Kanazawa University NanoLSI Podcast: Researchers define a nanopipette fabrication protocol for high resolution cell imaging

Researchers define a nanopipette fabrication protocol for high resolution cell imaging

Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Yasufumi Takahashi at the Kanazawa University NanoLSI.

The research described in this podcast was published in Analytical Chemistry in August 2023

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Researchers define a nanopipette fabrication protocol for high resolution cell imaging

Researchers at Kanazawa University report in Analytical Chemistry how to produce nanopipettes that reliably provide nanoscale resolution scanning ion conductance microscopy images of living cells.

A nanoscale view of living cells can provide valuable insights into cell structure and function. Over the years, various microscopy techniques have been enrolled to obtain a window into biological specimens at the nanoscale but all with their limitations and challenges. Although scanning ion conductance microscopy has demonstrated the capability to image living biological samples in solution with nanoscale resolution, it has been hampered by challenges in reliably producing nanopipettes with the optimum geometry for the job. Now researchers led by Yasufumi Takahashi at Kanazawa University’s Nano LSI and Nagoya University have devised a protocol for reproducibly fabricating nanopipettes with the preferred geometry for high quality imaging. 

So what is scanning ion conductance microscopy and what kind of nanopipette does it need?

Scanning ion conductance microscopy uses a nanopipette to control the distance between nanopipette and sample using an ion current as feedback signal. The shape of the nanopipette significantly influences the performance of the device. For instance, a wide aperture limits the possible resolution, a long shunt can lead to rectification effects that warp the ion current measurements, and if the glass of the nanopipette is too thick it can deform the sample before the proximity of the aperture has reached the point needed for constant ion current topographical mapping. As a result, the ideal nanopipette has a short shunt, small aperture and thin glass walls.

The standard procedure for fabricating the nanopipette is to pull a capillary tube with a laser puller that heats the capillary tube it is manipulating. The capillary then narrows where it lengthens until it is finally drawn into two separate pieces. Although quartz can allow a little more control in the process of drawing the capillary tube into shape it is hydrophobic, which raises complications in actually filling the nanopipette with the aqueous solution needed for the ion current. For this reason, the researchers developed a protocol by which they could draw nanopipettes from borosilicate glass capillaries with the required control and reproducibility.

Takahashi and his collaborators noted that ideally the starting capillary should have thick walls and a narrow inner diameter, however it is not easy to obtain capillary tubes to these requirements from commercial suppliers. Instead, they preheat the capillary for 5 s without pulling it, which causes the glass walls to the thicken and reduces the inner diameter. They also optimized the parameters for pulling the tube, such as the velocity.

So did it work? Apparently so

The researchers demonstrated the performance of the nanopipettes they produced by imaging a cell undergoing a type of endocytosis, where it engulfs and absorbs some external material. They were able to image the microvilli – that is, tiny cellular membrane protrusions – found on the cell surface, as well as the endocytic pits that form and the formation of a cap c

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