Live-Cell Nanoinjection

Instant Labeling and Imaging of Living Cells by Nanopipettes

  • Live-Cell Nanoinjection - Instant Labeling and Imaging of Living Cells by NanopipettesLive-Cell Nanoinjection - Instant Labeling and Imaging of Living Cells by Nanopipettes
  • Live-Cell Nanoinjection - Instant Labeling and Imaging of Living Cells by Nanopipettes
  • Fig. 1: (a) The principle of nanoinjection. Approaching the cytoplasm of single cells comprises the manual approach of the nanopipette by a manual positioning system about 20 μm above the surface (1.). In the next step (2.) a computer controlled approach begins with subsequent tracking of the ionic current. A decreasing ion current indicates the contact between the nanopipette and the cell surface. In step 3 the cell membrane is penetrated and the delivery of fluorescent probes into the cell begins by increasing the voltage. (b) White-light image of a single COS7 cell prior to nanoinjection. The targeted injection point is marked by the red arrow. (c) Time sequence of the labeling process in the fluorescence view. Step by step the actin structure is labelled with the ejected ATTO655-Phalloidin molecules. (d) Fluorescence image of the labeled cell after 86 s of injection. Scale bar, 7.5 µm
  • Fig. 2: Controlled delivery of Alexa647 attached to 30 bp DNA to the nucleus of a living HeLa cell. (a) White-light image of the cell prior injection. The nanopipette is already placed some microns above the cell nucleus. (b) Approach curve of the nanopipette for the nucleus of the cell. After penetrating the cell membrane with the accompanied decrease of the ionic current (1) a short period of stabilization (2) in the current can be detected followed by a subsequent decrease (3) due to the nuclear membrane indicates the final position of the nanopipette inside the nucleus. (c) Fluorescence image of the nanopipette, precisely inserted into the nuclear region. The small spot indicates the fluorophores confined into the tip of the nanopipette. (d) Fluorescence image of the nanoinjected nucleus. The fluorescence is limited to the area of the nucleus. Scale bar, 5 μm.
  • Fig. 3: Multitarget Nanoinjection. (a) A single living U2OS cell was labeled with three different functionalized fluorescent probes, delivered from a single nanopipette. The labeled cellular structures were visualized by wide-field fluorescence imaging. (b) Actin was visualized by ATTO 655−Phalloidin (2 μL,10−5 M), (c) β-tubulin was labelled by Paclitaxel−OregonGreen (4 μL, 10−4 M), and (d) the nucleus was stained with DAPI (4 μL, 10−4 M). First, staining of tubulin was carried out by applying a negative voltage of −1.0 V for 5 min; the nucleus and actin were stained subsequently by applying +1.0 V for 4 min. (a) Z-Projection of the three different fluorescently labeled structures. The injection position is indicated by the bright spot. The images were acquired, using an integration time of 500 ms for Paclitaxel-OregonGreen, 250 ms for ATTO 655-Phalloidin, and 150 ms for DAPI. The total acquisition time for the whole 3D image was 15 min including the labeling process. Scale bar, 5 µm.

A single nanopipette was used to deliver up to three fluorescent labels into individual living cells within a single injection step. The utilized nanoinjection system allows monitoring of the approach, selective staining of cellular compartments and also live tracking of the delivery process by wide-field fluorescence microscopy.

Introduction

In the field of optical microscopy, fluorescence imaging evolved to a powerful tool for the investigation of cellular structures and also allows insights into cellular dynamics [1,2]. With the recently developed super-resolution techniques, the limits of the spatial resolution were pushed down to a few tens of nanometers [3]. However, before being able to apply fluorescence imaging to living cells, the fluorescent labels for specific intracellular structures or molecules of interest have to be introduced into the cell without harming the cell or interfering with the cell-cycle [4]. Especially for super-resolution imaging techniques, often specialized fluorescent probes have to be used [5]. Also labeling of primary cells is often challenging due to the limited options regarding the labeling method. Various labeling strategies have been developed over the years, ranging from electroporation, lipofection, transfection and microinjection.

