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1. RERE 72 DAX Bigital tosArglogueX Ma ctis ns reta teretes enam Rc benk one Bex a usen b impcu ncn ska 74 E E Ae e ES 75 cre NepemT 91 Impedanceanalysis acoso reo dene ee cheer ner eei etae iesec tese see ere mer 93 Woo coo U 96 Fastlook Video Ta MR M 105 SIII Renee te eres IUE RR M RP A IM RNC E ROME CR NERONE 109 lg mec P ee 117 ANalySi m teaiaae 118 Analyze WNdOW H 118 ise E RR C RE E 129 lal di g Mee S 131 alo e T E 132 eso P M re 135 D ta load WNW EE S 136 ABF Search cee ae eon eee t a eie ee ee ee 137 Episode IAL Mel cae ce sacs rear 138 CC Data Plot 141 Analysis SCP T 143 gli e s 148 GV GII IEW TTE 148 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Hard Te 148 nE TT NN RT RT 149 Control EN TIER Pt ER 151 Vew DA TD 156 lup 159 cT DERE ERN RN Md rc c RE 161 Notes on CM calibration eeeerereennnnnn 165 Detrending for Cm Analysis esses eese 167 I 174 jClamp32 Whol
2. viewed with the CC data pletwindew After version 5 3 0em eal file is appended The dem save file Xem eapaeitance tracking data files that are opened and viewed with the CC data plot windew setter ee ininifile cal capacitance tracking calibration files Text file See Cm Tracking Heen Cell Censor data that is collected when save is enabled in the cell censor window all parameters e g Rs Rm Cm etc are saved These data files that are opened and viewed with the CC data plot window Text or binay set in inifile not these are note files that can be audio video or text If collected during saving of CC data cc then markers are visible in the CC data plot window that allow viewing upon clicking the marker Also accessible from the logger window Otherwise the appropriate viewing window must be opened from the CC window jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Update tips Automatic updates If your computer is connected to the internet j Clamp when run will automatically check for new updates on the web currently at www scisoftco com jclamp html if enabled in the ini file under the Script Tab Installation of new versions is also automatic There may be either of two kinds of updates a full update which requires running a Microsoft MSI installation file if you do it manually then you first run the file to uninstall the last j Clamp version and then run again to install th
3. Click on Player to open video player window 156 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Cree SER RI al C Mastlook dataX03 01 201103 01 2011 122 M Length 1720 frames Ww 640H 480 With Prosiica GE680 S N 02 2020C 06675 d View Live off Capture Player Theron Image Control Cntrst Brite E Rotate Flip Open file and view Press up down buttons to scroll through frames Press play button lt or gt to play backward or forward at the desired entered frame rate If frame are marked during recording when playing a green box will display when frame is selected Shift down on forward or reverse buttons to scroll to marked frames During real time viewing or playing you can do some rudimentary measures such as plot a line or box intensity profile or measure intensity of select points see measure tab in Control Panel 157 GH FastLook Player a Frame 1 Sifas s suo SE C astlook_data 03 01 2011 03 01 2011_12 2 ih Z Length 1720 frames W 640H 480 NEA Levee dass 7 1 960p Eno ps c With Prosica GE680 S N 02 20200 06675 T Eus hh ew 231 FastLook jc no camera attached 0 X Zz 3 1 2011 12 341 Rate 171 748 fps Elapsed time 1419852 ms X 0 41 i Fila Right click and drag to place red green blue yellow and orange areas to measure Double left click on them to remove O
4. Jum units Export data to file during play Refresh List Done You can save data to disk if Export is set on in the Contols window Measure tab You can draw a line or average a few lines by holding shift down and dragging mouse with left button down A plot of the intensity profile can be shown if Line Plot is checked Below is a plot of the sinusoidal voltage blue that drove an LED red made in Excel see below from the text output during a Forward play Whatever is plotted in the Analysis window is output Im Cm Rm Mag Phase etc Capture was a 30 FPS You can see that there seems to be a delay between the two It is not possible to get exact synchrony in Windows with DirectShow cameras This is only possible with triggerable cameras like Prosilica s see FastLook help You may try to better align video and data by using an alignment procedure posthoc in the Analysis window Move the mouse over the LX checkbox and a window pops up First place the cursor to a particular time where a fiduciary frame should be located Enter the frame number to associate with that time and press set Now the frame will be 101 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 1630 200 2037 750 ts Mohm ms Set 25 hoo do sila i bon m N aaa 142 aligned to that time and all other frames appropriately shift in time Check synchronization with an LED driven by voltage stimuli in a protocol You may w
