Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells

Summary

Here we present a protocol for the Hopping Probe Ion Conductance Microscopy (HPICM), a non-contact scanning probe technique that allows nanoscale imaging of stereocilia bundles in live auditory hair cells.

Abstract

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.

Introduction

Despite the fact that stereocilia bundles in the auditory hair cells are big enough to be visualized by optical microscopy and deflected in live cells in a patch clamp experiment, the essential structural components of the transduction machinery such as tip links could be imaged only with the electron microscopy in dead cells. In the mammalian auditory hair cells, the transduction machinery is located at the lower ends of the tip links, i.e., at the tips of the shorter row stereocilia¹ and regulated locally through the signaling at the tips of stereocilia^2,3. Yet, label-free imaging of the surface structures at this location in live hair cells is not possible due to the small sizes of stereocilia.

The mammalian cochlea has two types of auditory sensory cells: inner and outer hair cells. In the inner hair cells, stereocilia are longer and thicker compared to those in the outer hair cells⁴. The first and second row of stereocilia have a diameter of 300-500 nm in mouse or rat inner hair cells. Due to the diffraction of light, the maximum resolution achievable with a label-free optical microscopy is approximately 200 nm. Hence, visualization of individual stereocilia within first and second rows of the inner hair cell bundle is relatively easy with optical microscopy. In contrast, shorter row stereocilia in the inner hair cells and all stereocilia of the outer hair cells have diameters around 100-200 nm and cannot be visualized with optical microscopy⁵. Despite recent progress in super-resolution imaging, this fundamental limitation persists in any optical label-free imaging. All current commercially available super-resolution techniques do require some sort of fluorescent molecules⁶, which limits their applications. In addition to limitations due to the need for specific fluorescently tagged molecules, exposure to intense light irradiation has been shown to induce cellular damage and might influence cellular processes, which is a great disadvantage when studying live cells⁷.

Our current knowledge of the ultrastructural details of hair cell stereocilia bundles has been obtained mostly with various electron microscopy (EM) techniques, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), freeze-fracture EM, and recently with 3D techniques such as serial sectioning with focused ion beam or cryo-EM tomography^{8,9,10,11,12,13,14,15,16}. Unfortunately, all these EM techniques require chemical or cryofixation of the sample. Depending on the time scale of the phenomena, this requirement makes a study of the dynamic processes at the tips of stereocilia either impossible or very labor intensive.

Limited efforts have been made to image hair bundles of live hair cells with atomic force microscopy (AFM)^17,18. Since AFM operates in physiological solutions, it could, in theory, visualize dynamic changes in the stereocilia bundles of live hair cells over time. The problem lies in the principles of high-resolution AFM, which implies certain physical contact between the AFM probe and the sample, even in the least damaging "tapping" mode¹⁹. When the AFM probe encounters a stereocilium, it usually crashes against it, damaging the structure of the hair bundle. As a result, this technique is not suitable for visualizing live, or even fixed, hair cells bundles^17,18. The problem may be partially alleviated by using a large ball-shaped AFM probe that imposes only hydrodynamic forces to the surface of the sample²⁰. However, even though such a probe is ideally suited to test mechanical properties of the sample²¹, it provides only a sub-micrometer resolution when imaging the organ of Corti²² and still applies to the sample a force that may be substantial for the highly sensitive stereocilia bundles.

Scanning ion conductance microscopy (SICM) is a version of scanning probe microscopy that uses a glass pipette probe filled with a conductive solution²³. SICM detects the surface when the pipette approaches the cell and the electric current through the pipette decreases. Since this is happening well before touching the cell, the SICM is ideally suited for non-contact imaging of live cells in physiological solution²⁴. The best resolution of SICM is on the order of single nanometers, which allows resolving individual protein complexes at the plasma membrane of a living cell²⁵. However, similar to other scanning probe techniques, SICM is able to image only relatively flat surfaces. We overcame this limitation by inventing hopping probe ion conductance microscope (HPICM)²⁶, in which the nanopipette approaches the sample at each imaging point (Figure 1A). Using HPICM, we were able to image stereocilia bundles in live auditory hair cells with nanoscale resolution²⁷.

