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 ....

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

1. Create a program in the micropipette puller to obtain pipettes with a resistance between 200 and 400 MΩ, wh.......

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

Name	Company	Catalog Number	Comments
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.