2.1. System Setup
We conducted the intracellular pressure measurement experiment on the micropipette electrode system developed in our laboratory. The system setting is shown in
Figure 1. The 35 × 10 mm culture dish was placed on a fixed platform on the vibration isolation platform, in which the cell operating fluid and experimental samples were placed. A standard upright microscope (Eclipse FN1, Nikon, Tokyo, Japan) capable of movement in the XY plane with a repeatability of ±0.1 μm over an area of 2 × 2 cm
2 was used to observe cells in the intracellular pressure measurement experiment. The motorized focusing device on the microscope was used to automatically focus the cells with a repeatability of ±0.1 μm in the vertical direction. On the left arm was an X-Y-Z micromanipulator model MP285 (stroke space 2 × 2 × 2 cm
3, maximum speed 1 mm/s, repeatability ±0.1 μm) for mounting micropipette electrodes. The right arm was a scientific model X-Y-Z micromanipulator (stroke space 2.5 × 2.5 × 2.5 cm
3) for mounting the holding pipette (HP). An in-house developed pneumatic chamber [
18] was used to provide pressure inside the micropipette. A charge-coupled device (CCD) camera (IR-2000, DAGE-MTI, Michigan City, IN, USA) was mounted on the microscope to acquire images of the cells at 60 frames per second. A host computer was used for microscopic image processing, electrical signal acquisition, suction pressure control, and motion control of the microscope and manipulator. The entire robot system was covered with an electromagnetic shield to isolate electrical interference from the external environment. A human–machine interface based on Qt programming (see
Figure 2) was developed to provide visual feedback and display information about the process of measuring intracellular pressure, suction pressure, electrical signals, output pressure, etc. This interface allows the operator to update the pressure applied inside the micropipette and the distance the micropipette electrode moves at each step. The electrical signal detected by the micropipette electrode was amplified using an amplifier (MultiClamp 700B, Molecular Devices, San Jose, CA, USA), then converted into a digital signal, and finally transmitted to the host computer.
2.2. Fabrication of Micropipette Electrode and Oocyte Preparation for Intracellular Pressure Measurement
The micropipette electrode used in the intracellular pressure measurement experiment was made of a glass tube (BF150-117-10, Sutter, Sacramento, CA, USA) with an inner diameter of 1.17 mm and an outer diameter of 1.5 mm. The glass tube was pulled with a micropuller (P97, Sutter) to form a tapered tip with a diameter of ~2 μm (see
Figure 3a). If that micropipette were mounted directly on the robot arm, the micropipette electrode would penetrate the cell at an oblique 30°. This is because the electrode holder on the robot is installed at an inclination of 30° (see
Figure 3b) in consideration of the use limitation of space, the stroke of the robot, etc. Therefore, to reduce the torsional effect of micropipette penetration on cells, we used a micropipette forging instrument to bend the tip of the micropipette by 30° (see
Figure 3c). In this way, the tip can be horizontal after being installed on the robot. In the same way, we also bend the holding micropipette by the same degree before mounting it on the right arm. In this way, the axes of the cell, micropipette electrode, and holding pipette are in a horizontal state during the experiment.
Then, the micropipette was backfilled with 20 μL electrode liquid. Before the measurement, the cell culture medium was replaced by extracellular fluid (g/L: HEPES 0.6911, C16H18N2O4S 0.05, NaHCO3 2.2504, NaCl 1.7532, C21H39N7O12 0.06, M199 9.5, pH 7.0~7.2) for electrical signal recording. A silver electrode wire with a diameter of 0.2 mm was inserted into the micropipette to form the micropipette electrode. One section of the micropipette electrode was immersed in the extracellular fluid, and the ground wire connected at the other end was directly placed in the extracellular fluid, thus forming a complete circuit.
The oocytes we used in the experiment were obtained using the following methods. We collected pig ovaries from the local slaughterhouse and used a sterile 10mL syringe with an 18-gauge needle to aspirate ovarian follicles with a diameter of 3–6 mm to obtain cumulus–oocyte complexes (COCs). At this time, there are many tissue impurities in the COCs liquid. To obtain clean COCs, we poured them into Tyrode’s lactate (TL)–Hepes-PVA (polyvinyl alcohol, 0.1%) for washing. After washing, we waited for their precipitation for 20 min and then poured out the supernatant to wash and precipitate again, repeating three times. Then, 20~40 COCs were picked up and transferred to 100 μL mature medium (TCM-199 supplemented with 15% FBS, 10 ng/mL EGF, 10% porcine follicular fluid, 10 IU/mL of eCG, 5 IU/mL of hCG, 0.8 mM L-glutamine, and 0.05 mg/mL gentamicin) for 42 h at 38.5 °C, 5% CO
2, and saturated humidity. At the MII stage, we selected target oocytes that had a clear perivitelline space, integrated cell membrane, and a visible polar body (PB1) (
Figure 3d).
2.3. Resistance Analysis for Micropipette Electrode Out of the Cell
It is well known that when the glass micropipette is filled with electrolyte solution inside and outside, and an electromotive force is applied, the glass micropipette can form a stable resistance.
