Use of Combined Scanning Electrochemical and Fluorescence

Sep 18, 2013 - Release of ROS from prostate cancer (PC3) cells was studied using scanning electrochemical microscopy (SECM) and fluorescence microscop...
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Use of Combined Scanning Electrochemical and Fluorescence Microscopy for Detection of Reactive Oxygen Species in Prostate Cancer Cells S. Ehsan Salamifar and Rebecca Y. Lai* Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, United States S Supporting Information *

ABSTRACT: Release of ROS from prostate cancer (PC3) cells was studied using scanning electrochemical microscopy (SECM) and fluorescence microscopy. One-directional lateral scan SECM was used as a rapid and reproducible tool for simultaneous mapping of cell topography and reactive oxygen species (ROS) release. Fluorescence microscopy was used in tandem to monitor the tip position, in addition to providing information on intracellular ROS content via the use of ROSreactive fluorescent dyes. A unique tip current (iT) vs lateral distance profile was observed when the tip potential (ET) was set at −0.65 V. This profile reflects the combined effects of topographical change and ROS release at the PC3 cell surfaces. Differentiation between topographical-related and ROS-induced current change was achieved by comparing the scans collected at −0.65 and −0.85 V. The effects of other parameters such as tip to cell distance, solvent oxygen content, and scan direction on the profile of the scan were systematically evaluated. Cells treated with tert-butyl hydroperoxide, a known ROS stimulus, were also evaluated using the lateral scanning approach. Overall, the SECM results correlate well with the fluorescence results. The extracellular ROS level detected at the SECM tip was found to be similar to the intracellular ROS level monitored using fluorescence microscopy. While the concentration of each contributing ROS species has not been determined and is thus part of the future study, here we have successfully demonstrated the use of a simple two-potential lateral scan approach for analysis of ROS released by living cells under real physiological conditions.

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technique, has found widespread applications in biomedical research in recent years.19−31 It is well-suited for studying ROS release in living cells. In this work, we monitored the intercellular and extracellular ROS simultaneously using a hybrid SECM−fluorescence microscopy technique (SECMFluor) (Scheme 1). The PC3 cell line was chosen as the model system due to its high ROS content when compared to other prostate cancer cell lines.9 The SECM-Fluor setup is shown in Scheme 1 and Figure S1, Supporting Information. Prior to detecting ROS release and in order to maintain the distance between the tip and cell surface, the topographical image of the cell surface was first recorded by scanning in the Y direction using Ru(NH3)63+ as the solution mediator. In brief, 2 mM of Ru(NH3)63+ was first added to the cell media, and the SECM tip potential (ET) was set at −0.3 V to reduce Ru(NH3)63+. The approach curve was recorded by moving the tip in a vertical direction toward the bottom of the

s a result of oxygen (O2) metabolism, small amounts of reactive oxygen species (ROS), such as hydroxyl radicals (OH·), superoxide anions (O2·−), singlet oxygen (1O2), hydrogen peroxide (H2O2), etc., are constantly produced in aerobic cells.1,2 ROS can act as secondary messengers controlling various signaling cascades in living organisms.3 Cellular antioxidants detoxify the excess amount of generated ROS to keep their concentrations balanced inside of the cell.4 However, disruption of this balance creates a condition known as oxidative stress, which could lead to cell injury and pathological conditions, such as cancer.2,5−8 For example, recent studies have shown that a high amount of ROS contributes to the etiology and pathogenesis of the prostate cancer.9−12 While fluorescence techniques are the most widely used techniques to study cellular ROS,13−15 there are disadvantages that limits their applicability in real-time detection of ROS in living cells. These include rapid photobleaching, phototoxicity, and out-of-focus contributions blurring the in-focus image.14−18 Analytical techniques capable of monitoring transient release of ROS from the cell membrane under physiological conditions are thus sought after. Scanning electrochemical microscopy (SECM), a noninvasive scanning © XXXX American Chemical Society

