The overall goal of this procedure is to optically report the membrane potential changes with a genetically encoded voltage indicator. This method will describe the strengths and weaknesses of imaging with different genetically encoded fluorescent probes of voltage. For instance, FRET-based probes will offer the advantage of ratiometric imaging but will give a lower signal.
The main advantage of this technique is that we can visualize the neural activity in real time. Generally, individuals new to this method will struggle because the signal-to-noise ratio is not very large and there are several source of noise and multiple steps that can go wrong. New users are often confused and interference is taken as the real signals.
Visual demonstration of this method is critical to ascertain the validity of the probes being used and what it actually indicates. For the setup, an image splitter is installed between the slow and fast CCD cameras for the imaging of FRET-based GEVI. Before the experiment, insert a filter cube with a dichroic mirror and two emission filters in the image splitter.
Remove this second filter cube when imaging a GEVI with a single fluorescent protein. To begin the experiment, locate a healthy HEK293 cell that shows strong, localized membrane fluorescence compared to the internal fluorescence and avoid patching circular cells as they are either dividing or dying. Then apply a test pulse to check the current response.
Afterward, lower the patch clamp pipette to right above the cell's surface. Then, slowly lower the pipette until it gently touches the cell membrane. The membrane resistance should increase to one to two megaohm.
After that establish a gigaohm seal by gently applying negative pressure through the pipette. Once a gigaohm seal is achieved, set the pipette potential at a desired holding potential. Next, focus the high-speed CCD camera on the cell body.
For the FRET-based GEVI recording, adjust the size of the split images to result in an evenly spaced view for each emission wavelength using the knobs on the image splitter. Then rupture the cell membrane by applying negative pressure in order to form the whole cell configuration. Now, open the imaging software.
Then click acquire SciMeasure camera menu to open the CCD acquire page. Next, create a new data file to save the recording. Click analog output and then read an ASCII to open a pulse protocol file in order to conduct the imaging.
Next click on average the internal repetitions to average the number of trials. Then close the analogue output page. Set the specific acquisition parameters on the CCD acquire page.
After that input the specific values for number of frames for acquisition and number of trials. Then, click the take data optics plus BNC button to start the recording. While the voltage imaging is taking place monitor the oscilloscope to ensure stable whole-cell configuration throughout the recording.
To calculate the fractional fluorescence change click file and then read data file to open a data file that was recorded in the previous step. The cell image in its resting light intensity should be seen on the right side. Next, click on show BNC's to show the current end voltage values.
Change the page mode from RLI frame to frame subtraction in order to utilize the frame subtraction function to identify the pixels with responsive optical signals. Next, select two time points for subtraction and identify the cell area that shows signals in response to the membrane potential change. Next, designate the pixels that need to be analyzed by dragging or clicking each pixel.
The graphical representation of the average fluorescence intensity from the selected pixels should appear on the left side of the software window. Divide the subtracted pixels with resting light intensity by clicking divide by RLI's button to acquire the fractional fluorescence change values. To export the data, remove the current end voltage graphs by unclicking show BNC's menu.
Go to output, save traces as displayed ASCII to export the fluorescence trace in an ASCII file format for the curve fitting analysis. In this procedure, draw the fluorescence change versus voltage graph by plotting the fractional fluorescence changes in response to voltage in a data analysis program. After that fit the curve to a Boltzmann function in order to determine the voltage range of the optical signal by clicking analysis, fitting, sigmoidal fit then open dialog.
Replot the normalized fractional fluorescence changes in response to voltage by using the data analysis program. To calculate the speed of the optical response, open the ASCII file and plot the fractional fluorescence change trace versus time. Then click on data selector in the data analysis software and select one time point corresponding to the beginning of a stepped voltage pulse and a second time point where the optical signal has reached the steady state.
Fit this range to both a single and double exponential decay function by clicking analysis, fitting, exponential fit then open dialog and report the better fit. This figure shows the fluorescence change of an HEK cell expressing a single FP based GEVI Bongwoori. This is a typical fluorescence change in response to the stepped voltage pulses.
Here an HEK293 cell was imaged with a high-speed CCD camera. This image shows the resting light intensity of a cell expressing Bongwoori. And this is a frame subtraction image indicating the pixels where fluorescence change was observed.
Shown here is the optical recording of the induced action potentials from the mouse hippocampal primary neurons expressing Bongwoori. The action potentials were evoked under the whole-cell currents clamp mode. The fractional fluorescence change trace was selected from the pixels correlated to the soma.
In this figure, the fluorescence change of an HEK cell expressing FRET-based GEVI shows the responses to the stepped voltage pulses in two wavelengths. Recorded at one kilohertz with a high-speed CCD camera. While attempting this procedure it's important to remember to test variable fluorescence levels since overexpression of fluorescence can affect the health of the cell.
Following this procedure other methods like slicer codings can be performed in order to answer additional questions involving neuron activity of various circuits. After watching this video, you should have a good understanding of how to image membrane potential using different types of probe.
