Update Technical Note 3

SciSoft Company (www.SciSoftCo.com)

Notes on Calibration

Running the calibration parameter file, <accalib.prm>, generates a correction file for the real and imaginary components of the system for several time resolutions (which correspond to different frequencies in the frequency domain).

The file <accalib.prm> will produce corrections for six resolutions.

jClamp now uses two approaches for 2 sine stimulation, one being the original implementation where f2=2*f1 (index into the FFT array being 1 and 2), and the other f2<>f1*2 (index into the FFT being 2 and 3). By making the latter stimulus we avoid possible two-tone distortions such as sums and difference frequencies that can be generated in a nonlinear system. Thus, for the original implementation, f1 of 390Hz and f2 of 781 Hz could generate distortion components at f2-f1 which would sum with the f1 response and potentially interfere with measurement of cell parameters. Other distortion components, e.g. harmonics, could also interact with the primary frequencies causing problems. Now I introduce a 2-sine signal that is immune from these effects. Since the indices are not harmonically related, distortion components of an f1 of 390Hz and f2 of 585Hz will not interact as before. The new implementation will provide a few differences from the old one. For example, the old implementation at 10 usec, would produce six resolutions at 0.16, 0.32, 0.64, 1.28, 2.56, 5.12 ms or correspondingly an f1 at ~ 6250, 3125, 1562, 781, 390, and 195 Hz, with f2 at 2*f1. On the other hand, the new implementation at 10 usec, will produce the same six time resolutions at 0.16, 0.32, 0.64, 1.28, 2.56, 5.12 ms but correspondingly f1 at ~12500, 6250, 3125, 1562, 781, and 390 Hz, and f2 at ~18750, 9375, 4687,2343,1171, and 585 Hz. Two cycles of f1 and 3 cycles of f2 are used for each measurement of cell parameters.

The file <accalib.prm> is able to generate these corrections because it delivers episodes with incrementing frequencies that are used to obtain the corrections at each time resolution.

Thus, if you create a calibration file using a 10 usec clock, and a 10 kHz filter between the amp and A/D board, you may collect data at any of the 6 time resolutions noted above. The calibration file that is loaded will be used and automatically chooses the appropriate resolution correction factors for the data analysis. So, if you run only 10 usec data collections with that 10 kHz filter, then you only need this calibration file for all data collections. If you change the clock to 20 usec or any other rate, or if you change the

filter, then you need to create a new calibration file with these settings that you must use (have loaded) prior to collecting data with these new settings.

I suggest that you make a bunch of calibration files that are saved with different filenames, for example.

Amp1_10us_10kHz.cal -- Amplifier number 1 with 10 kHz filter collected with <accalib.prm> set to 10 usec clock

Amp1_5us_10kHz.cal -- Amplifier number 1 with 10 kHz filter collected with <accalib.prm> set to 5 usec clock

Amp2_10us_10kHz.cal -- Amplifier number 2 with 10 kHz filter collected with <accalib.prm> set to 10 usec clock

Amp1_10us_3kHz.cal -- Amplifier number 1 with 3 kHz filter collected with <accalib.prm> set to 10 usec clock

And so on. If you have any other changeable equipment that could alter the system response, such as an analog switch or multiplexer in the path, then any changes will require a new calibration file.

Each time you collect data at a given clock and filter frequency setting you must use the appropriate calibration file. The resolution of the measurement that you select does not dictate the calibration file to be used. This depends only on the clock and filter. jClamp automatically uses the appropriate corrections from the calibration file to analyze at the selected time resolution.

After you make a calibration file, you should load it either by default (set in the ini file), or within a script file (before running a command protocol requiring the calibration file) or manually during jClamp execution.

For command protocol data collections, the loaded calibration file is saved with the abf data file and is automatically used for correction in the Analysis window (if enabled in the Data load window). On the other hand, Cm data collected in the Cell Censor Cm track section uses the currently loaded calibration file to correct on-the-fly and corrected data are saved to disk. So, <abf> data files are saved raw and analyzed each time the file is opened, but <Cm> files recorded in the Cell Censor Cm track section cannot be reanalyzed. This means that for <abf> files, different calibration files can be used to analyze the data, and therefore better estimates of the system response can be used to correct the data post hoc.

Finally, be careful to understand how you might interfere with valid correction using a given calibration. The calibration simply flattens out an unflat system response; it removes the system response so you can accurately gauge the cells physiological response. Lets review the calibration procedure. When you place a pure resister between headstage and ground (and balance out the stray capacitance), the response that you measure which deviates from a purely resistive response should be due to the amplifier, filter, D/A characteristics and any other influential component that you have in the path between that resister and the recorded response. If everything is done correctly with the calibration (pure resistivity, accurate entry of resistance magnitude) then we get correction factors (for all the resolutions at the given clock rate) that can be used to remove the effect of the equipment, and thus measure the cells impedance correctly. In order to validly use the correction factors, you have to make sure that after the formation of a gigohm seal, stray capacitance is balanced out because the evaluation of the cells characteristics is model dependent and does not include a pipette capacitance (see the Help file). Additionally, any manipulation of the amplifier after whole cell configuration could potentially introduce changes to the state of the system that could invalidate the calibration. For example, series resistance compensation may involve (in some amplifiers) an introduction of a lag in the system. This could invalidate the correction values in the calibration file. You can check the effect of series resistance compensation or other amplifier manipulations with an electrical model cell. I routinely will not use this feature if I am doing capacitance measures. The best thing that I can say is that you should check everything you might do with a model cell before trying on a real cell.

Remember that commercial electrical model cells may not really let you balance out stray electrode capacitance. You should make your own so that you can better evaluate the above procedures (see the jClamp help file under Cm tracking for more on this).