Received: July 29, 2013 Accepted: September 18, 2013

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cell distance due to the increase of cell height will result in a decrease in the corresponding iT. The effect of tip−cell distance on iT is shown in Figure S2, Supporting Information. The amplitude of the two successive dips in the lateral scan was comparable, suggesting height and morphological similarity between the two cells along path B. The scan across path C showed a similar trend; however, the amplitude of the dips was significantly different. A larger decrease in iT was observed above the first cell, whereas a smaller decrease was evident when the tip was positioned above the second cell. This implies higher height for the first cell when compared to the second cell along path C. To demonstrate that this technique is reproducible, we collected extra scans along the same paths (gray curves in Figure 1B,C). The resultant scans were very similar to the ones obtained previously, verifying the reproducibility of this method for topographic mapping at cellular dimensions. Furthermore, the maximum cell height across paths B and C was calculated from the maximum change in iT during each scan. A maximum of 11% and 16% change in iT along paths B and C, respectively, was determined by comparing the initial iT value to its minimum value recorded on the top of the cells. Using the negative feedback theory for a 10 μm tip located at 8 μm from an insulating substrate, the calculated maximum topographical change in cell height was found to be 2.2 and 3.1 μm along paths B and C, respectively. It is worth noting that, prior to ROS mapping, we first obtained a topographical image of the scanned area to prevent potential tip−cell contact during the subsequent imaging steps. Also, to ensure that the distance between the tip and the cells was within the same range in every experiment, the maximum accepted change in iT was limited to −20% of the initial iT recorded at the beginning of each scan. The main focus of this study is to determine the flux of ROS species released from the cells using SECM.18,28,29,32,33 Prior to the SECM experiment, 1× PBS was added to the Petri dish after removal of the F-12K medium. ET was set at a potential capable of reducing both O2 and H2O2; the two main species originated from dismutation of ROS inside the cell. In this solvent, the reduction of both species occurs at ET ≤ −0.65 V, as verified in a separate experiment (Figure S3, Supporting Information). Hoechst 33342 was used to stain the cell nucleus, and 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) was used to detect ROS inside live cells. Details of the staining procedure and chosen wavelengths are described in the Supporting Information. Figure 2A shows a phase contrast image superimposed on the fluorescence image obtained in presence of Hoechst 33342. The fluorescence image resulted from carboxy-H2DCFDA staining superimposed on the Hoechst 33342 fluorescence image is shown in Figure 2B. The scans recorded above the cell, along the marked path, under different experimental conditions are shown in Figure 2C−E. As shown in Figure 2C, the scan collected in F-12K medium containing 1 mM Ru(NH3)63+ with ET set at −0.3 V showed a decrease in iT above the cell due to topographical change as previously described. However, when the same scan was recorded in 1× PBS with ET set at −0.65 V, the profile of the scan was significantly different (Figure 2D). The recorded iT decreased, reaching a minimum value above the cell, and increased sharply right above the center of the cell, reaching a maximum, followed by a decrease back to the initial baseline value. This unique profile, however, is not unexpected and can be explained. When the tip is directly above the cell, the recorded

Scheme 1. Schematic Representation of the SECMFluorescence Microscopy Setup for Detection of Both Extracellular and Intracellular ROS

Petri dish. Using the negative feedback current obtained in this way, the distance between tip and Petri dish was set at ∼8 μm. The tip was stopped and scanned laterally in the Y direction. Figure 1A shows the phase contrast image of the PC3 cells

Figure 1. Phase contrast image of the PC3 cells and 10 μm Pt SECM tip position (A). The resultant scans along marked paths B (B) and C (C). The red arrow shows the scan direction. The scans were collected in F-12K medium containing 2 mM Ru(NH3)63+ (pH = 7.4) with ET set at −0.3 V.

relative to the SECM tip. The two chosen scan paths used to obtain the tip current (iT) vs lateral distance plots are marked by arrows B and C (Figure 1A, right side). The chosen paths were separated by 10 μM, and the tip was moved from one path to another by moving 10 μm in the X direction. Figure 1B,C shows the resultant tip currents (iT) during two subsequent scans along paths B and C, respectively, in a cell medium containing 2 mM Ru(NH3)63+ with ET set at −0.3 V. In both cases, iT decreased significantly from its initial value when the tip moved above the cells, resulting in two dips in the scans. The concept behind this interpretation is described in detail elsewhere.21 In brief, given that Ru(NH3)63+/2+ is hydrophilic and cannot move across the hydrophobic cell membrane, the cell surface acts just like an inert substrate, which blocks the diffusion of Ru(NH3)63+/2+ to the tip at very short tip−cell distances. Thus, any decrease in iT during the scan is attributed to the decrease in the tip−cell distance, which corresponds to the increase in cell height. Similarly, any decrease in the tip− B