Another fundamental advantage of this technique is that HPICM is not only an imaging tool. In contrast to other scanning probe techniques, the HPICM/SICM probe is an electrode widely used in cell physiology for electrical recordings and local delivery of various stimuli. Ion channel activity does not usually interfere with HPICM imaging, because the total current through the HPICM probe is several orders of magnitude larger than the extracellular current generated by the largest ion channels²⁵. However, HPICM allows precise positioning of the nanopipette over a structure of interest and subsequent single-channel patch-clamp recording from this structure²⁸. This is how we obtained the first preliminary recordings of single channel activity at the tips of the outer hair cell stereocilia²⁹. It is worth mentioning that even a large current through the nanopipette cannot produce significant changes of the potential across the plasma membrane due to enormous electrical shunt of the extracellular medium. However, individual ion channels can be activated mechanically by flow of liquid through the nanopipette³⁰ or chemically by local application of an agonist³¹.

In HPICM, the image is generated when a nanopipette sequentially approaches the sample at one point, retracts, and then moves in lateral direction to repeat the approach (Figure 1A). A patch clamp amplifier constantly applies voltage to an AgCl wire in the pipette (Figure 1B) to generate a current of ~1 nA in the bath solution. The value of this current when the pipette is away from the surface of the cell is determined as a reference current (I_ref, Figure 1C). Then, the pipette moves in the Z axis to approach the sample until the current is reduced by an amount predefined by the user (setpoint), usually 0.2%-1% of the I_ref (Figure 1C, top trace). The system then saves Z value at this moment as the height of the sample, together with X and Y coordinates of this imaging point. Then, the pipette is retracted away from the surface (Figure 1C, bottom trace) at a speed defined by the user, usually 700-900 nm/ms. After retraction, the pipette (or, in our case, the sample – see Figure 1B) is moved laterally to the next imaging point, a new reference current value is obtained, and the pipette once again approaches the sample, repeating the process. The X-Y movement of the pipette is preferred in an upright microscope setup that is typically used for recordings of the hair cell mechanotransduction currents. In this setting, the HPICM probe approaches the hair cell bundles not from the top but at an angle³². However, the best resolution of HPICM imaging is achieved in an inverted microscope setup (Figure 1A,B), where the movement of the sample in X-Y directions is de-coupled from Z-movement of the nanopipette, thereby eliminating potential mechanical artefacts.

Using HPICM, we obtained topographic images of mouse and rat inner and outer hair cell stereocilia bundles, and even visualized the links between the stereocilia that are about 5 nm in diameter^26,27. The success of hair cell bundle imaging with this technique relies on several factors. First, the noise (variance) of the nanopipette current should be as small as possible to allow the lowest possible setpoint for HPICM imaging. A low setpoint allows HPICM probe to “sense” stereocilia surface at a larger distance and at any angle to the probe approach and, surprisingly, improves X-Y resolution of HPICM imaging (see Discussion). Second, the vibrations and drifts in the system should be decreased to less than 10 nm, since they contribute directly to the imaging artefacts. Finally, even though the HPICM probe and the specimen stage are moved in Z and X-Y axes by the calibrated feedback-controlled piezo actuators that have an accuracy of a single nanometer or better, the diameter of the nanopipette tip determines the spread of the current (sensing volume) and hence the resolution (Figure 1A). Therefore, before imaging live hair cells, it is vital to pull the adequate pipettes, reach the desired resolution with calibration samples, and achieve low noise in the recording system.

For at least a couple of decades, the SICM technique has not been commercially available and it has been developing by only few labs in the world with the leading lab of Prof. Korchev in the Imperial College (UK). Recently, several SICM systems became commercially available (see Table of Materials), all of which are based on the original HPICM principles. However, imaging stereocilia bundles in the hair cells requires several custom modifications that are technically challenging (or even impossible) in the closed “ready-to-go” systems. Therefore, some component integration is needed. Since HPICM setup represents a patch clamp rig with more stringent vibration and drift requirements and a piezo-driven movement of the HPICM probe and the sample (Figure 1D), this integration is relatively easy for any researcher, who is proficient in patch clamping. However, a scientist without proper background would definitely need some training in electrophysiology first. Despite remaining challenges such as increasing the speed of imaging (see Discussion), we have been able to image stereocilia bundles in live hair cells with nanoscale resolution without damaging them. 

This paper presents a detailed protocol to perform successful HPICM imaging of the live auditory hair cell bundles in young postnatal rat or mouse cochlear explants using our custom system. The integrated components are listed in the Table of Materials. The paper also describes common problems that can be encountered and how to troubleshoot them.

