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. Author manuscript; available in PMC: 2022 Feb 11.
Published in final edited form as: Methods Mol Biol. 2018 Jan 1;1721:135–153. doi: 10.1007/978-1-4939-7546-4_13

Wide-band Electrical Impedance Spectroscopy (EIS) Measures S. pombe Cell Growth in vivo

Zhen Zhu, Olivier Frey, Andreas Hierlemann
PMCID: PMC7612359  EMSID: EMS141047  PMID: 29423854

Abstract

This chapter describes a microfluidic device that enables immobilization and culturing of single rod-shaped S. pombe cells in a stand-up mode. The wide-band electrical impedance spectroscopy (EIS) has been integrated in the microfluidic device to continuously measure cell growth of single S. pombe cells. Cell growth curves showing cellular and intracellular features at high spatiotemporal resolution can be obtained from EIS signals. The features include longitudinal cell elongation in the G2 phase, mitosis, and cell division during an entire cell cycle of S. pombe cells. Microfluidics-based EIS systems provide, hence, a tool for dynamic single-cell studies.

Keywords: Schizosaccharomyces pombe, Microfluidics, Electrical impedance spectroscopy, Cell growth, Cell cycle, Nuclear division

1. Introduction

Monitoring cell growth at high-spatiotemporal resolution offers the potential to study cell dynamics and heterogeneity [1–3]. Optical microscopy is the most common method to simultaneously monitor multiple cellular processes in a single cell [4]. However, optical methods usually require fluorescence labels, which may impact cellular development [5, 6]. Label-free methods, combined with fluorescence microscopy, could provide additional information on a biological process.

A candidate method is electrical impedance spectroscopy (EIS) [7], which is noninvasive and label-free and can provide complementary information on cellular and subcellular properties. EIS relies on applying an external electrical field of variable frequency to measure the dielectric properties of a biological sample that interacts with that external field, while the sample is usually placed between electrodes or within the reach of the electric field [7, 8]. Two different parameters, the impedance magnitude and phase, are typically measured. Different cellular information can be extracted from the measured impedance signals depending on the frequency of the applied electric field [9]: at low frequencies between ~100 kHz and ~1 MHz, one can obtain information on the cell size and volume; at higher frequencies above 1 MHz, one can gain information related to the cell membrane and intracellular compartments, such as cytoplasm or vacuoles [10] and the cell nucleus [11]. EIS can also be used to detect cell motion [12, 13] or cellular mechanical (muscle cells) [14] and electrical (cardiac cells) activity [15].

To perform EIS measurement at single-cell and even subcellular resolution, EIS has been commonly implemented in microfluidic devices [16]. The majority of EIS-integrated microfluidic systems have been designed for microfluidic impedance cytometry [17–20], which is analogous to flow cytometry and allows for rapid multi-parameter analysis of large numbers of single cells. The cell samples can be classified according to cell size and dielectric properties. In cytometry systems, however, cells are detected at single time points (when they pass the detection unit) so that continuous monitoring of selected cells is usually not possible. For continuous monitoring, cells need to be individually trapped under precisely controlled culturing conditions by dedicated microstructures [21–23] that contain electrodes. One popular method relies on microwell arrays to trap single cells by sedimentation [24–26]. Another frequently utilized approach is to passively capture single cells with specially designed microstructures by using hydrodynamic forces [27–30]. Then, one can perform continuous EIS measurements on the immobilized cells at the traps by means of electrodes [11, 13, 31–34].

In this chapter, we describe an EIS-integrated microfluidic device featuring narrow cell-trapping orifices, which were designed to capture single Schizosaccharomyces pombe cells through hydrodynamic forces. The rod-shaped S. pombe cells can be reliably immobilized at the orifices in an upright “standing” mode. We then applied continuous medium perfusion for cell-culturing and time-lapse EIS measurements of the immobilized S. pombe cells. From the recorded EIS signals, we retrieved the dynamic cellular processes, including longitudinal cell growth in the G2 phase, mitosis, and cell division during an entire cell cycle of S. pombe cells (Fig. 1).

Fig. 1. Micrographs of an S. pombe cell during its cell cycle indicating the different processes occurring in the cell and the nucleus [11].