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obtained from the scan at ET ≤ −0.65 V should be highly dependent on the ROS concentration and thus can be used in the quantification of ROS released from live cells. These experiments are currently underway, and the results will be disseminated in a future publication. The effect of tip−cell distance on the profile of the lateral scan was evaluated in a separate experiment. The tip−cell distance was adjusted by moving the tip up by 1.0 μm in the Z direction during each scan. As shown in Figure S4, Supporting Information, a systematic decrease in the current amplitude was observed with increasing tip−cell distance during each scan. However, the same trend was evident even at the largest tip− cell distance where iT is normally independent of both substrate topography and activity. To further confirm this theory, we recorded a separate scan with ET set at −0.85 V (Figure 2E). Due to the increase in the background current, the total iT was two times larger even at the beginning of the scan. However, no increase in iT was observed throughout the scan. This is because, with ET set at −0.85 V, the total iT is less affected by the ROS current, owing to the large background current. This trend is reasonably reproducible, as verified by the gray line in Figure 2E. The effect of scan direction on the profile of the scan was also investigated (Figure S5, Supporting Information). Reversing the scan direction reversed the observed trend in iT when ET was set at −0.65 V; however, no major change in the scan profile or iT was evident when ET was set at −0.85 V. This further supports the above interpretation of the iT change during the scan. In this study, the use of two different ET is critical; it allows clear differentiation between iT contributed by the release of ROS and that attributed to the differences in cell topography and cellular respiration. While most experiments were conducted under ambient physiological conditions, we were interested in understanding the effect of solution O2 content on the experimental results. Here, we investigated the effect of solution O2 content by scanning the tip in the X−Y plane (Figure S6, Supporting Information). In a solution purged with nitrogen, the effect of ROS release from the cell was more pronounced due to the decrease in the background current. Nonetheless, a distinguishable decrease in iT prior to a sharp increase was evident. When ET was set to −0.85 V, a decrease in iT over the entire cell surface was observed (Figure S6D, Supporting Information). This effect is similar to that observed under ambient condition (Figure 2E). Overall, there is a strong correlation between the SECM and fluorescence results. It is worth noting that, when the tip was scanned across two neighboring cells, a large increase in iT was only observed with the cell showing strong green fluorescence (i.e., high ROS content). The second cell did not display substantial fluorescence; thus, only a slight increase in iT over the cell surface was observed. The consistency between the 1-D scan data in the Y-axis and the 2-D scan data in the X−Y plane highlights the merits of using lateral scan for this study. This 1-D method is not only fast and effective, it also minimizes the chance of tip fouling and tip crashing into the cell. In contrast, 2-D scanning is more timeconsuming; the use of repetitive tip rastering could also prevent the cells from returning to their equilibrium state, which could affect the accuracy of subsequent scans due to differences in the diffusion layer. To further demonstrate this method’s applicability in ROS detection, we incubated the cells in tert-butyl hydroperoxide (TBHP), a known ROS inducer (Figures 3 and S7, Supporting Information).35 As shown in Figure 3B, stronger fluorescence

Figure 2. Phase contrast image superimposed on the fluorescence image resulted from Hoechst 33342 staining (blue fluorescence) (A). Superimposed fluorescence images resulted from carboxy-H2DCFDA (green fluorescence) and Hoechst 33342 staining (B). Lateral scans collected along the marked path (red arrow) shown in panel A in F12K medium containing 1 mM Ru(NH3)63+ with ET set at −0.3 V (C) and in 1× PBS with ET at −0.65 V (D) and −0.85 V (E).