5. Use edit function to add new parameter file names to a rpd directory Double click on an entry field and a file box opens to allow addition of a parameter file script file or CMT file to the RPD directory In the file box window use the extension selector to display each of the three types of files Double click on the filename and it is inserted into the RPD field It is also possible to save and load different RPD directories RPD files are text files and have the extension lt rpd gt the file can also be edited in a text editor DAX Digital to Analog eXtra DAX not available anymore jClamp32 Whole cell voltage and current clamp 25 5 0 Manual oConvert digital outputs to analog outputs with the optional DAX D A eXtra hardware add on Control up to four more amplifiers Monitor seal formation set holding potentials motitors cell characteristics sychronous episodic delivery DAX hardware simply connects to the digital out BNCs The single male BNC connects to ch0 the attached white wire connects to ch1 and the other four BNC assemblies attach to any of the digital out channels ch2 5 A 4 5 5 volt power source central pin positive drives the DAX Output from each channel is 5 volts Cm Analysis Cm Analysis jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 3 Cell Censor vClamp with Axon Digidata 1440A 2216003abf 1 IX CCT Save 403 ms gre E 77085 12 CC inter oo File Cmd h
6. If Frame marker is set a pop up window will appear during recording to allow a button press to mark the frame for future identification in the video player Can be set in ini file 153 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual i Controls FastLook jc n Config Exposure Stats E Calibrate Measure File Info Awi Bimap Line Plot 2 delta length calibrate fi distance Lines to average 10 128 pixels um units Box Plot 7 Check box to view fiduciary line box li tical Enter distance and units of the dssdo line Uncheck box avg box lines horizontally Frame Marker X US 50 audio threshold aoe oe Sub none view 1 fr sec during capture 7 Export data to file during play Refresh List Done g DEN Frame Mark 5 E Capture Player Options Mark it Entrst Brite Rotate Flip fo B Pixel 1 3 1 2011 1 00 46 Rate 67 797 fps Xx 498 Y 0 Mag 53 X0 48 lt Recording You can view the header information of VDA files that are being viewed jClamp32 Whole cell voltage and current clamp 25 5 0 Manual ET Controls a lS x Config Image Acquisition Exposure Stats GigE Main Calibrate Measure Fi H Avi Bitmap date 02 27 2010 79861111 z time S Le 24 owner Fast Look Joe Santos Sacchi DAX 1 frames 15 height 240 7 width 320 3 d
7. Vpulse size of voltage or current pulse delivered to cell Steppable from 40 mV to 40 mV in 5 mV increments or from 10 to 10 nA in 0 1 nA increments A value can be entered directly in the text box as well nAvg number of pulses averaged to determine CC Censor data The number of points multiples of 512 for the stimulus is set in the INI file The maximum number of averages depends on the number of points 25 averages for 512 pt stimulus since the stimulus is delivered as one array and the maximum array is about 16k It is important that the current or voltage response reaches steady state in order for Cell Censor to calculate the proper cell clamp parameters This is especially important when averaging since the stimuli are delivered as a sequential train of pulses CC inter time between data collections With a 90 MHz Pentium and Stealth graphics accelerator data has been collected at 20 Hz with interval set to zero and no averaging Most intervals set in jClamp are estimates since in Windows multitasking will not permit precise time definitions Data collection timing however is exact since DMA is used If you place the cursor over the CC inter text box a popup appears which show the actual interval RPD opens the rapid parameter directory window Pressing insert opens and closes the window as well Vhold or Ihold sets the holding current or voltage for the CC channel or channel 1 Three predefined in the INI file
