Amplitude Modulation Mode of Scanning Ion Conductance Microscopy

Abstract

Live-cell imaging at the nanoscale resolution is a hot research topic in the field of life sciences for the direct observation of cellular biological activity. Scanning ion conductance microscopy (SICM) is one of the few effective imaging tools for live-cell imaging at the nanoscale resolution. However, there are various problems in existing scanning modes. The hopping and AC modes suffer from low speed, whereas the DC mode is prone to instability because of the DC drift and external electrical interference. In this article, we propose an amplitude modulation (AM) mode of SICM, which employs an AC voltage to enhance the stability and improve the scanning speed. In this AM mode, we introduce a capacitance compensation method to eliminate capacitance effect and use the amplitude of the AC current component to control the tip movement. Experimental results on polydimethylsiloxane samples verify the validity of the AM mode and demonstrate an improved performance of both speed and stability of this new mode.

Keywords

scanning ion conductance microscopy AM mode AC voltage capacitance compensation nanoscale imaging

Introduction

High-resolution imaging of live cells plays a key role in the cellular research of biological life. Traditionally, optical microscopy is extensively used in cellular studies such as bright field, phase contrast, and differential interference contrast illumination. However, owing to the restrictions imposed by Abbe’s limit, the resolution of optical microscopy is limited to approximately 200 nm. Although this fundamental limit has recently been overcome by advanced technology, including stimulated emission depletion (STED),¹ photoactivated localization microscopy (PALM),² or stochastic optical reconstruction microscopy (STORM),³ these techniques require the samples to be fluorescently labeled. One potential method to perform high-resolution imaging of live cells is scanning probe microscopy (SPM), in which a fine probe is used to scan over a surface to acquire 3D topography of a given sample by keeping the probe sample separation constant. Atomic force microscopy (AFM) is one such typical SPM technique that maintains the probe sample separation by monitoring the atomic force between the tip and the sample. AFM has been used to image various delicate biological samples, such as DNA,⁴ virus,⁵ proteins,⁶ and fixed or live cells.⁷ Unfortunately, AFM is not an ideal tool for imaging live cells because it inevitably exerts vertical and lateral forces on cells that cause sample damage while scanning. A more advantageous alternative to AFM for imaging live cells is scanning ion conductance microscopy (SICM). In 1989, Hansma et al.⁸ first introduced SICM that was specifically designed for biological and electrophysiology research. In SICM, the probe sample separation is maintained by monitoring the current through a nanopipette, and a decreased current accompanies the decreased probe sample distance. Unlike AFM, where the tip has direct contact with the sample surface, the probe of SICM is kept 30 to 100 nm above the sample surface, where it applies negligible force on the sample.⁹ Recently, Zhang et al¹⁰ revealed that AFM leads to vertical distortion of ﬁbrils proteins, whereas SICM provides accurate height measurements.

Currently, two kinds of working modes of SICM have been reported, a hopping mode⁹ and its variant,¹¹ which drives the tip up and down, and a continuous mode that maintains a constant tip sample separation such as DC mode,¹² AC mode,^13,14 and phase modulation (PM) mode.¹⁵ Although the hopping mode has been widely adopted in live-cell imaging because of its ability to avoid probe collisions with steep sample structures, its low speed has become the main hurdle to its widespread application. Even with some improvements such as adaptive hopping⁹ or the standing approach,¹⁶ its speed is far less than that of the DC mode. The DC mode usually offers high speed (e.g., Korchev et al.¹² achieved 5 min per frame using DC mode). Nevertheless, the DC mode is prone to instability, owing to the influences of external electrical interference and DC drift. In the AC mode, an AC current is generated through modulating the pipette up and down, which is then used to control the probe sample separation. The AC mode is a very robust approach such that the stable imaging of living cells can last in some cases more than 24 h.¹⁴ However, the AC mode has very slow scan speed, owing to the low modulation frequency of the piezo actuator. Recently, a state-of-the-art mode, the PM mode, was introduced by our group to solve these issues.¹⁵ By modulating the applied bias voltage instead of modulating the probe sample distance, the PM mode offers high modulation frequency and high stability and, as a result, a high scanning speed.

In this article, we present an amplitude modulation (AM) mode to replace PM mode, which uses the in-phase component of the AC current with applied voltage to control tip sample separation. The in-phase component is acquired by the lock-in amplifier instrument. Herein, we suppress the quadrature component of the AC current through capacitance compensation. Therefore, after suitable compensation, the amplitude of the AC current could be directly used for feedback control. The amplitude of the AC current signal can be obtained through a home-built RMS-DC converter, and therefore, the AM mode offers an economic alternative to the PM mode.