Protocol

The study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky (protocol 00903M2005). 1. Manufacturing and testing the nanopipettes Create a program in the micropipette puller to obtain pipettes with a resistance between 200 and 400 MΩ, wh…

Representative Results

The protocol presented in this paper can be used to visualize any live cells with complex topography. Following these steps, we routinely obtain images of live rat auditory hair cell bundles (Figure 6B,D). In spite of having lower X-Y resolution when compared to SEM images, our HPICM images can successfully resolve the different rows of stereocilia, the shape of the stereocilia tips, and even the small links (~5 nm in diameter) connecting adjacent stereocilia (<strong class=…

Discussion

To obtain successful HPICM images, users need to establish a low noise and low vibration system and manufacture appropriate pipettes. We strongly recommend the use of AFM calibration standards to test the stability of the system before attempting to perform any live cell imaging. Once the resolution of the system is tested, users can consider imaging fixed organ of Corti samples to get familiar with the imaging settings before attempting any live cell imaging.

The optimal setpoint for imaging …

Materials

Analog oscilloscope	B&K Precision	2160C	Analog oscilloscope for real-time monitoring of nanopipette current and Z-axis approach
AFM calibration standards	TED PELLA Inc	HS-100MG; HS-20MG	These 100 and 20 nm calibration standards are used to test the performance of HPICM system
Benchtop vibration Isolator	AMETEK/TMC	Everstill K-400	Active vibration isolation
Borosilicate glass capillaries	World Precision Instruments (WPI)	1B100F-4	Borosilicate glass capillaries for the nanopipettes
D-(+)-Glucose	Sigma-Aldrich	G8270	To be added to the bath solution to adjust osmolarity
Digitizer	National Instruments Corporation	PCI-6221	Multi-channel input/output digitizer
Fast analog Proportional-Integral-Derivative (PID) control for Z movement	Standford Research Systems	SIM900, SIM960, SIM980	Instrumentation modules integrated in an external PID controller for Z movement. It requires a fast response that is usually not implemented in commercial piezo amplifiers.
Faraday cage	AMETEK/TMC	Type II	Required to shield electromagnetic interference
Glass bottom dish	World Precision Instruments (WPI)	FD5040-100	Used as the dish for the chamber for the tissue
Hanks' Balanced Salt Solution (HBSS)	Gibco, Thermo Fisher Scientific	14025092	Extracellular (bath) solution
Instrumentation amplifier	Brownlee Precision	Model 440	Instrumentation amplifier provides required offsets, filtering, and secondary magnification or attenuation
Laser-based micropipette puller	Sutter Instrument	P-2000/G	Micropipette puller to fabricate the nanopipettes. Laser is needed for sharp quartz pipettes.
Lebovitz's L-15, without phenol red	Gibco, Thermo Fisher Scientific	21083027	Extracellular (bath) solution
Micromanipulator	Scientifica	PatchStar	Used for "course" positioning of the Z piezo actuator
Microscope	Nikon	Eclipse TS100	Inverted optical microscope
Patch amplifier	Molecular Devices	Axopatch 200B	The patch clamp amplifier measures the current through the nanopipette
Piezo amplifier (XY axes)	Physik Instrumente (PI)	E-500.00, E-505.00, E-509.C2A	Amplification and PID control for XY piezo translation stage
Piezo amplifier (Z axis)	Piezosystem jena	ENT 400 & 800	Custom amplifier consisting of ENT 400 power supply and two ENT 800 amplifiers in parallel to achieve max current of 1.6 nA
Plastic Coverslips	TED PELLA Inc	26028	Used in the fabrication of the chambers for the tissue
SICM controller & software*	Ionscope, UK (ionscope.com)	N/A	Custom controller based on SBC6711 digital signal processing board from Innovative Integration Ltd
Silicone elastomer (Sylgard)	World Precision Instruments (WPI)	SYLG184	Used to attach the flexible glass fibers to the chamber for the tissue
Silicon glue	The Dow Chemical Company	734	Used to glue the different parts of the chamber for the tissue
Tungsten rod	A-M Systems	717500	Used for holding the dental floss strands in the chamber for the tissue
XY piezo nanopositioner	Physik Instrumente (PI)	P-733.2DD	XY translation stage with capacitive sensors
Z piezo nanopositioner	Piezosystem jena	RA 12/24 SG	Ring piezoactuator with a strain gage sensor
*Ionscope does not sell separate SICM controllers anymore. There are few other commercial systems:  NX12-Bio and NX10 SICM,
Park Systems, Korea and SICM modules from ICAPPIC Limited, UK (icappic.com). All these systems are based on the original
HPICM principles. However, imaging stereocilia bundles in the hair cells requires several custom modifications that are technically
challenging (or even impossible) in the closed “ready-to-go” systems such as Ionscope or NX12-Bio/NX10. Currently, there is only one
modular system (ICAPPIC) that has the flexibility to suit any SICM/HPICM experiment but requires some component integration.