The tip diameter used in our experiment was ~2 μm pipette, whose geometric structure is similar to an infinite cone (
Figure 4). We chose KCl solution as the internal solution of the electrode and assumed that KCl diffusion, just like in isotropic homogeneous media, increases the KCl flux due to a large amount of flow:
where
C = KCl concentration (moles/liter),
D = KCl diffusion coefficient (cm
2/s),
r = distance from origin of cone (cm), and
v(
r) = fluid velocity at
r (cm/s).
It is assumed that the fluid velocity is the same on any given cross-section (“plug” flow), and the velocity passes through the origin of the cone (which allows a one-dimensional solution in a spherical coordinate system). For a given flow, the speed of each cross-section will be inversely proportional to the square of the radius of that cross-section (Equation (2))
where
k = a constant containing geometrical terms and pipet hydraulic resistance,
Pin = pressure applied to the inside of the pipet (mm Hg), and
Pout = outside pressure (mm Hg).
We can calculate the total pipette resistance by integrating the conductivity on the length of the pipette.
where
R = total pipet resistance (Ω),
ro = distance of the tip from the origin of the cone (cm), and
A(
r) = cross-sectional area at
r (cm
2).
The electrode resistance is affected by the solution concentration. When the solution concentration decreases, the measured electrode resistance increases. When the solution concentration (less than the solubility of the solution itself) increases, the measured electrode resistance decreases.
When the micropipette electrode is out of the cell, the measurement micropipette electrode circuit is shown in
Figure 5a. The micropipette electrode measurement resistance
RM is composed of the silver wire electrode resistance
RE, the solution resistance
RL, and the resistance
RI caused by the concentration gradient field of the electrode solution and the extracellular solution near the micropipette electrode opening, namely
Among them,
RE and
RL can be considered to be constant when the volume of the liquid enters or comes out of the micropipette is negligible in comparison to the whole volume of liquid inside the micropipette and the environment. For
RI in Equation (4), the ion concentration gradient field corresponding to
RI outside of the cell is formed by the combined action of the following two factors: capillary pressure
PC between the tube wall and the liquid in the electrode and the injection pressure
PI in the microtubule, as shown in
Figure 5b. When
PI is small, capillary pressure
PC will press extracellular fluid (dilute electrolyte solution) into the electrode to form a stable ion concentration gradient and
PI value. When the
PI increases, the ion concentration distribution at the tip will shift to the opening, resulting in an increase in the concentration of KCl at the tip, which will lead to a decrease in the resistance of the electrode. When
PI is larger enough, the concentration gradient area will be pushed out of the micropipette opening. In that case,
RM will reach a stable state with a further increase in
PI if the deposition volume of the solution into the environment is negligible in comparison to the whole volume of solution inside the micropipette.
In summary, a “downslope” section of measurement resistance of the micropipette can be obtained with an increase in PI. When the micropipette enters intracellular space, the intracellular pressure leads to a shift of concentration gradient inside the micropipette and subsequently changes the measured resistance of the micropipette electrode. Thus, the variation of the measured electrode resistance can be utilized to measure the intracellular pressure.
2.4. Prepare the Required Electrode Internal Liquid and Measure the Working Range of the Probe Micropipette Electrode
As aforementioned, the variation in the measured resistance of the micropipette electrode can be utilized to measure intracellular pressure. In the experiment, we chose KCl solution as the electrode solution. To guarantee measurement accuracy, an appropriate concentration of the solution producing a steep downslope in the
RM-
PI relationship is preferred because the
RM is more sensitive to the variation of
PI in that case, which improves the measurement accuracy of
PI. An increase in KCl concentration increases the height of the downslope, according to the analysis in
Section 2.3. However, a too-large KCl concentration will increase the crystallization of KCl molecules and increase the blocking issues of the micropipette opening. Thus, an appropriate KCl concentration needs to be determined through tests.
The concentration gap of the KCl solution starts to increase from 0.1 mol (pure water) to 3 mol with an interval of 0.1 mol until the experiment is stopped due to the crystal blocking of the nozzle due to the excessive concentration of the solution. According to testing results, we found that the decreasing speed of
RM increases in the slope area as the concentration of KCl solution increases at the beginning. After the concentration is larger than 1 mol, the blocking incidences start to occur, and its occurrence rate grows as the concentration increases. Therefore, in the following experiment, we chose KCl with a concentration of 1 mol/L as the electrode internal solution.
Figure 6 shows the obtained
RM-
PI curve. It can be found that a steep downslope section, which is the ideal working section for intracellular pressure measurement, exists in the obtained
RM-
PI curve.