Fig. 1

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2. Materials

Store materials and reagents used for device fabrication in a cleanroom (22 °C). Prepare and store all other materials and reagents at room temperature (unless indicated otherwise). Prepare all solutions using ultrapure water. Diligently follow all waste disposal regulations when disposing materials.

2.1. Device Fabrication

  1. Wafer: 4-in. silicon wafer (0.5 mm thickness). 4-in. glass wafer (Borofloat 33, 0.5 mm thickness).

  2. 5-in. chromium (Cr) mask: Cr mask 1 to define the dimensions of the Poly(dimethylsiloxane) (PDMS) covers and the positions for punching inlet and outlet holes. Cr mask 2 to pattern the metal electrodes and to define the alignment marks and dicing marks. Cr mask 3 to define the regions for dry etching of SiN x . Cr mask 4 to pattern the SU-8 fluidic network.

  3. Photoresist: S1813 (MICROPOSIT, Dow Electric Materials). LOR 3B (MicroChem). SU-8 3025 (MicroChem).

  4. Developer: MF 319 (MICROPOSIT, Dow Electric Materials) for developing S1813 and LOR 3B photoresist. Remover 1165 (MICROPOSIT, Dow Electric Materials) for stripping photoresist after metal deposition. mr-Dev 600 (micro resist technology) for developing SU-8 photoresist.

  5. Poly (dimethylsiloxane) (PDMS) (Sylgard 184 Silicone Elastomer, Dow Corning).

  6. Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane (97% Sigma-Aldrich).

  7. (3-Aminopropyl) triethoxysilane (APTES) (99% Sigma-Aldrich).

  8. Acetone (Electronic grade is fine for all processes).

  9. 2-Propanol (IPA) (Electronic grade is fine for all processes).

  10. Deionized (DI) water.

  11. Kapton tape (20 mm wide).

  12. Hole puncher: 1.0 mm diameter (Harris Uni-Core).

2.2. Experimental

  1. YES medium: 0.5% w/v Yeast Extract (YE) and 2% w/v glucose. Dissolve 5 g Yeast Extract and 20 g glucose in 1 L water. Autoclave to sterilize. Store at room temperature.

  2. Cell-culturing medium: 150 mM NaCl and 0.05% w/v Pluronic F127 (Sigma-Aldrich) in YES medium. Dissolve 500 mg Pluronic F127 in 1 L YES medium and then add 100 mL 1.5 M NaCl to 900 mL medium. Filter through a 0.22 μm syringe filter to sterilize. Store at 4 °C.

  3. Plate culture of S. pombe cells.

  4. Inoculating loop.

  5. Glass test tube with lid.

  6. Centrifuge tube (1.5, 15 mL).

  7. 70% Ethanol.

  8. Glass syringe (2.5 mL TLL PTFE Luer Lock, ILS).

  9. Metal syringe needle (Gauge 25, NLL Metal Luer-Screw, ILS).

  10. PTFE tubing (I.D. 0.5 mm, O.D. 1.0 mm, BOLA).

  11. Custom-made aluminum (Al) holder for microfluidic devices and peripheral parts (Fig. 2a). The holder has the same size as a 96-well plate so that it can be placed on the stage adapter of the microscope. It includes a window for optical observation and thread holes for fixing peripheral parts with screws.

  12. Custom-made poly(methyl methacrylate) (PMMA) cover with through holes (Fig. 2b).

  13. Custom-made printed-circuit board (PCB) for electrical interfacing (Fig. 2c).

  14. M3 plastic screws.

Fig. 2. Photographs of the custom-made Al holder (a), PMMA cover (b), and PCB (c).

Fig. 2

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3. Methods

Fabricate and assemble microfluidic devices in a cleanroom (22 °C). Carry out all the other procedures at room temperature unless otherwise specified.

3.1. Fabricate PDMS Covers

An SU-8 master is required in order to cast the PDMS covers (Fig. 3a). This SU-8 master is also used to define the size of each PDMS cover and the position of each inlet and outlet on the PDMS cover, which will be aligned and bonded to the SU-8 fluidic network.

Fig. 3. Photograph and schematic of the microfluidic EIS device.

