabilize the membrane of millions of cells simultaneously (14-16, 19). Instrumentation that can be used for electroporation of a small number of cells in suspension (20, 21) and for a small number of adherent cells grown on a substratum (22, 23) also has been described. In the present work, electroporation with subcellular spatial resolution is accomplished by applying the electric field through carbon fiber ultramicroelectrodes (»5 µm in diameter). In addition to the high spatial resolution achieved by using microelectrodes, this technique avoids the use of expensive high-voltage pulse generators and complicated microchamber mounts. The method can in principle be battery-operated because the spacing between the electrodes is small, typically 20 µm or less, which results in a high electric field strength with a small amplitude voltage pulse.