Introduction of Membrane-Impermeant Dyes into Single Cells by Electroporation. Fig. 1 is a schematic setup of the experiment, which depicts the geometrical arrangement of the electrodes with respect to a cell undergoing permeabilization. Because the electrodes are movable in steps of 0.2 µm in three-dimensional space, a high degree of freedom to focus in on a single cellular structure of choice in a complex multicellular network is offered. This is in contrast to fixed-electrode poration chambers in which the dimensions of the electrodes typically extend the size of a cell severalfold. Ideally, a cell is approached with the electrodes at an angle from above, and electrode contact with the cell membrane is avoided, which otherwise can lead to electrochemical transformation of membrane-bound proteins and other structures. Highly focused electrical fields were produced by application of single or multiple rectangular waveform voltage pulses by using a low-voltage pulse-generator.
The high spatial resolution achieved with this technique is demonstrated in Fig. 2, in which fluorescein, a largely cell-impermeant highly fluorescent dye, is electroporated into single cells. The bright-field images in Fig. 2 A and C shows examples of populations with a small number of cells in which one cell was electroporated in a fluorescein-supplemented Hanks'-Hepes buffer. Application of voltage pulses results in formation of pores and diffusion of extracellular fluorescein into the cell. Resealing of the pores after electroporation then traps the dye inside the cell. Fig. 2 B and D shows fluorescence images of the same fields as in Fig. 2 A and C. The electroporated cells exhibit strong fluorescence from fluorescein entrapped in the cytosol.
The success rate for electroporation of progenitor cells as well as individual cells in a kidney 293 cell line for the introduction of fluorescein (n = 28) by using 3 to 10 pulses of 0.99 ± 0.1 V at 0.5 Hz repetition rate in flawless experiments was 86%. We found that, below these plasma membrane threshold potentials, it was impossible to detect any dye accumulation in the cells. Reversible membrane breakdown can nonetheless occur at lower potentials but would require more sensitive detection means to observe than those used in the present study.
Electroporation of Intracellular Organelles and Cellular Processes. Further experiments were performed with fluorescein to see whether organelles of single progenitor cells could be electroporated in situ. Specifically, fluorescein was introduced into single cells by 10 0.5-Hz superthreshold field applications, resulting in potential shifts at the cell membrane of 1.6 V ± 0.07 V (range 1.5 to 2.4 V). After this, the extracellular dye-containing media was replaced by a Hanks'-Hepes solution. The result of this electroporation scheme is shown in Fig. 3A, in which punctuate fluorescence is observed in the exonuclear cytoplasmic region but not in the nuclear region, which contains a smaller number of organelles. In total, 18 of 20 cells electroporated in this protocol displayed a similar staining pattern, reminiscent of what is observed by using an organelle-specific dye such as rhodamine 123, which labels the mitochondrial fraction of organelles (Fig. 3B). In comparison, cells electroporated at the plasma membrane threshold potential generally displayed a diffuse fluorescence from fluorescein entrapped in the cytosol (Fig. 3C). This observation of a differential localization of fluorescein comparing application of low and high electric-field strengths is consistent with the higher breakdown potential of intracellular organelles as predicted by Eq. 1 because of their smaller dimensions. It has been shown that 2.5- to 10-fold higher electric-field strengths are required for electroporation and fusion of isolated mitochondria compared with 10-µm diameter cells (17, 18). The results are also in accord with morphological observations in giant squid axons following mild, moderate, and severe electroporation protocols (26). It should be stressed, however, that it is unclear what organelles accumulated fluorescein and that electroporation at the plasma membrane threshold potential, in particular by using high-frequency multiple-pulse protocols, might result in permeabilization of organelles. These issues were not dealt with in the present study but warrant further investigation.
To find functional evidence of organellar electroporation, single progenitor cells were electroporated with 10 µM fluo-3, a dye that becomes fluorescent when chelating Ca
2+ (
27). Because the concentration of free intracellular Ca
2+ is extremely low (»100 nM), the cytosol only becomes weakly fluorescent on addition of fluo-3. Specifically, fluo-3 supplemented to a Ca
2+-free Hanks'-Hepes buffer is first introduced into the cells with a sequence of 1- to 5-, 0.5- to 10-ms rectangular waveform pulses at the plasma membrane threshold potential. This pulse protocol only induced a slight increase in cytoplasmic fluorescence. After this, the fluo-3-containing buffer was replaced with a Ca
2+-free Hanks'-Hepes buffer, and additional pulses of 1.3 to 2.8 V (1 ms) were applied. Of a total of 47 cells, this protocol resulted in increased cytoplasmic fluorescence in 31 cells, most likely because of membrane breakdown of intracellular calcium stores.
In experiments using multiple pulse protocols with plasma membrane superthreshold field applications, cells frequently displayed a swollen and damaged appearance. Presumably, such cells enter a hyper-leakage state with deterioration of many cellular functions (26). Of importance, high-voltage applications appear to affect organellar structures to a lesser extent (17) and were found in single-pulse applications for gene transfer not to affect considerably cell viability in COS 7 cells (see below). Electroporation of organelles in situ might become useful for probing and selective labeling of organelles for functional studies and for recently emerging single-organelle analysis schemes in conjunction with chemical separations (10).
Experiments were conducted to electroporate individual submicrometer diameter cellular processes as a further demonstration of the high spatial resolution of this technique. Specifically, fluo-3 was electroporated into individual processes of thapsigargin-treated neuronal progenitor cells. Thapsigargin releases »50% of total Ca2+ from intracellular stores in Jurkat cells (28) to the cytoplasm by inhibition of a Ca2+-ATPase of the endoplasmic reticulum (29). Fig. 4 shows a time sequence of the spreading of a fluorescence wave emanating from the electroporation loci in two cells. These experiments were performed in quadruplicate and consistently showed the same pattern.
Transfection of Single COS 7 Cells. In a third category of experiments, individual COS 7 cells were electroporated to investigate the feasibility of selective gene transfer by using the presented technique as well as to investigate the survival rate of cells after electroporation. The plasmid vector pRAY 1 was used as a reporter gene and was electroporated into individual COS 7 cells. A Hepes buffer with 10 µg plasmid/ml and 0.75% dimethyl sulfoxide was used throughout the experiments. In initial experiments, using a plasmid-supplemented Hanks'-Hepes buffer, it was noted that it was difficult to achieve a high transfection yield. Addition of dimethyl sulfoxide at low concentrations to the electroporation buffer, however, facilitated the electroporation-induced uptake of plasmid into cells. This is in accordance with observations made by other researchers (30). For experiments, single 1-ms rectangular waveform voltage pulses of »1.9 V were applied followed by careful washing of the cells with Hepes buffer. Transfected cells (n = 50, five cell dishes) were detected 36 hours after electroporation by using the same laser-induced fluorescence microscope setup as for the other dyes. The transfection yield and survival rate was 97%. Individual electroporated cells generally exhibited strong fluorescence from expression of green fluorescent protein (Fig. 5). Control cells (n > 50, five cell dishes) exposed to plasmid at the same concentration and time did not exhibit any fluorescence over background level under the same detection conditions (Fig. 5).
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