Fig. 3

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(a) 3D schematic of a PDMS cover with punched holes as inlets and outlet. (b) Photograph of the device with the fluidic connections. (c) Schematic top view of the microfluidic cell-trapping and impedance measurement area without PDMS cover for better illustration. A common stimulus microelectrode and individual recording microelectrodes were used for EIS implementation. (d) 3D closeup of a cell-trapping orifice with a vertically immobilized S. pombe cell [11]

  1. Treat wafer: Dehydrate a new 4-in. silicon wafer in an oven at 200 °C for 2 h, then immediately clean the wafer in an O2 plasma asher at 200 W for 5 min. Cool down the wafer with a N2 gun.

  2. Spin coat SU-8: Pour sufficient SU-8 3025 photoresist onto the wafer (see Note 1). Place the wafer on the chuck of a spin coater. Turn on the vacuum to fix the wafer. Start the spin coater. Ramp to 500 rpm at a rate of 1000 rpm/s, and hold for 5 s. Then ramp to 2000 rpm at a rate of 2000 rpm/s and hold for 60 s. Slow down the spin coater to 0 rpm at a rate of 500 rpm/s.

  3. Soft bake: Bake the wafer on a hotplate at 95 °C for 12 min (see Note 2).

  4. Expose: Place Cr mask 1 in a mask aligner. Expose the wafer in a hard-contact mode to about 200 mJ/cm2 of exposure dose (see Note 3).

  5. Post-exposure bake (PEB): Place the wafer on a hotplate. Start the hotplate. Ramp to 95 °C linearly in 8 min, and hold for 2 min. Cool down to 22 °C by switching off the hotplate.

  6. Develop: Pour about 1 cm height of SU-8 developer (mr-Dev 600) into a beaker. Immerse the wafer in the developer for 6 min, and stir the beaker gently. Take the wafer out of the developer by using tweezers and immediately rinse with IPA and DI water. Carefully blow dry with a N2 gun.

  7. Hard bake: Bake the SU-8 wafer on a hotplate at 150 °C for 1 h.

  8. Silanize: Place the SU-8 wafer and a glass slide in a vacuum chamber. Pipette several drops of trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane on the glass slide and close the chamber with its lid. Apply vacuum to the chamber and hold for 2 h. Afterward the wafer with SU-8 master is ready for PDMS casting.

  9. Mix PDMS solution (10:1 w/w): Weigh 40 g of silicone elastomer base in a plastic cup on a scale, and then add 4 g of curing agent. Use a plastic stick to thoroughly mix the solution.

  10. Degas: Place the plastic cup in a vacuum chamber and close the chamber with its lid. Apply vacuum to the chamber and hold for 30 min (see Note 4).

  11. Casting: To make the SU-8 wafer as a container for the PDMS solution, tape all the sides of the wafer with Kapton tape. Attach half of the tape width on the backside of the wafer and leave the other half width of the tape standing up. Pour the degassed PDMS solution on the wafer and place it on a hot-plate at 68 °C for 3 h.

  12. Cut the cast into chips and punch fluidic holes: Peel the solid PDMS off the SU-8 master wafer, and cut out each PDMS cover along patterns replicated from the SU-8 master. Punch fluidic holes (inlets and outlets) with the hole puncher. Positions of the holes are patterned on each PDMS cover through the SU-8 master.

  13. Clean: Rinse each PDMS cover as well as the punched holes with IPA and DI water and blow dry with a N2 gun. Store PDMS covers in a closed clean petri dish for further assembly of the microfluidic devices (Fig. 3a).

3.2. Fabricate Microfluidic Devices

A lift-off metallization process was used to pattern microelectrodes on a glass wafer, followed by plasma-enhanced chemical vapor deposition (PECVD) of a SiNx passivation layer and reactive-ion etching (RIE) to reopen the sensing regions and contact pads of the microelectrodes. Then, the SU-8 fluidic channels were directly patterned on the glass wafer, and the wafer was diced into single chips, followed by an irreversible bonding with the PDMS covers to conclude the fabrication of the microfluidic devices (Fig. 3).

  1. Treat wafer: Treat a new 4-in. glass wafer as in step 1 of Subheading 3.1.

  2. Spin coat LOR 3B: Place the wafer on the chuck of a spin coater. Turn on the vacuum to fix the wafer. Pipette 3 mL LOR 3B photoresist in the center of the wafer. Start the spin coater. Ramp to 500 rpm at a rate of 500 rpm/s, and hold for 5 s. Then ramp to 4000 rpm at a rate of 2000 rpm/s and hold for 45 s. Slow down the spin coater to 0 rpm at a rate of 500 rpm/s.

