Anal. Chem. 2003, 75, 4148-4154
Scanning Electrochemical Microscopy of Living Cells. 5. Imaging of Fields of Normal and Metastatic Human Breast Cells Wenju Feng, Susan A. Rotenberg, and Michael V. Mirkin*
Department of Chemistry and Biochemistry, Queens CollegesCUNY Flushing, New York 11367
Scanning electrochemical microscopy (SECM) was used to image fields of different types of human breast cells in monolayer culture. The goal of these experiments was to demonstrate the possibility of distinguishing between nontransformed human breast epithelial cells (MCF-10A) and metastatic breast cells (MDA-MB-231) by their redox activities. Imaging of densely packed cells by SECM requires approaches that differ from previously reported experiments with well-separated single cells. The combination of SECM with optical and fluorescence microscopies was used to locate individual cells in a homogeneous or heterogeneous field of cells. To establish that metastatic breast cells can be detected against a field of normal cells, the former were preloaded with fluorescent nanospheres and plated together with unlabeled MCF-10A cells. By matching SECM and fluorescence images of a selected group of metastatic cells, the level of discrimination and fidelity of the SECM signal could be shown. Several factors (distance between the electrode and the cells, cell density, choice of mediator, and its concentration) were identified that can be used to maximize the contrast between images of metastatic and nontransformed cells. These studies provide a framework for future analysis of malignant cells in human breast tissue samples. A number of recent publications have been focused on studies of biological cells by amperometric or potentiometric scanning probe techniques.1,2 The objectives of such studies ranged from detection of ion channels3 to topographic imaging4,5 to probing redox (“reduction-oxidation”) activity in a single cell6-8 to * Corresponding author. E-mail:
[email protected]. (1) Horrocks, B. R.; Wittstock, G. Biological systems. In Scanning Electrochemical Microscopy; Bard, A. J., Mirkin, M. V., Eds.; Marcel Dekker: New York, 2001; pp 445-519. (2) Yasukawa, T.; Kaya, T.; Matsue, T. Electroanalysis 2000, 12, 653-659. (3) (a) Korchev, Y. E.; Negulyaev, Y. A.; Edwards, C. R. W.; Vodyanoy, I.; Lab, M. J. Nat. Cell. Biol. 2000, 2, 616-619. (b) Schar-Zammaretti, P.; Ziegler, U.; Forster, I.; Groscurth, P.; Spichiger-Keller, U. E. Anal. Chem. 2002, 74, 4269-4274. (4) (a) Lee, C. M.; Kwak, J.; Bard, A. J. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1740-1743. (b) Liebetrau, J. M.; Miller, H. M.; Baur, J. E.; Takacs, S. A.; Anupunpisit, V.; Garris, P. A.; Wipf, D. O. Anal. Chem. 2003, 75, 563-571. (5) Korchev, Y. E.; Bashford, C. L.; Milovanovic, M.; Vodyanoy, I.; Lab, M. J. Biophys. J. 1997, 73, 653-658. (6) Liu, B.; Rotenberg, S. A.; Mirkin, M. V. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9855-9860. (7) Cai, C.; Liu, B.; Mirkin, M. V.; Frank, H. A.; Rusling, J. F. Anal. Chem. 2002, 74, 114-119.