Introduction to AM-SICM

The schematic of the AM-SICM system is considerably similar to PM-SICM reported in our previous work¹⁵ and is illustrated in Figure 1 . We applied a sinusoidal voltage instead of a DC voltage between two Ag/AgCl electrodes. The resulting AC current is detected and processed by a commercial patch clamp amplifier (EPC800USB; Heka, Lambrecht/Pfalz, Germany). The AC current, instead of being detected through the lock-in amplifier in the PM mode, is delivered to an RMS-DC convertor circuit (AD536; Analog Device, Norwood, Massachusetts).¹⁷ Similar to the PM mode, the tip sample system can be simplified by the combination of a solution resistance R_sol and total capacitance C_total. During scanning, the capacitance is kept almost constant and remains insensitive to the sample surface; however, the resistance monotonically changes with tip sample separation. Current passes through the solution resistance I_sol adapted in the PM mode for the control input of the feedback loop. I_sol makes a minor contribution to the overall AC current I_ac as most of the current will pass through the capacitance path. Consequently, the current will be maintained at a constant value because of the constant capacitance value, which is the so-called capacitance effect. Moreover, this effect will get worse when the modulated frequency increases. A built-in function of the patch clamp amplifier is to provide a compensation capacitance C_comp to eliminate capacitance effect in the circuit elsewhere, as shown in Figure 1 . Compensation capacitance provides an extra input current I_comp, increasing the I_sol component in the I_ac. When applying an appropriate compensation value, the capacitance effect can be suppressed, resulting in the amplitude of the AC current I_ac becoming sensitive to the tip sample separation, and it is required for controlling tip movement.

Figure 1.

Schematic of scanning ion conductance microscopy in the amplitude modulation mode.

Core Units of AM-SICM

Capacitance Compensation of AM-SICM

As described in PM-SICM, the current passing through the capacitance I_cap is considerably larger than I_sol, making the total AC current I_ac insensitive to the tip sample separation. Here, the capacitance compensation function is introduced to solve this issue. This function adds an extra current path I_comp, together with I_ac, to feed I_sol and I_cap. The current I_ac now becomes

I_{a c} = I_{c a p} + I_{s o l} - I_{c o m p} .

The emerging I_comp increases the component part that I_sol takes in I_ac, consequently making I_ac more sensitive to tip sample separation. Moreover, the compensation ratio can be regulated by the capacitance C_comp value. When under the same applied voltage U_ac and tip sample separation, I_sol and I_cap will be constant. Based on the value of I_comp, which changes along with the manual adjustable C_comp, the current analysis will be divided into three situations: undercompensation, full compensation, and overcompensation, as shown in Figure 2 . In the undercompensation scenario ( Fig. 2a ), I_comp is lower than I_cap. At this instance, raising C_comp will result in I_comp increasing, and I_ac will decrease until full compensation has been achieved ( Fig. 2b ). For full compensation to occur, I_comp must be equal to I_cap, whereas I_ac is minimal and equals I_sol. In this situation, the I_sol component dominates I_ac, and therefore, the sensitivity of I_ac with regard to tip sample separation is at its highest. If C_comp further increases, I_comp will become larger than I_cap, resulting in the overcompensation scenario ( Fig. 2c ). Here, the sensitivity to tip sample separation will decrease with the increase of C_comp.

Figure 2.

Different situations of the current analysis in amplitude modulation–scanning ion conductance microscopy: (a) undercompensation, (b) full compensation, and (c) overcompensation.

Only in the full-compensation situation is the sensitivity of I_ac to tip sample separation optimal; otherwise, sensitivity reduces when C_comp moves beyond the optimal value. These results are verified by the approach curves showing I_ac versus tip sample separation in Figure 3 . As shown in Figure 3 , the optimal C_comp is 5.65 pF, where I_ac versus tip sample separation (cyan line) is close to I_sol versus tip sample separation. For undercompensation (green line and red line) or overcompensation (magenta line and blue line) scenarios, the sensitivity of I_ac is lower than that of I_sol. Moreover, when the compensation value is closer to the optimal value (5.65 pF), the sensitivity will be higher. For example, in the undercompensation scenario, the sensitivity of I_ac when C_comp is 5.4 pF (red line) is higher than when C_comp is 5.1 pF (green line); in the overcompensation scenario, the sensitivity of I_ac when C_comp is 6.2 pF (magenta line) is higher than when C_comp is 6.5 pF (blue line).

Figure 3.

Approach curves for the relationship of AC current I_ac amplitude versus tip sample separation with different capacitance compensation values. When the C_comp is in the undercompensation situation (green line and red line) and overcompensation situation (magenta line and blue line), the sensitivity of I_ac versus tip sample separation is less than the one of I_sol. Only in the full-compensation situation (red line) is the sensitivity of I_ac versus the tip sample separation close to the one of I_sol (black line). The modulated frequency is 30 kHz.