  3. Soft bake: Bake the wafer on a hotplate at 200 °C for 3 min.

  4. Spin coat S1813: Place the wafer on the chuck of a spin coater. Turn on the vacuum to fix the wafer. Pipette 3 mL S1813 photoresist in the center of the wafer. Start the spin coater. Ramp to 500 rpm at a rate of 500 rpm/s, and hold for 5 s. Then ramp to 3000 rpm at a rate of 2000 rpm/s and hold for 60 s. Slow down the spin coater to 0 rpm at a rate of 500 rpm/s.

  5. Soft bake: Bake the wafer on a hotplate at 116 °C for 5 min.

  6. Expose: Place Cr mask 2 in the mask aligner. Expose the wafer in a hard-contact mode to 100 mJ/cm2 of exposure dose (see Note 5).

  7. Develop: Pour about 1 cm height of MF 319 developer into a beaker. Immerse the wafer in the developer for 45 s, and mean-while stir the beaker gently by hand. Immediately take the wafer out of the developer by using tweezers and rinse with DI water. Spin dry with a spin coater at a speed of 2500 rpm. Inspect the undercut under a microscope (see Note 6).

  8. Deposit metal: Deposit 20-nm-thick TiW and then 200-nm-thick Pt on the wafer with metal-deposition equipment (see Note 7).

  9. Strip: Pour about 1 cm height of Remover 1165 into a beaker. Immerse the wafer in Remover 1165 overnight. Take the wafer out of the beaker by using tweezers and rinse with IPA and DI water. Spin dry with a spin coater at a speed of 2500 rpm.

  10. Deposit SiN x : Deposit 500-nm-thick SiN x on the wafer in the PECVD process tool (see Note 8).

  11. Spin coat S1813: Place the wafer on the chuck of a spin coater. Turn on the vacuum to fix the wafer. Pipette 3 mL S1813 photoresist in the center of the wafer. Start the spin coater. Ramp to 500 rpm at a rate of 500 rpm/s, and hold for 5 s. Then ramp to 4000 rpm at a rate of 5000 rpm/s and hold for 60 s. Slow down the spin coater to 0 rpm at a rate of 5000 rpm/s.

  12. Soft bake: Bake the wafer on a hotplate at 116 °C for 5 min.

  13. Expose: Place Cr mask 3 in the mask aligner and align the mask with the wafer by using alignment marks. Expose the wafer in hard-contact mode to 100 mJ/cm2 of exposure dose.

  14. Develop: Pour about 1 cm height of MF 319 developer into a beaker. Immerse the wafer in the developer for 75 s, and meanwhile stir the beaker gently by hand. Take the wafer out of the developer by using tweezers, and rinse with DI water. Spin dry with a spin coater at a speed of 2500 rpm.

  15. Etch SiN x : Dry etch the SiN x layer in the RIE machine (see Note 9).

  16. Remove S1813: Place the wafer in an O2 plasma asher. Turn on the asher at 400 W for 30 min to remove the resist.

  17. Pattern SU-8 channels: Follow the process steps 1–7 of Subheading 3.1. Here, use Cr mask 4 and align the mask to the microelectrodes on the wafer by using alignment marks under the mask aligner, and expose the wafer in hard-contact mode to about 300 mJ/cm2 of exposure dose (see Note 10).

  18. Dice: Protect the SU-8 structures on the wafer with a blue tape. Dice the wafer into chips along the dicing marks patterned in the metal (see Note 11).

  19. Clean: Peel the chips off the blue tape. Rinse the chips with IPA and DI-water. Carefully blow dry with a N2 gun.

  20. Silanize: Treat PDMS covers with O2 plasma in the plasma asher at 50 W for 15 s. Immerse PDMS covers and chips in a 5% v/v APTES water solution for 20 min. Rinse with DI-water and blow dry with a N2 gun.