2.5. Intracellular Pressure Measurement Based on Electrode Resistance Model
To measure the intracellular pressure, the measurement resistance of the micropipette electrode is modeled, as shown in
Figure 7a. When the micropipette electrode enters the cell, the zona pellucida resistance
RZP and the cytoplasmic resistance
RC also become part of the measurement resistance
RM, namely
Among them,
RE,
RL, and
RZP can be regarded as constants in the experiment. The cytoplasmic resistance
RC can be regarded as constant on the premise that the micropipette electrode is sealed with the ZP and the amount of injected material is far less than the cytoplasmic volume during the puncture process. After the micropipette electrode is inserted into the cell before the intracellular pressure is released, the ion concentration gradient field corresponding to
RI is formed by the joint action of the intracellular pressure
PIn, the injection pressure
PI, and the capillary pressure
PC, as shown in
Figure 7b. Adjusting the
PI,
RI, and
RM will also change accordingly. When the applied
PI remains unchanged and the change amplitude of
RM within a certain period of time does not exceed 3% of the current value, it is considered to have reached a quasi-stable state. Record the
PI1 at this time and retreat the micropipette electrode. Then, the micropipette electrode enters intracellular space again after the release of intracellular pressure. The ion concentration gradient field corresponding to
RI is formed again by injection pressure
PI and capillary pressure
PC. Then, a new stable state of
RM similar to the former one before intracellular pressure release can be achieved through adjustment of
PI again. The difference between the two injection pressures
PI forming the stable states of
RM before and after the release of intracellular pressure separately can be considered as the released intracellular pressure value.
2.6. Robotic Measurement Procedure of Intracellular Pressure Measurement
A robotic measurement procedure of intracellular pressure was developed based on the above work.
Figure 8 summarizes the robotic measurement procedure.
Before measurement, the micropipette electrode and the holding micropipette were moved into the field of vision manually. Then, one oocyte was put into the field of view. The system automatically focuses and 3D localizes the micropipette electrode, holding micropipette, and the target oocyte [
19,
20], corresponding to the frame “localization of microelectrode and cell” in
Figure 8. Then, the system controlled the holding micropipette to approach the target oocyte and immobilize it with aspiration pressure [
21].
Before cell membrane (zona pellucid (ZP) for porcine oocyte in this paper) penetration, the injection pressure inside the micropipette electrode was adjusted to make the resistance of the micropipette electrode within the working range (downslope section). After that, the micropipette electrode was controlled to penetrate the cell membrane along the central axis of the holding micropipette, and the system automatically detected the resistance of the micropipette electrode, corresponding to the frame “microelectrode moving” in
Figure 8. Once the micropipette electrode contacts the cell, the detected resistance will start to rise, and the resistance will continue to rise as the micropipette electrode advances. When the detected micropipette electrode resistance begins to decrease, which means the micropipette has entered the intracellular space, the system assumes that the cell membrane has been punctured, corresponding to the frame “resistance begins to drop?” in
Figure 8. To reduce the squeezing of micropipette electrode cells, which usually generates a false larger intracellular pressure, the system controls the micropipette electrode to retreat until the cytoplasm contour recovers its sphere shape, which means the deformation resulting from cell membrane penetration fully recovers, corresponding to the frame “microelectrode retreat” in
Figure 8. The contour detection method for cytoplasm has been reported in our previous research [
22].
After the cell membrane penetrates, the system increases
PI, and
RM decreases accordingly, corresponding to the frame “apply injection pressure” in
Figure 8. This is because a 1 mol/L KCl solution is a concentrated electrolyte solution compared to the intracellular fluid. When the applied
PI remains unchanged and the amplitude of
RM change within 5 s does not exceed 3% of the current value, it is considered to have reached a quasi-stable state, corresponding to the frame “the quasi-stable state has been reached?” in
Figure 8.
PI1 was recorded at this time, and the micropipette electrode was withdrawn from the cell along the axial direction of the holding micropipette and waited for 60 s to release the intracellular pressure, corresponding to frames “record the injection pressure
PI1” and “withdraw the microelectrode and wait” in
Figure 8. Then, the system controlled the holding pressure of the holding micropipette to first spit and then hold the cell again. In this way, the cell rotated by some degrees relative to the original position. Then, the micropipette electrode entered the cell again along the axis direction, retreated to let the cell recover its shape, increased the injection pressure again to reach the quasi-stable state, and recorded the required injection pressure
PI2 at this time. The intracellular pressure of cells is then determined by the difference in injection pressure before and after the release of intracellular pressure. The corresponding
PI2 is considered valid only when the difference in resistance values between two quasi-stable states does not exceed 100 Ω, which means that the measurement results can be adopted. We have established a flowchart for the robotic measurement of intracellular pressure (see
Figure 8).
The reason why we make the cell rotate relative to the original position before entering the cell again is given as follows. The micropipette electrode usually releases a small amount of electrode fluid (concentrated electrolyte solution) around the point it stays after the first puncture. Due to the poor diffusion of the KCl solution inside the cell in comparison to that outside the cell, there may still be some KCL solution around the previous point. If the micropipette tip stays at the same position after the second entry, the existence of the left KCl solution may weaken the concentration gradient and the variation of RM along PI, increasing the difficulty of obtaining PI2. Therefore, cell rotation before the second entry is required to make the position it stays inside the cell different from the first one. This ensures that the micropipette electrode tip is filled with KCl solution on the inner side and pure intracellular liquid on the outside during the two penetration processes of the cell.