KANAZAWA, Japan , Aug. 22, 2023 /PRNewswire/ -- 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 (SICM) 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. Scanning ion conductance microscopy (SICM) uses a nanopipette to control the nanopipette-samples distance 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. In 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.

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 – cellular membrane protrusion - on the cell surface, the endocytic pits that form and the formation of a cap closing up the pit. Previously attempts to image the cap formation have been inhibited by limitations in the spatial resolution.

The researchers were even able to resolve extracellular vesicles as small as 189 nm released in the process. As they point out in their report there is increasing evidence that these extracellular vesicles play an important role in communication between cells and homeostasis, with diagnostic and therapeutic applications focused on the smaller extracellular vesicles between 40 nm and 150 nm in particular. The researchers conclude in their report, "We envision this protocol will help to reproducibly fabricate borosilicate nanopipettes for high-resolution topographical mapping using SICM."

Image link

https://nanolsi.kanazawa-u.ac.jp/wp/wp-content/uploads/fig1-6.jpg



© 2023 American Chemical Society

Caption: Fig 1. New method for fabricating nanopipette and ultra-high-resolution imaging of its shape. Upper left: Glass capillary after preheating to deform glass. Lower left: Electron micrograph of a nanopipette tip; Right: SICM image of the surface of a fixed HeLa cell.

Glossary

Scanning ion conductance microscopy

The basic principle behind scanning ion conductance microscopy (SICM) relies on monitoring the ion current that flows through a nanopipette containing some kind of electrolyte. It can map the topography of a sample by adjusting the nanopipette height to maintain a constant ion current as it is scanned over the sample surface. The proximity to the sample surface has a measurable effect on the ion flow and hence current before the tip makes contact with the sample, making it useful for biological specimens that might be easily damaged. It also works well in solution whereas techniques such as electron microscopy require the sample to be fixed.

SICM has been used in hopping mode – where the pipette approaches and retracts from the sample, further lowering the potential for sample damage, particularly where the sample surface is not flat. However, it takes longer due to the vertical distances travelled by the nanopipette and can suffer from more noise. In this report the authors showed that their high performance nanopipettes eliminated the need for hopping mode measurements for their samples.

Extracellular vesicle

These are particles bound by a lipid bilayer that all cells produce, although unlike a cell they do not themselves reproduce. There has been a recent surge of interest in them as studies reveal their likely role in transferring signals, which has led to them being described as a "novel -crine system" in analogy to the endocrine system that secretes hormones to send signals around the body.

Reference



Yasufumi Takahashi , Yuya Sasaki , Takeshi Yoshida , Honda Kota, Yuanshu Zhou, Takafumi Miyamoto , Tomoko Motoo, Hiroki Higashi , Andrew Shevchuk , Yuri Korchev , Hiroki Ida , Rikinari Hanayama, Takeshi Fukuma . Nanopipette Fabrication Guidelines for SICM Nanoscale Imaging, Analytical Chemistry August 20, 2023 .



DOI：10.1021/acs.analchem.3c01010

URL: https://doi.org/10.1021/acs.analchem.3c01010

Contact

Hiroe Yoneda

Senior Specialist in Project Planning and Outreach

NanoLSI Administrative Office

WPI Nano Life Science Institute (WPI-NanoLSI)

Kanazawa University

Kakuma-machi, Kanazawa 920-1192, Japan

Email: [email protected]

Tel: +81 (76) 234-4550

About Nano Life Science Institute (WPI-NanoLSI)

Nano Life Science Institute (NanoLSI), Kanazawa University is a research center established in 2017 as part of the World Premier International Research Center Initiative of the Ministry of Education, Culture, Sports, Science and Technology. The objective of this initiative is to form world-tier research centers. NanoLSI combines the foremost knowledge of bio-scanning probe microscopy to establish 'nano-endoscopic techniques' to directly image, analyze, and manipulate biomolecules for insights into mechanisms governing life phenomena such as diseases.

https://nanolsi.kanazawa-u.ac.jp/en/

About Kanazawa University

As the leading comprehensive university on the Sea of Japan coast, Kanazawa University has contributed greatly to higher education and academic research in Japan since it was founded in 1949. The University has three colleges and 17 schools offering courses in subjects that include medicine, computer engineering, and humanities.

The University is located on the coast of the Sea of Japan in Kanazawa – a city rich in history and culture. The city of Kanazawa has a highly respected intellectual profile since the time of the fiefdom (1598-1867). Kanazawa University is divided into two main campuses: Kakuma and Takaramachi for its approximately 10,200 students including 600 from overseas.

http://www.kanazawa-u.ac.jp/e/

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