4148 Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
measuring concentration profiles of various species consumed or ejected by cells.9-11 A few groups applied electrochemical imaging techniques to practical problems such as evaluation of the viability of a bovine embryo based on its consumption of oxygen12 and investigation of the resorption of bone by osteoclasts.13 Probing molecular transport through the nuclear membrane was reported recently.14 We have used scanning electrochemical microscopy (SECM) to measure kinetics of charge-transfer reactions in individual cancer cells and to map cellular redox activity with micrometer or submicrometer resolution.6-8,15 In a feedback mode SECM experiment, the tip ultramicroelectrode is placed in a solution containing the oxidized (or reduced) form of a redox mediator. The reduction (or oxidation) of mediator species at the tip surface produces steady-state current (iT,∞). If the tip is positioned near an immobilized living cell, the product of the tip reaction enters the cell and can be reoxidized (or rereduced) there (Figure 1). This process produces an enhancement in the current flowing at the tip electrode (positive feedback) depending on the normalized tip/cell distance (d/a, where d is the separation distance and a is the tip radius). If mediator regeneration by the cell is slow, the tip current decreases when the ultramicroelectrode is moved toward the cell because the cell surface blocks the diffusion of redox species to the tip (negative feedback). The mediated electron transfer between the SECM tip and intracellular redox centers (Ocell/Rcell) can be presented as follows:
O + ne- ) R
(at the tip electrode)
k
R + Ocell 98 O + Rcell
(inside the cell)
(1) (2)
Alternatively, some mediator species, such as N,N,N′,N′-tetra(8) Liu, B.; Rotenberg, S. A.; Mirkin, M. V. Anal. Chem. 2002, 74, 6340-6348. (9) Tsionsky, M.; Cardon, Z. G.; Bard, A. J.; Jackson, R. B. Plant Physiol. 1997, 113, 895-901. (10) Hengstenberg, A.; Blochl, A.; Dietzel, I. D.; Schuhmann, W. Angew. Chem., Int. Ed. 2001, 40, 905-908. (11) Yasukawa, T.; Kondo, Y.; Uchida, I.; Matsue, T. Chem. Lett. 1998, 8, 767768. (12) Shiku, H.; Shiraishi, T.; Ohya, H.; Matsue, T.; Abe, H.; Hoshi, H.; Kobayashi, M. Anal. Chem. 2001, 73, 3751-3758. (13) (a) Berger, C. E. M.; Horrocks, B. R.; Datta, H. K. J. Endocrinol. 1998, 158, 311-318. (b) Berger, C. E. M.; Horrocks, B. R.; Datta, H. K. Electrochim. Acta 1999, 44, 2677-2683. (14) Amemiya, S.; Wazenegger, T. Pittcon 2003, 2003; Absract No. 1300-3. (15) Liu, B.; Cheng, W.; Rotenberg, S. A.; Mirkin, M. V. J. Electroanal. Chem. 2001, 500, 590-597. 10.1021/ac0343127 CCC: $25.00
© 2003 American Chemical Society Published on Web 07/17/2003
Figure 1. Schematic representation of the feedback mode SECM experiment.
methyl-1,4-p-phenylenediamine dihydrochloride (TMPD), can be oxidized electrochemically and then undergo reduction by the cell. From SECM current versus distance (IT vs d/a, where IT ) iT/ iT,∞ is the normalized tip current) curves, one can extract the value of the effective heterogeneous rate constant (k) that expresses the overall rate of the mediator regeneration by the cell (reaction 2). Substantially different values of mediator regeneration rate constants were measured for different types of mammalian cells, in particular for nonmetastatic MCF-10A human breast cells and overtly metastatic MDA-MB-231 human breast cells.6 These findings opened the possibility of distinguishing between normal and metastatic breast cells based on differences in their redox response. Most recently, we showed that discrimination based on redox activity was significantly improved by the proper choice of redox mediator and its concentration.8 A major departure of the present study from our earlier experiments is that, instead of working with well-separated individual cells, we address the problems and potential use of the SECM with high-density cell populations. For these studies, images were obtained in a constant-height mode in which the tip is scanned laterally above the cells at a constant vertical distance from the dish surface. The objective of this analysis was to discriminate between cell types in a heterogeneous field of breast cells, which is a necessary intermediate step toward detecting single cells or cell clusters in breast tissue samples. EXPERIMENTAL SECTION Chemicals. Menadione (General Biochemicals, Chagrin Falls, OH), 1,2-naphthoquinone (Aldrich, Milwaukee, WI), and TMPD (Eastman Kodak, Rochester, NY) were used as received. All other chemicals were reagent grade. All aqueous solutions were prepared from deionized water (Milli-Q, Millipore Corp.). Culture media, serum, and antibiotics (fungizone, penicillin, streptomycin) were purchased from Invitrogen Corp. (Rockville, MD). Electrodes. A two-electrode setup was employed with a 0.25mm AgCl-coated Ag wire serving as a reference electrode. Carbon fibers (5.5-mm radius) and Au wires (5-mm radius) were heatsealed in glass tubes under vacuum and beveled to produce SECM tips, as described previously.16 The tip was polished with 0.05mm alumina before each experiment. (16) Bard, A. J.; Fan, F.-R. F.; Kwak, J.; Lev, O. Anal. Chem. 1989, 61, 132138.
