High-speed scanning ion conductance microscopy for sub-second topography imaging of live cells

Simeonov, Stefan; Schäffer, Tilman E.

doi:10.1039/c8nr10162k

Cited by 48 publications

(40 citation statements)

References 37 publications

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“…The speed of approach curve measurement can be made much faster by employing instrumentation developed for fast scanning ion-conductance microscopy. 35 For instance, an ionic current of ~2.7 nA was measured with a precision of ±0.5 pA (i.e., ±0.02% of the ionic current) when an ~100 nm-diameter water-filled nanopipet traveled 2 μm in 4–40 ms, i.e., 50–500 μm/s. 36 In comparison with our setup, not only was the similar current measured more precisely despite much faster sampling, but also the approach velocity was ~100–1000 times faster.…”

Section: Resultsmentioning

confidence: 99%

Nanoscale Intelligent Imaging Based on Real-Time Analysis of Approach Curve by Scanning Electrochemical Microscopy

et al. 2019

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Scanning electrochemical microscopy (SECM) enables high-resolution imaging by examining the amperometric response of an ultramicroelectrode tip near a substrate. Spatial resolution, however, is compromised for non-flat substrates, where distances from a tip far exceed the tip size to avoid artifacts caused by the tip-substrate contact. Herein, we propose a new imaging mode of SECM based on real-time analysis of approach curve to actively control nanoscale tip-substrate distances without contact. The power of this software-based method is demonstrated by imaging an insulating substrate with step edges using standard instrumentation without combination of another method for distance measurement, e.g., atomic force microscopy. An ~500 nm-diameter Pt tip approaches down to ~50 nm from upper and lower terraces of a 500 nm-height step edge, which are located by real-time theoretical fitting of experimental approach curve to ensure the lack of electrochemical reactivity. The tip approach to step edge can be terminated at <20 nm prior to the tip-substrate contact as soon as the theory deviates from the tip current, which is analyzed numerically afterward to locate the inert edge. The advantageous local adjustment of tip height and tip current at the final point of tip approach distinguishes the proposed imaging mode from other modes based on standard instrumentation. In addition, the glass sheath of Pt tip is thinned to ~150 nm to rarely contact the step edge, which is unavoidable and instantaneously detected as an abrupt change in the slope of approach curve to prevent the damage of fragile nanotip.

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Section: Resultsmentioning

confidence: 99%

Nanoscale Intelligent Imaging Based on Real-Time Analysis of Approach Curve by Scanning Electrochemical Microscopy

et al. 2019

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“…This v z value attained here is more than 300 times improvement over the v z value used in a recent SICM imaging study on biological samples (50 nm/ms for r a = 50 nm) with a conventional design of SICM Z-scanner [34]. Very recently, the Schäffer group successfully increased v z up to 4.8 µm/ms for r a = 80-100 nm using their sample stage scanner and a step retraction sequence called 'turn step' [40]. Our v z value achieved for even 3-4 times smaller r a still surpasses their result.…”

Section: Evaluation Of Improved Vzmentioning

confidence: 98%

“…Since we have not yet introduced other devices proposed previously for increasing the temporal resolution, there are still room for further speed enhancement. One of candidates to be added is (i) the 'turn step' procedure (applying a step function to the Z-piezodriver) developed by Simeonov and Schäffer for rapid pipette retraction [40]. Other candidates would be (ii) further current noise reduction of the transimpedance amplifier in a high frequency region, and (iii) lock-in detection of AC current produced by modulation of the pipette Z-position with small amplitude [12].…”

Section: G Outlook For Higher Spatiotemporal Resolutionmentioning

confidence: 99%

Development of high-speed ion conductance microscopy

Watanabe

Kitazawa

Sun

et al. 2019

Review of Scientific Instruments

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Scanning ion conductance microscopy (SICM) can image the surface topography of specimens in ionic solutions without mechanical probe-sample contact. This unique capability is advantageous for imaging fragile biological samples but its highest possible imaging rate is far lower than the level desired in biological studies. Here, we present the development of high-speed SICM. The fast imaging capability is attained by a fast Z-scanner with active vibration control and pipette probes with enhanced ion conductance. By the former, the delay of probe Z-positioning is minimized to sub-10 µs, while its maximum stroke is secured at 6 µm. The enhanced ion conductance lowers a noise floor in ion current detection, increasing the detection bandwidth up to 100 kHz. Thus, temporal resolution 100-fold higher than that of conventional systems is achieved, together with spatial resolution around 20 nm.

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“…Since piezo actuators used to move the probe have finite time response, the imaging rate depends on the image size and the number of pixels comprising the image. It has been demonstrated that it is possible to design and construct SICM that could be operated in high speed (HS) regime sufficient to follow highly dynamic processes in living cells (Ida et al, 2017;Simeonov & Schäffer, 2019a, 2019b.…”

Section: Sicm-fcmmentioning

confidence: 99%

Release of insulin granules by simultaneous, high-speed correlative SICM-FCM

Bednarska

Novák

Korchev

et al. 2020

Preprint

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SummaryExocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label-free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM-FCM) to study the exocytosis of individual granules of insulin from the top, non-adherent, surface of pancreatic β-cells. Using SICM-FCM, we were first to directly follow the topographical changes associated with physiologically-induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal.

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High-speed scanning ion conductance microscopy for sub-second topography imaging of live cells

Abstract: High-speed scanning ion conductance microscopy (HS-SICM) reveals ultra-fast morphodynamics of live cells at a rate of 0.6 s per frame.

Cited by 48 publications

References 37 publications

Nanoscale Intelligent Imaging Based on Real-Time Analysis of Approach Curve by Scanning Electrochemical Microscopy

Nanoscale Intelligent Imaging Based on Real-Time Analysis of Approach Curve by Scanning Electrochemical Microscopy

Development of high-speed ion conductance microscopy

Release of insulin granules by simultaneous, high-speed correlative SICM-FCM

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