iT is a summation of the background current that originates from the reduction of dissolved electroactive species such as O2 and the current from the reduction of ROS species released from the cells. The magnitude of the background current is dependent on the tip−cell distance as well as the respiratory function of the cell which is known to deplete nearby O2. Thus, the decrease in iT in the earlier portion of the scan is presumably due to the decrease in the tip−cell distance, which reflects the increase in topographical height (i.e., tip was on top of the cell). At shorter tip−cell distances, the background current decreases due to limited diffusion of dissolved O2. In addition, respiratory function of the cell as well as the reduction of O2 at the SECM tip further depletes O2 within the tip−cell gap, which sets up a concentration gradient of O2 between the intracellular medium and the electrolyte solution close to the cell surface.34 This concentration gradient could act as a force in driving ROS from inside the cell toward the SECM tip. In a system where no extra ROS is released, the compounded effect generally leads to a sharp decrease in the observed iT. However, in this case, as the tip−cell distances decreases, higher flux of ROS could reach the tip before diffusing into the bulk solution. Thus, at short tip−cell distances, as the background current decreases, the ROS current increases. At the highest point of the cell (i.e., shortest tip−cell distance), the background current and ROS current should reach their corresponding minimum and maximum. On the basis of this theory, the sharp increase in total iT observed in the latter portion of the scan suggests that the decrease in background current is outcompeted by the increase in ROS current. It also alludes to the diffusion of ROS from inside the cell, presumably due to O2 depletion within the tip−cell gap, which could occur very quickly. Reproducibility of the observed trend was confirmed by repeating the same scan after allowing the cells to reach equilibrium for about ∼1 min (gray line in Figure 2D). This result agrees well with the fluorescence data; the scanned cell displayed strong green fluorescence, indicating high ROS content (Figure 2B). More importantly, the peak current C

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treatment at ET of −0.65 and −0.85 V. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +1 402 472 9402. Tel: +1 402 472 5340. E-mail: rlai2@ unl.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Army Research Office (W911NF-09-2-0039) and National Science Foundation (CHE-0955439). The authors would like to thank Prof. David B. Berkowitz and David Nelson for the PC3 cells and Anita J. Zaitouna for the helpful discussions.

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Figure 3. Phase contrast image of the cells superimposed on a fluorescence image resulted from Hoechst 33342 staining after a 90min incubation in 100 μM TBHP (A). Superimposed fluorescence images resulted from carboxy-H2DCFDA and Hoechst 33342 staining (B). Also shown are lateral scans collected along the marked path (red arrow) in panel A in 1× PBS with ET set at −0.65 V (C) and −0.85 V (D).

was evident from the cells post-TBHP treatment. We observed two peaks in the scan when ET was set at −0.65 V, suggesting substantial release of ROS from both cells. However, unlike previous experiments, no decrease in iT was recorded prior to the sharp increase in iT above the cells (Figure 3C). This could be attributed to the enhanced release of ROS from the cells after the TBHP treatment; the increase in current generated by ROS reduction outcompetes the decrease in background current. The change in membrane properties and cell topography could, in part, contribute to this drastic change in the scan profile. However, when ET was set at −0.85 V, two dips were observed in the scan, reflecting the topographical image of the cells. The fluorescence intensity agrees well with the recorded iT above each corresponding cell. For the cell that exhibited lower fluorescence intensity (Y Distance ∼ 40 μm), the recorded iT minimum was lower than that observed with the cell that showed higher fluorescence (Y Distance ∼ 65 μm) (Figure 3D). This effect was further verified by the 2D images obtained from scanning the tip in the X−Y plane (Figure S7, Supporting Information). In conclusion, our results highlight the advantages of using dual-potential SECM lateral scan and fluorescence microscopy to analyze both extracellular and intracellular ROS content in live cells. Quantitative studies including the use of approach curves and simulations, however, are necessary, and both aspects are currently under investigation in the lab. The use of nanoelectrodes to minimize the effect of O2 depletion, as well as the quantification of intercellular ROS concentration, will also be part of the future studies.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental conditions; SECM-Fluor setup; effect of tip−cell distance during lateral scan in topographical imaging; detection of O2 and H2O2 at Pt tip; effect of tip−cell distance during lateral scan in ROS detection; effect of scan direction at different ET during lateral scan in ROS detection; 2D scan of cells at ET of −0.65 and −0.85 V; 2D scan of cells after TBHP D

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