Cell Culture. Mid-passage MCF-10A cells, a human breast epithelial cell line, were cultured in DMEM/F12 media (1:1) supplemented with 5% equine serum, insulin (10 µg/mL), epidermal growth factor (20 ng/mL), cholera toxin (100 ng/mL), and hydrocortisone (0.5 µg/mL) and maintained with penicillin (100 units/ml), streptomycin (100 µg/mL), and fungizone (0.5 µg/mL). Cells were redistributed at 1:3 every 3-4 days. MDA-MB-231 cells were cultured in Iscove’s Modified Dulbecco’s Medium with L-glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin. To carry out kinetic measurements with cells at low density, the cells were plated the previous day at (1-3) × 103 cells/60mm plate. For imaging experiments with cells at high density, cells were plated at 60-80% confluence ((1-5) × 104 cells/60mm plate) on the day before the SECM experiment. Similarly, to culture mixed fields of cells at high density, MCF-10A cells were plated to approximately 60-80% confluence on the same day with MDA-MB-231 cells (104 cells/plate) in complete DMEM/F12 medium. Prior to each SECM experiment, the cells were washed with phosphate-buffered saline (PBS; 153 mM Na+, 4 mM K+, 1 mM Ca2+, 1 mM Mg2+, 144 mM Cl-, and 10 mM phosphate, pH 7.4). For fluorescent labeling of cells, monolayers of MDA-MB-231 cells were incubated with fluorescent nanospheres (63-nm diameter) (Polysciences, Warrington, PA) in serum-free Iscove’s Modified Dulbecco’s Medium for 30 min at 30 °C and 5% CO2. During this period, >95% of cells were fluorescently labeled, as could be demonstrated with a fluorescence microscope. Instrumentation and Procedures. A 60-mm culture dish in which a monolayer of cells was immersed in PBS (containing the chemical mediator) was mounted on the horizontal stage of an Axiovert-100 inverted fluorescence microscope (Zeiss) that was set on an optical table. A video camera (IK-TU40A, Toshiba) was attached to the microscope to capture optical and fluorescence images. The SECM was set on the same optical table as the microscope so that the SECM tip could be positioned above the cell culture plate. The SECM apparatus and procedures employed for single-cell imaging and kinetic experiments were described previously.6-8,15,16 All measurements were performed at ambient temperature. A twoelectrode setup was employed for imaging and approach curve experiments. The approach curves were obtained by moving the tip vertically toward the cell surface at a scan rate of 0.5 mm/s. In an SECM imaging experiment, the tip current (iT) was recorded as a function of the tip position as the tip was scanned laterally in a horizontal (x-y) plane above the cell surface at a scanning rate of 10 mm/s. In this way, one-dimensional current profiles and gray scale constant-height images of cells were obtained. As described previously,15 a single cell or a group of low-density cells suitable for imaging can be found by scanning the tip quickly along x or y axis a few micrometers above immobilized cells. This approach, however, is not suitable for imaging of densely packed cells and especially mixed fields of different cells because the number and nature of cells present in a specific microscopic region cannot be determined from a linear scan. To resolve this problem, transmitted light video microscopy was used to locate the area of interest in the cell culture. The center of the selected rectangular area was moved to the center of the field of view using an eyepiece reticle. The SECM Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
4149
Figure 2. SECM image of a 230 µm × 160 µm densely packed field of MCF-10A cells. The tip was 5.5-µm-radius carbon disk. The mediator was 30 µM 1,2-naphthoquinone. One of the cells is highlighted to emphasize the honeycomb pattern.
