This protocol enables researchers to precisely heat a droplet interface bilayer, or DIB, model membrane, which allows for studying a variety of temperature-related effects that occur in cellular environments. The core advantage stems from the ability to locally measure and control bath temperature in a low-volume reservoir without obstructing access for the electrical or optical characterizations of the DIB. The ability to precisely heat the fluid bath unlocks the possibility to study the transport and signaling properties in DIBs formed from a much wider variety of membrane constituents, including natural cellular extracts.
This is a multi-step protocol leveraging multiple equipment that must be followed closely to achieve sufficient experimental accuracy, thus any ambiguity in the written process can be clarified via visual demonstration. To begin gather two pieces of one-millimeter thick insulative rubber trimmed to 25 by 40 millimeters in length, two pieces of a six-millimeter thick rubber that are also 25 by 40 millimeters, a prepared aluminum base fixture assembly, and an acrylic oil reservoir that fits in the viewing window of the aluminum base fixture. Place the thinner rubber pieces onto the stage of the microscope such that the long edge of each piece is tangential to the stage opening.
Position the aluminum base fixture on top of the insulative pads with the viewing window of the fixture centered above the objective lens. Place a thicker piece of rubber on top of each resistive heating element, then use a microscope stage clip to hold it in place to protect the heating elements from damage caused by the stage clips and to insulate against accidental electrical shorting. Carefully bend the measurement end of a thermocouple to achieve a 90-degree angle at about four millimeters from the end.
Insert the bent tip of the thermocouple into the lower left corner of the aluminum fixture and gently secure it with a locking screw. Place the acrylic reservoir into the well of the aluminum fixture prior to adding hexadecane oil to the well of the aluminum fixture to minimize the risk of trapping air bubbles between the viewing window and the bottom of the acrylic reservoir. Dispense about 1, 000 microliters of hexadecane oil into the well of the aluminum fixture to provide a maximal surface area for heat transfer to occur while not allowing oil to spill over the edges of the fixture onto the microscope stage or objective lens.
Dispense about 1, 000 microliters of hexadecane oil into the acrylic reservoir without overfilling it. Measure the nominal capacitance of the membrane while lowering the temperature of the oil bath from a set point that permits bilayer formation to identify thermotropic phase transitions of the lipids in the membrane. Right-click the temperature graph on the GUI and clear the display data to ensure that sufficient space in the buffer is available for subsequent recordings.
Using the waveform generator connected to the patch clamp amplifier, apply a triangular voltage waveform across the droplet interface bilayer, or DIB, electrodes and record the induced current response through the bilayer. Cool the bilayer by reducing the set-point temperature in five-degree increments, waiting a minimum of five minutes at the new steady-state temperature between temperature changes until the desired temperature is achieved. After the oil bath and cooling the bilayer to the desired minimum temperature, right-click the temperature graph in the GUI again and export the temperature data versus time to a spreadsheet software.
Stop the current recording. From the measured current, calculate the nominal capacitance of the square wave current response versus time during the cooling period. Plot nominal capacitance versus temperature to observe how membrane capacitance changed, then locate non-monotonic changes in capacitance versus temperature to identify melting temperature.
Similarly, assess the quasi static-specific capacitance of the bilayer at fixed temperatures by successively incrementing the temperature of the oil bath and the bilayer area. Change the set-point temperature in 10 degree-Celsius increments using the GUI and allow the system to equilibrate to the new temperature. Perform previously described steps to initiate the measurement of capacitive current and recording.
Change the bilayer area by carefully adjusting the positions of the electrodes using the micro manipulators. Allow for the square wave current to reach a steady state amplitude and collect images of the DIB to enable calculation of membrane area versus time. Use a camera mounted to the microscope to image the bilayer as seen from the aperture.
Simultaneously add a digital tag to the current recording software to mark the corresponding time point for image collection. Obtain a total of five DIB images and steady-state regions of bilayer current, then reset the temperature and repeat the imaging. Analyze the current recordings and DIB images at the tagged time points corresponding to steady state bilayer areas, extract bilayer capacitance and area for each temperature.
Plot capacitance versus area for each temperature and compute the slope of the first order regression, which represents the specific capacitance of the bilayer at each temperature. Then plot values of specific capacitance versus their respective temperatures. Examine the specific capacitance versus temperature data for non-monotonic variations to identify melting temperatures.
Assess the dynamics of voltage-dependent ion channel formation by generating a DC voltage step input across the bilayer. Set initial voltage to the desired step value in millivolts and the final voltage and step size to a value higher than the desired step. Set a desired duration time for the step input in seconds, then choose the desired polarity for the step input.
Switch the patch clamp amplifier to send the command voltage originating from lab view or voltage output module to the head stage, turn on the voltage and record the induced current response, which should inhibit an S-shaped response to a critical voltage. Separately obtain dynamic current voltage relationships for a membrane at desired temperatures to reveal voltage-dependent relationships such as ion channel behaviors. Switch the patch clamp amplifier to send the command voltage originating from the waveform generator to the head stage and initiate current recordings.
On the waveform generator, output a continuous sinusoidal waveform with a desired amplitude, offset, and frequency. Record the induced current response across one or multiple cycles and repeat as desired for different sine wave amplitudes, frequencies, and temperatures. The temperature control system was used to showcase the temperature dependence of a DIB formed from brain total lipid extract, or BTLE, lipids.
Measurements of capacitive current and temperature versus time are shown during a heating cycle from room temperature to approximately 60 degrees Celsius. Changes in nominal capacitance versus temperature across one complete cooling/heating cycle, after initial bilayer formation at 60 degrees Celsius, were documented. Quasi-static measurements of specific capacitance at different temperatures can be used to identify lipid-melting temperature.
Plotting capacitance of a bilayer versus bilayer area allows for a linear regression, where the slope represents the value of specific capacitance. The DIB image shows that, when temperature is below the melting temperature, the membrane adopts a highly adhesive state, even under tension from stretched droplets caused by well-separated electrodes. The current versus voltage traces shown were obtained by applying sinusoidal membrane voltages, measuring the induced current at two different temperatures.
The arrows and subsequent numbers aid in visualizing the successive quarters of the sinusoidal voltage with respect to time. The measured current density for a monazomycin-doped BTLE membrane at the same voltage level and two different temperatures is shown here. It is crucial to dispense the hexadecane oil into the well of the aluminum fixture correctly.
If this is done out of sequence or not carefully, air bubbles will form under the acrylic well, which will obstruct the bottom-up view of the DIB. The user must also remember to clear the data buffer in the temperature-control software prior to each measurement to ensure full recording. This procedure allows for characterizing biomimetic membranes across a range of temperatures, which is needed to study temperature dependence of membrane structure and transport.
In addition, this capability could be used to reveal nanoscale effects of other membrane-active species, such as biological ion channels and engineered nanomaterials.