Units Experiment Verification and Discussion

System Setup

The system of SICM is composed of a scan head, feedback control module, host computer, and software system. The actual system setup is presented in Figure 4 . The scan head is in the inner core part of the system and is in charge of XYZ axis movement while also applying a sinusoidal voltage and obtaining the current. The feedback control module is controlled by a computer (control PC) in a real-time (RT) Linux operating system, which assists in sampling the amplified current and calculating the feedback output. A host computer (host PC) provides a user interface, parameters or command modifications to the system, and the display or storage of images.

Figure 4.

Photograph of home-built amplitude modulation–scanning ion conductance microscopy.

The scan head is mounted on an inverted fluorescence microscope (Ti-s; Nikon, Tokyo, Japan). With the assistance of the optical microscope, we control the coarse positioning of the tip sample separation through a motorized stage (9062-XYZ-PPP; New Focus Corporation, Irvine, CA). The nanoscale movement is carried out by two piezo structures. One is a Z-axis piezo actuator (P-753.31C; Physik Instrumente, Karlsruhe, Germany) with a travel range of 38 µm. The other is an XY-axis stage (P517.3CD; Physik Instrumente) with a 100 × 100–µm travel range. The Z-axis piezo actuator is driven by a 280-W peak power, high-voltage amplifier (E504; Physik Instrumente), while the XY nanostage is controlled by a digital piezo controller (E710; Physik Instrumente). Two Ag/Agcl electrodes are immersed in a solution of fine nanopipette and a Petri dish, and then, a sinusoidal voltage generated by an external function generator (AFG3022B; Tektronix, Beaverton, OR) is applied between those two electrodes, producing a weak AC current. The weak current is then amplified via a commercial patch clamp current amplifier (EPC800USB; HEKA Instruments, Lambrecht/Pfalz, Germany) while the gain is set to 0.1 mV/pA and the cutoff frequency of the low-pass filter is set to 100 kHz. The amplified current is then sent to an RMS-DC convertor circuit to obtain the RMS amplitude and is finally sent to a feedback input for controlling tip sample separation. In addition, a capacitor is added in series with the input of an RMS-DC converter to reject any DC components of the amplified current.

The control PC is equipped with a DAQ card (PCI-6251; National Instruments, Austin, TX) and is in charge of the feedback mechanism. The control PC uses the sampling current as feedback input and controls the output voltage of the Z-axis piezo actuator. As scanning along the Z-axis movement will be in real time, this task is loaded as a kernel module to make the control predictable and fast. Meanwhile, the XY-axis scanning does not require real-time quality and can be set to a lower priority. The XY-axis stage is actuated using a digital piezo controller, controlled by the control PC equipped with a GPIB card (PCI-GPIB; National Instruments, Austin, TX).

The software in the control PC is written in C language, and the program in the host computer is in the graphic language (Labview; National Instruments, Austin, TX).

Experiments on Polydimethylsiloxane Samples

To investigate the performance of the AM mode, we initially used this mode to image the polydimethylsiloxane (PDMS) samples. A PDMS microgrid is prepared using a soft lithographic approach¹⁸ and based on the master copy of Pt Coated Calibration Gratings (P/N 498-000-026; Digital Instruments, NY), which has a 10-µm pitch and 200-nm step depth. Figure 5a , b displays the topography and error images of the PDMS microgrid, respectively. In this experiment, the modulated frequency is set to 30 kHz and the scan rate to 0.75 Hz. The time of a complete scan is approximately 7 min. This is similar to the result in PM mode,¹⁵ which is much faster than a traditional AC mode scanning of the similar sample.¹⁰ The line profile and 3D topography of the microgrid, as shown in Figure 5c , d , also infers that AM-SICM provides good quality comparable to PM-SICM.¹⁵ Moreover, continuous imaging of the PDMS microgrid can last several hours, even with continuous modification of the DC bias voltage offset during scanning. These results indicate that we can greatly improve the efficiency and robustness of SICM with this new AM mode.

Figure 5.

Polydimethylsiloxane microgrid images with the amplitude modulation mode: (a) height image, (b) error image, (c) line profile from red line in a, and (d) 3-dimensional image. (Modulated frequency: 30 kHz; scan rate: 0.75 Hz; scan time: 6 min 48 s; and capacitance compensation C_comp: 6.2 pF; bar: 10 µm.)

In summary, we developed an AM mode of SICM. This mode not only contains the advantages of the PM mode, including high stability and high scan speed, but also offers a more economical replacement to the PM mode. Experimental results demonstrate the high scan quality and fast scan speed of the AM mode. This new mode holds great potential for live-cell imaging applications with SICM in the future.

Footnotes

Acknowledgements

The authors express their appreciation to Peng Yu and Zhu Liu for their generous technical assistance.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research work was partially supported by the National Natural Science Foundation of China (Project Nos. 61433017 and 61327014), the Instrument Developing Project of the Chinese Academy of Sciences (Project No. YZ201245), and the CAS FEA International Partnership Program for Creative Research Teams.

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