micropositioner was used to bring the tip close to the dish surface and place it above one of the corners of the selected area. The x-y tip position with respect to the selected area was checked microscopically. This arrangement facilitated the acquisition of optical, electrochemical, and fluorescence images of the same group of cells. A scale bar with smallest scale of 10 µm and a total range of 1 mm was used to calibrate the optical and SECM images. In this way, specific groups of cells were located and imaged without patterning the cells or making marks on the underlying plastic surface. The vertical distance between the tip and the dish surface (typically, e20 µm) was adjusted based on the theoretical IT versus d/a curve.17 If the cell density was very high, such that there was no unoccupied plastic surface, the vertical position of the tip was adjusted relative to the cell surfaces. A redox mediator was added to the buffer solution for amperometric feedback mode experiments. Because of the negative standard potentials of menadione and naphthoquinone redox couples, deoxygenation of the solution was accomplished by purging the buffer with nitrogen gas. The nearly complete removal of O2 was verified by cyclic voltammetry. To preserve the nearly oxygen-free environment during cell locating, a stream of nitrogen gas was directed above the surface of the buffer. Unlike quinone mediators, the TMPD2+/+ couple is not air-sensitive because of its more positive formal potential (E1/2 ) +380 mV vs Ag/AgCl in PBS). The effective heterogeneous rate constants measured for MCF-10A cells in the presence of O2 (k ) (6.9 ( 0.9) × 10-3 cm/s; average of seven cells) and after the removal of oxygen (k ) (6.6 ( 0.8) × 10-3 cm/s; average of seven cells) were essentially identical. However, partial oxidation of TMPD to TMPD+ by oxygen can affect imaging. Therefore, in imaging experiments with TMPD as mediator, oxygen was also removed. RESULTS AND DISCUSSION Imaging Fields of Similar Cells. A 230 mm × 160 mm SECM image of a dense field of MCF-10A immobilized on a 60-mm culture dish (Figure 2) was obtained with a 5.5-mm-radius carbon tip in PBS containing 30 mM 1,2-naphthoquinone mediator. Naphthoquinone was reduced at the tip electrode to produce a (17) Kwak, J.; Bard, A. J. Anal. Chem. 1989, 61, 1221-1227.
4150
Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
diol, which was reoxidized inside the cell. As discussed previously,6 this image is essentially a map of intracellular redox activity. Unlike SECM images of single mammalian cells,6,15 a lower spatial resolution in Figure 2 caused by a larger tip radius and a comparatively large size of the imaged area does not allow one to see fine details of cell topography or redox reactivity distribution. A typical cell appears as a polygon with a darker region in the middle surrounded by a brighter halo. (To make this clear, one of the cells in Figure 2 is highlighted.) Brighter regions correspond to higher local rate of mediator regeneration, while a dark area in the center of each cell is a nucleus. The redox activity in the nucleus is very low either because it is impenetrable to the mediator species,6,7 or due to the low concentration of redox centers in the nucleus. The cellular “polygons” form a characteristic honeycomb pattern, which is not the case with more motile and less closely packed metastatic breast cells tested with the same quinone mediator (Figure 3A). The importance of choosing the right vertical separation distance between the tip and the cell field can be seen from Figure 4. In Figure 4A, a 5.5-mm tip was scanned laterally at a ∼21-µm distance above the bottom of the dish. This distance was too long for successful imaging of immobilized MCF-10A cells. Hence, only one “tall” cell (or a multilayered cluster of cells) can be seen. After the tip was moved 2 µm closer to the surface (Figure 4B), a number of brighter spots appeared in the image of the same area, which correspond to several MCF-10A