8. jClamp32 Whole cell voltage and current clamp Version 25 5 0 User Manual Copyright O 2015 SciSoft Co www SciSoftCo com jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Table of Contents jClamp for Windows Introduction 4 OV OR VOW 4 Clamp Features xiii eei otra cca oui udi d ues esas se ner nee EE 4 Getting Started oce eerie a E usan cn iis en eei eamus uM 19 Supported Hardware 1 2 2 2 ttti tan ee dicatas eenn neee 23 Graphics Speed 2 2 22 12 deni coco amabo dada un cere depou qusc dugdus uso cde AE 30 Bu mete c M 33 Daae pee M 35 JC readme Utility T etas EEEE 35 jClamp for Windows Details sss 37 Data Collection mien T 37 15 37 Menu Bcc noue ddr ees mr meter iste Dent eraser mn RR Ico TR otn ten et pn 38 IST CS INS OM eI 41 LO ol NDIES E ee ne ented E E ee ene eee ee ML eee ee eT 46 cse ic m M 48 ic me PROCURE NUESTRAS 48 RZ s l Tele O 49 Cien etc emer eon ete cocum emule e m MEE eue 53 Ee OS EE T TE 54 Command Witility eds coc ate tecto otros e ee eee eee vee ere eee E 54 MultiClamp sc RET s E PO 65 Rapid Parameter Directory de
9. jClamp32 Whole cell voltage and current clamp 25 5 0 Manual cm trk clock 10 set clock cm trk flmult 2 cm trk f2mult 2 cm trk time 3 amount of time to record cm trk gain 1 hardware gain select cm trk analog false true cm trk digital true false cm trk digi 1101 on 1 off 0 digital out for digital out 0 3 cm_trk_dur 500 duration of voltage stimulus em trk durinc 50 increment in duration every repeat time cm trk vstart 150 start voltage of step command stimulus if ramp then start voltage cm trk vinc 100 increment of voltage stimulus every repeat id step if ramp end voltage of ramp cm trk stim ramp pulse choose type of command stimulus cm trk repeat true false repeat stimulus cm trk repeat time set cm tracking repetition time in seconds hold step true initiate hold stepping in Cell Censor hold step video true false enable for saving video clip at end of each step hold step trace true false enable for saving CC trace clip at end of each step comm 1 2 3 4 choose serial out channel serial command any commands in ascii supply command send serial true send it now script data directory path eg C script_data data files will be saved as first five leters of directory and filenumber scrip001 abf scrip002 abf in that directory kill files from filename list of files to delete uses script data directory that was previously set kill file lt filename gt one file to delete must state
10. EAC J Program Files x86 Viclamp32 Param Search Path breakdown jesper Episodic w iotech v 22 8 Drive free 150 189 Mb iClamp file prm Y amp J c Windows8_05 Switch from ms to pts by clicking on pts ms Power of 2 up down button shows necessary fro chirp amp Parameter File w axdd144x v 22 Command Utility vClamp with model c NjcdataV515112 none jclamp 9 30 11 Author J Santos S acchi private comment password Analog Chan s YC clamp Out In sci Dea nia 02 073 nChan Sync 0 EE N 1 Partition Design gt abf template z h RARA Properties Partition 1 Copy 1 1 Start 30 CIR EM MD nels eCm Chirp Time 102 ac stair m ramp abf step user Pre P N Digi Axon 700 mode D3 Dp ix TT chi a Pre B Epi Points K us Hold Number E enira s x4 4 sys ho 0 mV 1 is M5 seen dd In Analysis mode place mouse over command out selection and the holding level set in the CC window just prior to data collection will be shown Dd X password Analog Chan YC cli Out In 0 C1 CC Hold Chan 0 was 80 mVan D ync 4 m N Axon 700 mode jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Command design is similar to Axon Instrument s pClamp However the jClamp utility is more powerful and user friendly A user who knows pClamp will find that many build options ar
11. contains a list of notes one per line that will automatically load into the Quick Note window Selecting an item by mouse double click or by pressing lt enter gt will load the text into the log see log window below If the item has the extension lt txt gt it is treated as a text file and all text in that file is loaded into the log For example at the jClamp32 Whole cell voltage and current clamp 25 5 0 Manual beginning of the day you could insert the methods solutions etc into the log by double clicking on the appropriate text file item At the bottom of the window is a text entry field where you can write any thing and by pressing the command button insert the text into the log QuickNoteFile D jClamp source 2001 quicknotes intra_solutions txt E formula for intracellular solution MagCl2 2 mM EGTA 10 mM KCI 140 mM HEPES 10 mM pH 7 2 300 mOsm Time 10 57 23 41 QuickNote CIB soultion started Time 10 57 30 18 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Lat gt cel 4 x Time ts y Em pF Qm 0 254 Cm 30 39 0 264 0 18 6 20 9 21 12 23 15 24 18 25 21 26 24 27 30 29 B Hour 09 54 24 X 26 422 Y 30 602 Time s There is a new stopwatch feature that can be accessed by clicking on the time display in the lower left of the jClamp main window More than one stop watch can be opened and remain floating on top The executable stopwatch exe