  21. Bond: Place a chip under a stereo-microscope. Align the inlet and outlet holes of the PDMS cover with the channel inlets and outlets of the chip, and press down the PDMS cover onto the chip. Place the contacted device on a hotplate at 90 °C for 30 min. Then, the fabrication of microfluidic devices is concluded. Store the devices in a closed clean plastic petri dish (Fig. 3).

3.3. Prepare S. pombe Suspension

To prepare a cell suspension of S. pombe, the pre-cultured cells are diluted in the cell-culturing medium, and the cell suspension is then loaded in a glass syringe.

  1. Use a inoculating loop to scrape adequate S. pombe cells from a plate culture, and mix with about 1.5 mL cell-culturing medium in a glass test tube. Fix the tube in a shaker and incubate overnight.

  2. Dilute a cell suspension of S. pombe with cell-culturing medium to a concentration of about 105 cells/mL in a centrifuge tube.

  3. Rinse two glass syringes and two metal needles with 70% ethanol and DI water. Blow dry with compressed air.

  4. Load cell suspension and cell-culturing medium in respective syringes. Fix a needle to each syringe. Remove any air in the syringe by pushing the plunger.

  5. Cut two sections of PTFE tubing to adequate length (about 40 cm). Fix the tubing on each needle (see Note 12). Remove any air in the tubing by pushing the plunger. At this stage, the culturing medium and the cell suspension are ready for experiment.

3.4. Setup of the Experimental System

In the experimental setup, the microfluidic device is assembled by using the custom-made Al holder, PMMA cover, and PCB, and is connected to peripheral instruments for optical imaging, cell culturing, and EIS measurements (Fig. 4).

Fig. 4. Schematic of the microfluidic device for optical imaging, cell culturing, and EIS measurements [11].

Fig. 4

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  1. Place the microfluidic device on the Al holder. Place the PMMA cover on the microfluidic device so that the microfluidic device is sandwiched between the Al holder and the PMMA cover. Insert the plastic screws into the corresponding thread holes on the Al holder and tighten the screws to fix the device and the cover on the holder.

  2. Place the PCB on the PMMA cover. Let the spring probes pass through the corresponding holes and contact the electrode pads on the microfluidic device. Insert the plastic screws into the corresponding thread holes on the Al holder and tighten the screws to fix PCB on the holder (see Note 13).

  3. Connect the PCB to an impedance spectroscope (HF2IS, Zurich Instruments AG, Switzerland) and a current amplifier (HF2TA, Zurich Instruments AG, Switzerland) via the corresponding cables.

  4. Fix a stage adapter for the 96-well plate on an inverted confocal microscope (Nikon A1R+, Nikon Instruments Inc., Japan). Fix the Al holder on the stage adapter. In order to perform live-cell imaging, the microscope has to be enclosed in an environmental box (Life Imaging Services GmbH, Switzerland) to control the environmental temperature for cell culturing.

  5. Insert tubing (which has been affixed to the two syringes in step 5 of Subheading 3.3) into the inlet holes of the microfluidic device so that the cell suspension and cell-culturing medium are connected to the microfluidic device. The syringe with a cell-culturing medium should be connected with the upper-left inlet hole, while the syringe with cell suspension should be connected with the lower-left inlet hole (see Note 14 and Fig. 3).

  6. Place a syringe pump (neMESYS, Cetoni GmbH, Germany) in the environmental box. Affix the syringes to the syringe pump.

  7. Cut a piece of PTFE tubing (about 15 cm) and insert one end into the outlet hole of the microfluidic device (Fig. 3b). Place the other end of tubing into a centrifuge tube for waste collection.

  8. Connect the inlet hole for applying pressure to a pressure controller (DPI 520, Druck Ltd., UK) via a gas pipe.

  9. Control the microscope, the syringe pump, the pressure controller, and the impedance spectroscope by using the corresponding PC user interfaces (see Note 15).

3.5. Trap Single S. pombe Cells

  1. Switch on the syringe pump and run its user interface. Set the flow rates of both, cell-culturing medium and cell suspension, to 5 μL/min. Perfuse the whole fluidic network with liquid. Hold for 10 min to stabilize the system.

  2. Switch on the pressure controller and run its user interface. Set the pressure to about 2000 Pa (negative and positive pressures are relative to atmospheric pressure if not specified otherwise) (see Note 16). Hold for 10 min.