cells. However, at this height the tip has scratched the tallest cell, as signified by a very bright spot in the image. This collision has somewhat affected the tip surface, so the bottom part of Figure 4B is a little darker. In the next scan, the tip was lowered by another 3 µm to obtain Figure 4C. At this separation distance, the resolution improved significantly and a characteristic MCF-10A cell pattern can be seen. This outcome was expected because the highest resolution in SECM is obtained at close tip-substrate separations. The number of scratched cells in Figure 4C has also increased, and at an even shorter distance (Figure 4D, the tip/plastic separation of ∼13 mm) the multiple tip/cell collisions degraded the image. An obvious problem with multicell imaging is that different cells (and especially species of different types) are different with respect to their size and shape. Because some cells are taller than others (due to cell spreading or roundedness), it is not easy to image a heterogeneous field of cells without scratching any of them. (Similar difficulties recently encountered by Liebetrau et al. during constant-height imaging of a single neuron were caused by significant variations in height between different parts of such a cell.4b) If the tip radius is smaller than the difference between the heights of two cells in the field, either the taller cells will be scratched in the process of constant-height imaging or the shorter cells will not be imaged clearly. One way to obviate this difficulty is to use a relatively large tip (radius g5 µm) and to scan it within one to two tip radii above the cell surface. Even with a sufficiently large tip, the choice of the proper scan height is not trivial, and a very good alignment of the tip with respect to the substrate is required. Moreover, the use of a larger tip decreases spatial resolution (as compared to single-cell imaging with smaller tips).15 The above problems could be largely eliminated by using constant-distance mode SECM imaging. Different ways to maintain
Figure 3. SECM (A) and transmitted light (B) images of the same field of metastatic breast cells. The optical and electrochemical images of the same cell are labeled by the same number. For experimental parameters, see Figure 2.
Figure 4. Four successive images (from A to D) of the same field of MCF-10A cells obtained at different vertical distances between the 5.5-mm-radius carbon tip and the dish surface: (A) 21, (B) 19, (C) 16, and (D) 13 µm. The mediator was 30 µM 1,2-naphthoquinone.
a constant distance between the scanning probe and the imaged object were proposed including constant-current mode,3a,18,19 tip position modulation,20,21 use of shear force-based feedback,22 a combination of SECM and atomic force microscopy,23 and tuning (18) Wipf, D. O.; Bard, A. J.; Tallman, D. E. Anal. Chem. 1993, 65, 1373-1377. (19) Lee, Y.; Ding, Z.; Bard, A. J. Anal. Chem. 2002, 74, 3634-3643. (20) Wipf, D. O.; Bard, A. J Anal. Chem. 1992, 64, 1362-1367. (21) Bruckbauer, A.; Ying, L.; Rothery, A. M.; Zhou, D.; Shevchuk, A. I.; Abell, C.; Korchev, Y. E.; Klenerman, D. J. Am. Chem. Soc. 2002, 124, 88108811. (22) Hengstenberg, A.; Kranz, C.; Schuhmann, W. Chem. Eur. J. 2000, 6, 15471554. (23) Gardner, C. E.; Macpherson, J. V. Anal. Chem. 2002, 74, 576A-584A.
forks.19,24,25 Although all of these approaches are promising, few successful applications to cell imaging have been reported to date. A noticeable exception is constant-current imaging of cell topography5 and mapping of ion channels3a by ion conductance microscopy. However, this technique is different from constantdistance electrochemical imaging, and the latter may be somewhat harder to implement.19 Figure 5 shows an image of a MCF-10A cell field, which was obtained in PBS containing 100 µM TMPD as redox mediator. (24) Karraı¨, K.; Grober, R. D. Ultramicroscopy 1995, 61, 197-205. (25) James, P. I.; Garfiasmesias. L. F.; Moyer, P. J.; Smyrl, W. H. J. Electrochem. Soc. 1998, 145, L64-L66.
Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
4151
Figure 5. SECM image of the MCF-10A cell field obtained with 100 µM TMPD mediator in PBS and a 5-µm-radius gold tip.
TMPD2+ produced at the tip can permeate the mammalian cell membrane and undergo intracellular reduction:8
TMPD2+ + 1e ) TMPD+
(inside the cell)
(3)
The image in Figure 5 is clearer than that obtained with quinone mediators. Although the cells are plated close to each other, individual cells are easy to identify and a characteristic honeycomb pattern can be observed. The reason for the better quality of the image is the higher concentration of redox mediator in the solution (100 mM as compared to 30 mM 1,2-naphthoquinone in Figures 2-4). As discussed earlier,6 the optimal concentration of redox mediator in solution should not exceed the concentration of intracellular redox moieties capable of oxidizing (or reducing) that mediator. The concentration of intracellular redox centers capable of reducing TMPD2+ (a fairly strong oxidizing agent) is much higher than the concentration of redox moieties oxidizing menadiol or naphthoquinol.8 Thus, imaging with TMPD at 100 µM results in a greatly improved signal-to-noise ratio and an enhanced image quality. Another advantage of TMPD over quinone mediators is the lower degree of interference by dissolved oxygen, which is difficult to remove completely under these experimental conditions. Identification of Cells in SECM and Optical Images. To interpret SECM images of cell fields, one has to be able to select a desired region and to identify a specific cell within the field. This can be achieved by combining SECM with optical microscopy. With low-density cell cultures, the correlation of optical and electrochemical images is straightforward, as one can see from Figure 6, which shows the electrochemical (A) and bright field (B) images of two metastatic breast cells (MDA-MB-231). Although for high-density fields this procedure poses certain difficulties, it is possible to identify corresponding individual cells in an electrochemical image. For example, electrochemical and optical images of the same MDA-MB-231 cells are labeled by corresponding numbers in panels A and B of Figure 3. Clearly, the cells shown in Figure 3 are different in their shapes, sizes, and electrochemical activities. In some cases, the cell shape in the SECM image obtained with a relatively large (11-µm-diameter) tip is different from that in Figure 3B. However, almost every cell can be unambiguously identified based on the similarity of patterns in both images. The exceptions are cells 19 and 22, which exhibited no significant redox activity and may have not been alive at the time of imaging. 4152
Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
Combination of SECM with Fluorescence Microscopy. To use fluorescence microscopy in combination with SECM, one has to make sure that the addition of a fluorescent agent does not alter cellular redox activity. When an alkylating reagent such as Vybrant CFDA SE (Molecular Probes, Eugene, OR) was used to label the cells, the resulting rate of menadiol oxidation by labeled cells became too low to measure by SECM. This result suggests that alkylation of one or more molecular targets inhibited the redox reactivity. In contrast, when the cells were labeled by accumulation of fluorescent nanospheres, as described in the Experimental Section, there was no interference with the redox reactivity. Thus, the average rate constant of menadiol oxidation by tagged cells, k ) (1.59 ( 0.10) × 10-3 cm/s was virtually identical to that measured for unlabeled control MDA-MB-231 cells, k ) (1.57 ( 0.13) × 10-3 cm/s (eight measurements were made for each group of cells). The comparison of a bright field micrograph of MDA-MB-231 cells (Figure 7A) with a fluorescence micrograph of the same area (Figure 7B) shows that all cells incubated with fluorescent nanospheres were actually labeled. However, the nanospheres do not fill the entire cell volume, so that the fluorescence image of a labeled cell may appear as just a small bright spot. Imaging of Mixed Fields of Cells. Here we combine SECM with optical and fluorescence microscopies to identify individual cells in the field. In a mixed field of cells, one cell type (e.g., MDAMB-231) can be fluorescently tagged