This practical can help us study dynamic changes of the nano structural components of the mechanical electrical transduction machinery at the surface of the hair cell stereocilia bundles. The main advantage of this technique is the time-lapse imaging of the surface of living cells with complex topography at single nanometer resolution, and without making physical contact with the sample. This technique can be applied to nearly any living cell.
We previously matched the surface of lung epithelial cell lines infected with viruses, muscle cells, in My Image Bacteria Soon. Test the nanopipettes at the beginning of each imaging session. After fabricating a nanopipette, check for bubbles, remove any bubbles if present, and mount the nanopipette onto the hopping probe ion conductance microscope pipette holder.
At four milliliters of bath solution to the chamber and place the chamber onto the HPICM stage. Introduce the ground electrode to the bath solution and make sure that the voltage being applied to the pipette through the patch clamp amplifier is zero. After placing the micro manipulator in the vertical position, move the pipette in Z until it touches the liquid and set the amplifier offset to zero, before adding plus 100 millivolts to check the pipette current.
Use silicon glue to attach the AFM calibration standard to the chamber and cover the sample with four milliliters of HBSS. Use double-sided tape to secure the chamber to the X Y stage of the HPICM setup, and load a new nanopipette onto the holder as demonstrated. After testing the nanopipette resistance and diameter, set the current to one nano amp.
Use a coarse patch clamp manipulator to position the nanopipette approximately above the center of the calibration standard and increase the set point while monitoring the signal from the sensor of the Z piezo actuator on an oscilloscope in real time. After establishing a stable repeatable Z approach cycle, decrease the set point to the value just above the point of instability and move the pipette down at a speed of approximately five microns per second, until it reaches the sample. The bottom level of the real-time Z positioning signal will increase, indicating that the nanopipette is withdrawn due to sensing the sample surface.
Due to uneven mounting on the AFM standard, the highest point of the area of interest may be unknown. Therefore, set the amplitude of the pipette retraction to at least 200 to 500 nanometers. Taking care not to exceed the upper limit of the Z piezo actuator movement, begin imaging at low resolution.
Once the highest point of the sample in the imaging area has been identified, decrease the hop amplitude and retract the pipette about 200 microns along the Z axis to prevent any undesired collision with the sample before moving it to a new X Y location. When the area of interest has been located, begin imaging at a higher resolution. For auditory hair cell imaging, firmly secure a freshly isolated organ of Corti to a chamber using either dental floss, or flexible glass pipettes.
Use double-sided tape to firmly secure the chamber to the X Y piezo stage and load a new nanopipette onto the holder as demonstrated. After checking the nanopipette resistance, use the patch clamp micro manipulator to position the nanopipette over the hair cell region while observing the organ of Corti X plant in an inverted microscope. Record, the real-time current and Z positioning signal on the oscilloscope to check if the system is stable with a set point of 0.5%6or lower, and determine the optimal set point and approach the sample as demonstrated.
Perform low resolution imaging of the sample as demonstrated using a hop amplitude of at least six to eight microns. If the nanopipette needs to moved to a new X Y location, retract the pipette about 500 nanometers to avoid a collision with any tall features within the tissue and repeat the low resolution HPICM imaging until the region of interest where the hair cells is located. Then image the region of interest at a higher resolution in 15 minutes or less.
The HPICM protocol can be used to visualize any live cells with a complex topography, such as live rat auditory hair cell bundles. Despite demonstrating a lower X Y resolution compared to scanning electron microscopy images, HPICM images can successfully resolve the different rows of stereocilia. The shape of the stereocilia tips and even the small five nanometer links connecting the adjacent stereocilia.
Given the non-contact nature of HPICM imaging, continuous time-lapse imaging of the same hair cell bundle can be performed for several hours without damaging the bundle cohesiveness. Note that with a very low set point, the system might interpret small fluctuations in the current as encountering the cell surface, leading to white dot noise in the image. Similarly, large hop amplitudes might increase the lateral resonance of the pipette, also resulting in the production of noisy pixels.
In contrast, if the hop amplitude is too small, or the setpoint is too high, the nanopipette might collide with the sample resulting in imaging artifacts or hair bundle damage. The most important thing to remember when attempting this procedure is to spend less than 15 minutes per hair cell bundle when working with live cells. After getting an image of the hair bundle, one could attempt to obtain single channel recordings from specific locations on the surface of stereocilia to answer questions about mechanotransduction channel properties.