  3. Set the flow rates of both, cell-culturing medium and cell suspension, to 0.5 μL/min. Position the field of view and focus on the first cell-trapping orifice. If there is any cell immobilized at the orifice, release it by raising the pressure gradually (see Note 17).

  4. Set the pressure to –5000 Pa. Hold on until observing a cell captured at the orifice (see Note 18).

  5. If the cell is not vertically trapped or a cell cluster is trapped, release it by raising the pressure gradually. Then repeat step 4.

  6. If a vertically immobilized S. pombe cell is observed at the orifice, immediately increase the pressure to a higher value of around 500 Pa (see Note 19). Hold this pressure for the entire culturing and measurement of this immobilized cell.

3.6. Electrical Impedance Spectroscopy (EIS)

To perform the EIS measurements in the microfluidic device, the long microelectrode in the cell-culturing channel is used as the stimulus electrode, and the individual microelectrodes at each cell-trapping orifice work as the respective recording electrodes (Fig. 3). All ten microelectrodes of the microfluidic device are connected to the impedance spectroscope via ten spring probes and a PCB (Fig. 2). On the PCB, there are ten “on-off” switches to select each individual microelectrode manually. When an orifice has been selected for cell immobilization, culturing, and EIS measurements, the respective switch on the PCB is on and the others are off.

  1. Switch on the impedance spectroscope and run its user interface.

  2. Set parameters on the software interface. We set the stimulus signal as an alternating current (AC) signal with an amplitude of 1 V. This stimulus signal sweeps over a frequency range from 10 kHz to 10 MHz including 92 sampling frequencies during each EIS measurement.

  3. Run a single sweep on the software interface. The recorded signal will be displayed on the software interface (see Note 20).

  4. Save the EIS data.

3.7. Calibrate Impedance Measurement

The electrical impedance values of the employed orifice of each microfluidic device are calibrated by correlating them to the measured lengths of several vertically immobilized S. pombe cells by using confocal imaging.

  1. Start the impedance spectroscope and its user interface, and set parameters for measurement as steps 1 and 2 in Subheading 3.7.

  2. Capture a cell as described in the steps in Subheading 3.6.

  3. Carry out the confocal imaging of the immobilized cell. Save the optical data (see Note 21).

  4. Carry out the EIS sweep of the immobilized cell as during steps 3 and 4 in Subheading 3.7.

  5. Release the cell by raising the pressure gradually.

  6. Carry out the EIS sweep of the empty orifice as detailed in steps 3 and 4 in Subheading 3.7.

  7. Repeat steps 2–6 until an adequate number of cells are measured.

  8. After the calibration, extract the cell length of all measured S. pombe cells manually from the z-stacks of confocal micrographs (see Note 22 and Fig. 5).

  9. Extract the relative EIS magnitude signals at 1 MHz of all the measured S. pombe cells from the saved EIS data (see Note 23).

  10. Calculate the correlation between the cell length and the relative EIS magnitude signals at 1 MHz by linear regression (Fig. 6).

Fig. 5.

Fig. 5

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Length measurements of S. pombe cells using confocal imaging: Fluorescence micrographs of three exemplary cells represented as XZ- and YZ- plane intersections of the cells [11]

Fig. 6. Linear relationship between cell length measured by optical images and the corresponding relative EIS magnitude at 1 MHz [11].

Fig. 6

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3.8. Measure Growth of Single S. pombe Cells In Vivo

In vivo measurements of cell growth of single S. pombe cells were carried out in the microfluidic system by using EIS every 30 s and concurrent confocal imaging every 20 min.

  1. Switch on and set up the impedance spectroscope as detailed in steps 1 and 2 in Subheading 3.7.

  2. Start a self-made user interface to control the impedance spectroscope (see Note 24). Set 30 s as the time interval of time-lapse EIS measurement, and the path and filename for saving EIS data.

  3. Carry out the calibration as in Subheading 3.8.

  4. Set up the user interface of the confocal microscope for time-lapse imaging. Use a time interval of 20 min, an xy-resolution of 0.4 μm, an image size of 64 × 64 pixels, a z-stack height of 25 μm, and a z-stack interval of 0.5 μm (see Note 25).