and plated together with unlabeled MCF-10A cells. A redox activity map obtained by SECM can then be compared with a fluorescent map and an optical micrograph captured for the same field of cells to check the fidelity of electrochemical discrimination between the two cell types. The three panels of Figure 8 show images of the same mixed field of MCF-10A and MDA-MB-231 cells: a bright field micrograph (A), a fluorescence micrograph (B), and an SECM image (C). These images are of the same area, thereby making it possible to identify individual cells and similar cell patterns in all three images. The density of MCF-10A cells is high thereby making it difficult to discern the cell boundaries. The field of such cells can be seen in Figure 8A, and it corresponds to a similarly shaped large bright area in Figure 8C. In contrast, MDA-MB-231 cells were plated at a lower density and made fluorescent by preloading them with fluorescent nanospheres. MDA-MB-231 cells can be seen at the periphery on the left and top edges or within the cluster of MCF-10A cells in Figure 8A and C. In agreement with previous observations with quinone mediators,6 metastatic cells exhibit lower redox activity than do nontransformed breast cells and thus appear as darker spots in Figure 8C. Although most MDA-MB-231 cells found in the SECM image can also be detected in the bright field micrograph (Figure 8A), the shapes of both MCF-10A and MDA-MB-231 cells are not sufficiently well-defined to distinguish between the two cell types. In contrast, the comparison of the SECM image (Figure 8C) with Figure 8B, in which only fluorescent MDA-MB-231 cells can be seen, allows unambiguous identification of metastatic cells. It is notable that some gray spots on the SECM image do not have counterpart signals in the fluorescent image; these spots may correspond to blank spaces on the plate (verifiable in Figure 8A) or dying MCF10A cells.
Figure 6. SECM (A) and transmitted light (B) images of two MDA-MB-231 cells. The tip was scanned 12.5 µm above the plastic surface. For other parameters, see Figure 2.
Figure 7. Transmitted light (A) and fluorescent (B) images of the same group of MDA-MB-231 cells showing that the fluorescence of labeled cells conforms with the true positions of these cells on the plate.
Figure 8. Transmitted light (A), fluorescence (B), and SECM (C) images of a mixed field of MDA-MB-231 and MCF-10A cells. The mediator was 40 µM menadione in PBS, and the tip was a 5.5-µm-radius carbon fiber.
Detection of Metastatic Cells in a Field of Normal Breast Cells. Selection of the most effective chemical mediator at its optimal concentration is the key determinant in distinguishing nontransformed cells from metastatic cells. The relevant findings can be summarized as follows:6,8 (1) rates of regeneration of quinone mediators and TMPD by MCF-10A cells were higher than the corresponding rates exhibited by MDA-MB-231 cells. (2) The ratio of effective rate constants, k10A/k231, which indicates how well a mediator at its optimal concentration discriminates between the two cell types, was significantly higher for menadione (k10A/k231 ) 2.4) than for 1,2-naphthoquinone (k10A/k231 ) 1.7). For quinones, these ratios represent maximum values that were achieved with 60 µM. The maximum k10A/k231 ratio obtained for TMPD as
mediator was similar to those of the quinones but was essentially concentration-independent over a wide range (30-300 µM). An image of a mixed field of MCF-10A/MDA-MB-231 cells in Figure 8C is consistent with the above findings. The MCF-10A cells appear brighter than MDA-MB-231 cells due to their higher redox activity with the quinone mediator. By contrast, metastatic cells produced brighter signals with TMPD as mediator (higher SECM feedback current) than nontransformed breast cells. For example, in Figure 9A, a very dense field of MCF-10A cells near the lower right corner appears uniformly gray in the corresponding SECM image (Figure 9C), while several brighter spots in the upper half of the image correspond to MDA-MB-231 cells. This electrochemical pattern was observed to be in register with the Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
4153
Figure 9. Transmitted light (A), fluorescence (B), and SECM (C) images of a mixed field of MDA-MB-231 and MCF-10A cells in 120 µM TMPD.