12. feature of jClamp is enabled 170 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 456 The response is in accord with the set model parameters I should note that the generation of currents by the model is independent of the analysis procedure so it is unlikely that the effect of ramp rate is due to model evaluation with the difference equation technique Furthermore the clock was sufficiently fast and the same for all ramps 10 us I have looked at real data and the effects of ramp speed are not so obvious as in model data but to play it safe I implemented detrending several years ago in jClamp Recently jClamp V 10 5 I have exposed detrending to the user so that you may enable or disable it It is probably a good approach for analysis of data obtained with stimuli of rapid rates but I have found that it may introduce some noise into the measurements Below I have added noise to the current response prior to analysis If detrend is used then the noise depends on the magnitude of the capacitance however if detrend is off then the noise is small and uniform in the face of capacitance increase 171 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual With detrending DtR check box checked 172 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual No detrending DtR checkbox unchecked You can now decide whether to use detrending If the underlying stimulus rate is fas
13. if enabled for use in the Data load window So lt abf gt data files are saved raw and analyzed each time the file is opened This means that for lt abf gt 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 from the equation so you can accurately gauge the cell s physiological response Let s 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 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual resolutions at the given clock rate that can be used to remove the effect of the equipment and thus measure the cell s impedance correctly In order to validly use the correction factors you have to make sure that after the formation of a gigohm seal
14. in the jClamp32 directory is shelled from jClamp or can be run outside jClamp Toggle between seconds only or minutes and seconds by single clicking on any dark part of the watch Set time in minutes and or seconds in text box Left button counts up right button counts down to zero and beeps mddle button pauses Double clicking on the watch closes the watch Mouse down on the dark part and drag to move Seal Seal jClamp32 Whole cell voltage and current clamp 25 5 0 Manual me Seal Ea ope ese HIG Pel R T Close Rin 11 438GQ HzeMa 10 Hz dl P Zap Auto L 20 Ga Displays input resistance during seal formation Zap is a parameter file which can be modified by the user The scroll bar dynamically increases or decreases the amplitude of the signal Pressing the cartoon button delivers the zap stimulus which hopefully will establish whole cell configuration If not suck One zap can be delivered automatically when a given seal value is reached if auto is enabled Settings in the ini file Display channel is selectable Enabling audio produces a repeating beep at a particular frequency set with the Hz up dn arrows The change in sound frequency Hz Mohm is set in the text box If you set thigs right you can hear a difference when the electrode touches the cell Upon reaching 1Gohm a sound can be played The file must be named lt giga wav gt a Microsoft wav file placed in the jclamp32 directory lt sounds gt
15. method a single sinusoidal voltage or current stimulus is delivered and the parameter values Rs Rm Cm of the patch cell model shown above are estimated This method of Cm estimation is based on evaluation of the changes of the capacitive current component of IRs and arose from the initial work of Marty and Neher 1981 who tracked current at a phase angle that approximated the angle of OY OCm This method was subsequently modified by Joshi and Fernandez 1988 to use the angle of OY ORs See their paper for the reasoning behind this choice The method I employ here I devised by careful inspection of the phase tracking techniques above and determination that the equivalent results are obtained by evaluating the imaginary component of Ir B Cm is estimated by using the gain factor H of 1 OY OCm which is approximated Gillis 1995 by H 1 tan 90 Y where Y is the admittance at the angular frequency o Cm BH A resistive measure Rs is estimated from the real component of Y namely A and scaled with abs Y 2 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Unlike typical phase tracking paradigms a point by point analysis is performed However since the gain is approximated the Cm estimate suffers from interference from resistive changes in the model This interference is alleviated by two additional methods that I have devised the dual sine and eCm methods listed below Phase Sensitive Det