  5. Carry out the EIS sweep of the empty orifice as described in steps 3 and 4 in Subheading 3.7.

  6. Capture a single S. pombe cell as described in the steps in Subheading 3.6.

  7. Run the self-made software and the confocal imaging software. Now start recording the cell growth by using EIS every 30 s and concurrent confocal imaging every 20 min.

  8. Stop running the software after the recording has been done (see Note 26).

  9. Release the cell by raising the pressure gradually.

  10. Carry out the EIS sweep of the empty orifice as described in steps 3 and 4 in Subheading 3.7 (see Note 27).

  11. Repeat steps 5–10 to record from more S. pombe cells.

  12. Use the calibration result obtained in step 3 and EIS magnitude signals at 1 MHz to plot the cell growth curve as relative cell length versus time (Fig. 7).

  13. Plot the EIS phase signals at 5 MHz versus time to show dynamic cellular processes, including longitudinal cell growth in the G2 phase, mitosis and cell division during an entire cell cycle of S. pombe cells (Fig. 8).

Fig. 7.

Fig. 7

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Cell growth curves (G2 phase) retrieved from relative-magnitude EIS signals at 1 MHz and by using the corresponding calibration equations. Immobilized S. pombe cells were cultured and recorded from at 32 °C (n = 10) and 25 °C (n = 4) over different time periods. Five cells did not grow at all. [11]

Fig. 8. Monitoring of nuclear division and cytokinesis of S. pombe cells by using EIS.

Fig. 8

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(a) and (b) show optical and EIS results for one exemplary single S. pombe cell, whereas (c) show EIS recordings of two additional cells. (a) Time-lapse confocal micrographs and corresponding cell cartoons at 40, 60, and 80 min after trapping of the cell. (b) Growth curve displayed as relative magnitude signals at 1 MHz and relative phase signals at 5 MHz versus time, respectively. Green-shaded area indicates the time period of nuclear and cell division. The red arrows indicate the moment when the tilting of daughter cells occurred. (c) Phase curves obtained from EIS recordings of two additional cells showing nuclear division and daughter cell tilting after division [11]

4. Notes

  1. Hold the wafer at its rim with fingers. Use the other hand to grasp the bottle of SU-8 resist, and directly pour SU-8 resist onto the wafer from the bottle. Immediately tilt and rotate the wafer. Make sure that the resist covers at least 2/3 area of the wafer.

  2. There might be SU-8 resist stuck on the backside of wafer after the spin-coating process. Clean it by the following protocol: Place a cleanroom tissue on the bench. Spray some acetone on the tissue. Place the wafer on the tissue, and sweep it slowly over the tissue by using tweezers. The SU-8 resist on the backside is wiped away.

  3. We used a SUSS microTec MA/BA8 mask aligner in the photolithography process. The intensity of the broad-band i-line exposure is about 31 mW/cm2. The exposure time has to be calculated according to the exposure intensity.

  4. To prevent overflow of the PDMS solution, stop pumping when bubbles are approaching the rim of the plastic cup.

  5. S1813 photoresist is optimized for g-line exposure. The intensity of the broad-band g-line exposure in our mask aligner system is about 50 mW/cm2. The exposure time has to be calculated according to the exposure intensity.

  6. The underetch should be 0.5–1 μm wide. Please optimize the development time to adjust the width of the underetch.

  7. We used an Oxford Instruments Ionfab 300Plus system with tungsten (W), titanium (Ti), and platinum (Pt) targets for metal deposition.

  8. We used an Oxford Instruments Plasmalab 80Plus PECVD system for SiN x deposition.

  9. We used an Oxford Instruments Plasmalab 100 ICP-RIE system for SiN x etching.

  10. To improve the resolution of SU-8 structure, we installed an i-line (365 nm) filter in the mask aligner. The intensity of the band-pass i-line exposure in our mask aligner system is about 24 mW/cm2. The exposure time has to be calculated according to the exposure intensity.

  11. We used a dicing saw 8003 from ESEC AG, Switzerland.

  12. We used the needle with OD of 0.51 mm and PTFE tubing with an ID of 0.5 mm, so that the needle can be tightly fixed in the tubing.

  13. Screw the plastic screws deep enough into the Al holder so that the spring probes get good contact to the microelectrode pads.