Table 1. Effective Rate Constants of TMPD Regeneration Measured for Well-Separated and Confluent Cellsa rate constant, cm/s × 10-3 cell type
concn of TMPD, µM
well-separated cells
confluent cells
MCF-10A MCF-10A MDA-MB-231 MDA-MB-231
50 200 50 200
8.6 ( 0.9 (8) 8.2b 5.8 ( 0.7 (8) 6.4 ( 1 (8)
3.8 ( 0.5 (8) 3.5 ( 0.7 (7) 8.1 ( 1 (8) 8.7 ( 0.6 (6)
a The number of experiments with different cells of the same type is indicated in parentheses. b From ref 8.
fluorescent pattern for the same field of cells that identified the true positions of the MDA-MB-231 cells for the same field of cells (Figure 9B). The higher redox activities of MDA-MB-231 cells relative to MCF-10A cells obtained with TMPD seem to contradict previous kinetic measurements made with single cells in which their relative redox activities were reversed. To investigate this discrepancy, current versus distance curves were obtained with a tip approaching an individual cell in a densely packed MCF-10A or MDA-MB-231 field. From Table 1, which presents rate constants obtained with different concentrations of TMPD, it is evident that the redox activities of individual cells change when they are in contact with each other. The rate of regeneration of TMPD by densely packed MCF-10A cells is lower than that exhibited by well-separated MCF-10A cells. In contrast, confluent MDA-MB-231 cells, which do not pack together as closely as MCF10A cells, regenerate TMPD faster than similar cells at low density. Clearly, the SECM images are greatly affected by the choice and concentration of the redox mediator and by the density of cells in the field. Although the origin of the latter effect is not known, one explanation may be that densely packed MCF-10A cells have a lower rate of metabolism than do low-density cells (due to contact inhibition), thus enhancing the observed difference in redox reactivity with MDA-MB-231 cells. This finding suggests that metastatic cells embedded in intact breast tissue may provide a higher signal differential than with monolayer breast cells. 4154 Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
CONCLUSIONS We have used the SECM to image the topography and redox reactivity of dense fields of cultured human breast cells. Compared with imaging of single cells, constant-height SECM imaging of densely packed cells presents new challenges. A thorough alignment of the tip and the sample is required, and the correct choice of the vertical separation distance between the tip and the cells is critical. Furthermore, the redox activity of mammalian cells in a high-density field may be significantly different from that exhibited by well-separated cultured cells of the same type. Consequently, the results of single-cell kinetic measurements do not reflect the relative redox activities of different cell types plated at high density. SECM can be used to identify individual cells in fields of similar confluent cells and heterogeneous fields of cells with reference to images obtained by transmitted light and fluorescence. The ultimate goal of this projectsthe development of the SECM as a tool for discriminating between cancerous and normal cells in situ srequires further search for better mediators that would maximize the differences in redox responses. At present, the combination of electrochemical imaging with fluorescence microscopy is very useful because it provides unambiguous verification of a cell pattern produced by SECM. Use of this combination to establish the optimal conditions for imaging fluorescently labeled tumor cells implanted in normal breast tissue should be attainable soon. ACKNOWLEDGMENT The support of this work by grants from the National Institutes of Health (CA91341), NSF Division of International Programs (INT0003774), and PSC-CUNY is gratefully acknowledged. The software for SECM measurements was generously provided by Prof. D. O. Wipf (Mississippi State University). We thank Dr. Biao Liu for his assistance with early imaging experiments, and Professor Sorin Kihara (Kyoto Institute of Technology) and Dr. Kenji Kano (Kyoto University) for helpful discussions.
Received for review March 28, 2003. Accepted May 22, 2003. AC0343127