The Principle of Nanoinjection

Based on a scanning ion conductance microscope (SICM) a nanoinjection system was developed [6], which employs a small hollow glass capillary with a tip diameter of 100 nm. It allows the precise approach to cells and the delivery of molecules into single cellular targets. Figure 1 shows the principle of nanoinjection. After approaching and subsequent penetration of the cellular membrane the molecules of interest are ejected out of the nanopipette into the cell, resulting in a rapid and dense labeling (fig. 1a). In contrast to the similar microinjection tool, nanoinjection works with (di-)electrophoretic forces, preventing significant volume displacement originating from the pipette during the delivery process. Exploiting (di-)electrophoretic forces also allows the use of finer tip diameter. By tracking the ionic current flowing between two electrodes through the tip of the nanopipette it enables the precise axial control of the nanopipette.

A sudden current drop indicates the penetration of the membrane during the approach process. The delivery of molecules begins, by increasing the voltage between the electrodes, causing the ejection of molecules. The number of molecules ejected out of the pipette per time interval can be controlled by tuning the voltage. By monitoring the fluorescence intensity with (wide-field) fluorescence microscopy, the labeling density and thus the total quantity of delivered molecules into the cell can be adjusted precisely. Nanoinjection allows the labeling of a single cell within a dense ensemble of cells. Due to the practically infinite reservoir of molecules inside the nanopipette, the nanoinjection process can be repeated multiple times, to label a multiplicity of cells within an ensemble of cells.

Results

The precise and stepwise approach by continuously monitoring the ionic current enables the direct targeting of cellular compartments with the nanopipette. We inserted an oligonucleotide, consisting of Alexa Fluor 647 attached to 30bp DNA directly into the nucleus of a living HeLa cell. Placing the nanopipette manually some microns above the nucleus of the cell, the software-controlled approach was started afterwards. The tracked ion current shows a second decrease due to the nuclear membrane indicating, that the nanopipette is now directly located inside the nucleus (fig. 2b). Switching to the fluorescence view after the approach, the tip of the nanopipette becomes visible by fluorescence of the Alexa fluorophores located inside the pipette tip (fig. 2c). On increasing the voltage, the fluorescent dyes are ejected and after a few seconds the entire nucleus is stained (fig. 2d). Due to the nuclear membrane, the oligonucleotides are confined to the nucleus of the cell.

By filling the nanopipette with more than one type of functionalized fluorophores, nanoinjection is able to perform multiple labelings inside a single cell within one injection step. We were able to apply up to three different labels using a single nanopipette. The threefold labeling was carried out by first loading a mixture of Atto655-Phalloidin, Paclitaxel-OregonGreen and DAPI into the pipette. After approaching and penetrating the cell, we applied a sequence of voltages to deliver the different fluorophores. After the injection process the imaging of each label was performed by wide-field fluorescence imaging. Figure 3 shows the actin structure (red), the tubulin structure (green) and the nucleus (blue) of a living U2OS cell during the proliferation process.

Outlook

Due to the (di-)electrophoretic driven delivery of molecules, nanoinjection has the capability to use even smaller nanopipettes. As the survival rate seems to be directly connected to the tip diameter of the pipette, the cell survival can be increased by applying smaller pipettes. A decreasing tip size also allows the injection of smaller targets e.g. yeast cells. Recently the quantitative characterization of nanopipette deposited spots was introduced [7]. By using the quantitative delivery of molecules as a calibration step prior nanoinjection, the absolute number of delivered molecules into a single cell becomes accessible.

Acknowledgements
Dr. Idir Yahiatene is gratefully acknowledged for proofreading of the manuscript.

References
[1]    Stephens D.J. and Allan V.J.: Science 300 (5616) 82–86 (2003)
[2]    Chen B-J. et al.: Science 346 (6208) 1257998 (2014)
[3]    Heilemann M. et al.: Angew. Chem. 47 (33) 6172–6176 (2008)
[4]    Schnell U. et al.: Nat. Methods 9, 152–158 (2012)
[5]    van de Linde S. et al.: Nat. Protoc., 6 (7) 991–1009 (2011)
[6]    Hennig S. et al.: Nano Lett., 15 (2) 1374–1381 (2015)
[7]    Hennig S. et al.: ACS Nano, 9 (8), 8122–8130 (2015)

Author
Dr. Simon Hennig
(corresponding author via e-mail request)
Bielefeld University
Department of Physics
Biomolecular Photonics    
Bielefeld, Germany

Contact

University of Bielefeld
Universitätsstr. 25
33615 Bielefeld
Germany
Phone: +49 521 106 00

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