  14. The cell-culturing medium is infused from the upper-left inlet and flows along the trapping sites in the upper half of the cell-culturing channel. The cell suspension is infused from the lower-left inlet and flows along the lower half of the channel (Fig. 3). This flow arrangement keeps the cell stream away from the cell-trapping orifices during cell recordings. We modulated the parallel flow profile by adjusting the pressure applied to the suction channel [11].

  15. We used a Nikon A1R+ confocal microscope, a neMESYS syringe pump and a HF2IS impedance spectroscope in our experiment. The three instruments have their own user interfaces (PC software) provided by the manufacturers: NIS-Elements AR, neMESYS UserInterface, and ziControl, respectively. For the DPI 520 pressure controller, we designed a LabVIEW program as a user interface, which can communicate with the pressure controller and set the pressure values.

  16. If there is any bubble in the fluidic network, set the pressure to a higher value, for example, 20,000 Pa. The air bubble will be dissolved in liquid or permeate through the PDMS under high pressure, since PDMS is gas permeable.

  17. We usually use the first orifice of each microfluidic device. If the first one is blocked by any cell or dust in the medium and cannot be emptied by raising the pressure to a high value, use the second orifice for the experiments.

  18. If the cells accumulate rapidly at the orifice when –5000 Pa is applied, raise the pressure to a higher value, for example, –4000 Pa, so that one cell can be trapped at a time. But if you have to wait for minutes to capture a cell, decrease the pressure to a lower value, for example, –6000 Pa. Users have to optimize the value of the negative pressure in the beginning of every experiment.

  19. This pressure is highly dependent on the applied flow rates of the cell-suspension and cell-culturing medium as well as on the channel geometry. Users have to optimize this pressure value at the beginning of every experiment. In our experiments, we set the pressure to a value between 0 and 800 Pa. At this pressure, the immobilized cell was retained reliably and no additional cells were trapped during the culturing and measurements.

  20. The recorded signal is displayed in terms of magnitude and phase at each applied frequency of the impedance spectroscope. Since the measured impedance is inversely proportional to the recorded signal, the values of magnitude and phase are directly used as characteristic signals in the measurements [11].

  21. We used a Nikon A1R+ confocal microscope (CFI S Plan Fluor ELWD ADM 40× objective, 0.6 NA, correction ring adjusted to 0.5 mm) to scan the entire cell. On the software interface of a microscope, we set the xy-resolution to 0.1 μm, the image size to 256 × 256 pixels, we used a z-stack height of 25 μm and a z-stack interval of 1 μm. Users can adjust these parameters, scan speed, pinhole, HV, offset, and laser intensity based on their microscope and cell strains.

  22. We used the following methods to optimize accuracy of cell length extraction: (1) The bottom boundary of the cell was defined by a sharp increase in fluorescence intensity and the appearance of a ring-shaped structure that is caused by the cell plasma membrane; (2) for an undivided cell, the total length of the cell can be defined as two times the distance from the bottom boundary to the plane of brightest fluorescence through the nucleus, as the nucleus is maintained in the cell center by cytoplasmic microtubules; (3) all cells, no matter at which stage of division, are geometrically symmetric along the z-axis; (4) after division, the upper nucleus is visible due to the enhanced fluorescence of the nuclear membrane, even though the intensity may be still lower compared to the lower nucleus.

  23. The relative magnitude is calculated by dividing the signal magnitude upon trapping a cell by the signal magnitude of the empty orifice.

  24. We designed a LabVIEW user interface to control the impedance spectroscope. On this software interface, we can set parameters for time-lapse EIS measurement, including the time interval, and the filename and path for saving data.

  25. To avoid the photobleaching, we decreased the xy-resolution and image size compared to the corresponding settings in Subheading 3.8. Users can adjust these parameters, scan speed, pinhole, HV, offset, and laser intensity based on their microscope and cell strains.

  26. We used MATLAB software to read the saved EIS signals in real time. Once a steep change of signals or no change of signals was observed, we usually continued to record for another 20 or 40 min and then stopped the recording.

  27. We measured EIS signals before and after recording of the cell growth in order to verify that there was no signal drift during the recording.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 61404027 and No. 61774036), the National Key Basic Research Program of China (No. 2015CB352100), and the Swiss SystemsX.ch program within the RTD project “CINA.”

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