16. subfolder In the lt sounds gt directory there is a file lt no_sound wav gt that can be copied and renamed to lt giga wav gt so that no sound is played jClamp now has a stand alone version of lt jc_audio exe gt which can be used to record or play wav audio files since Windows Vista does not have a sound recorder that does this It is accessible in the jClamp directory or the jClamp start menu The time resolution of plot updates is the same as the History plot which is set in the ini file default 1 Hz Telegraph Telegraph jClamp32 Whole cell voltage and current clamp 25 5 0 Manual D Cell Censor vClamp with model IOTech board selected SIDi1034abf 19 Vhold mV cam 2 IX CCl Save 11ms Passhold S Coir orc Clamp Type ix vg x vi ix v2 ix v3 40 H EN B X wc x wel X Model b X TeleShow Dis dual Mh Trk a T k 5 ET Dual Singe Fes 256 C etm C MD n 390 6 E m ffset mv 2 ne D on Telegraph under the Cell Censor window telegraph information is shown in a separate window if the TeleShow box is checked Only those channels enabled in the ini file are shown that is they must be enabled in the ini file Up to four channels are available for monitoring of Channels 0 0 and up to four channels are available for monitoring of Channels 1 1 Same for chans 2 2 and 3 3 The channel numbers and functi
17. windows system You must disable User Account Control in Vista and Windows 7 In Windows Vista you may need to actually unregister some files that j Clamp installs This is a very strange bug for Vista A batch file named vista unreg bat will be in the jclamp directory and you should only need to double click on it You may look at the contents with a text editor like notepad If you are using an old Labmaster DMA board then jClamp is useable without a license Remember the free license is only available when the ini file is set to use the Labmaster board under gain tab Set the proper Port address The Labmaster board is controlled exclusively through Port I O with the jClamp VXD or SYS driver The only differences with the hardware setup required by pClamp is that the ports A0 A7 should all be configured for output A7 is scope trigger See the hardware manual BO is trigger input C channels are not used Cm tracking and gap free are functional only in Win 95 98 although 2 sine command protocols are supported Win 2000 performance is superior Normally if there is no hardware license the registration window will appear indicating that the license is software only or that so many days are left in the hardware fulldemo license For the free labmaster support there is no license so the register window will open If you want to bypass that window you may create a shortcut on the Windows desktop to jclamp32 exe as you would do for any other
18. with Axon Digidata 1440A none 10503 Vhold mV clk us 10 z N amp X CCl Save Pass hold S e ET k Nowe N fe 0 D 1 C 6 6 40 IET base o oT CCinter Disable File Cmdhid Chan Watch tau ms Vpul mV Zero R Fel R 0 1805 8530 en B Rm Ma YSR 311 48 s Cm pF nter ms 17 076 0 Ih n r 0 121 Offset mV Vm mV on 3 4 40 mS Freq 2 n 195 Hz Io 4 5813 n Cm pF vom ica acu a RE 512 EM Calib f Dither on Time Dual Single Pe es v i45 SES Y Freq C etm MD f1 1953 F 1 v m fc 100kHz d wj Hz Cm G n 17 09015 f2 17 08204 f3 17 09669 f4 17 17024 t5 17 72771 15 Capacitance Compensation Do rat guess fit j0 pF Rcs 10 Cs1 Res jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Cell Censor vClamp with Axon Digidata 1440A none 10537 Vhold mV C10 z clk us IN C S S Passhold Passi amp S L Ams E 80 X Auto fe i i e 40 9S L3 fo of 40 a 0 CCinter Disable File e E Cmdhid Chan Watch tau ms Vpul mV Zero ie Fix B 0 1803 z Rs M1 10 9 Rm MQ 311 70 Cm pF 17 157 th n r 0 121 Vm mV 3 4 21 7882 n Cm pF 40 v Freq 2 n 195 Hz CC Domain Freq options Fi2 3 512 wjrtM Calib 7 Dith a f Time Dual Single Hes 5 12 H G Zp KS ither on Y Freq C etm MD f1 1953 F 1 j mm fc 100kHz dl Haji Cm f
19. 0 Manual S oea User Files Gain ro T1 T2 T3 CC Hist CC Script 7 Gain D A A D Scroll then select unit How Scroll then select unit How many volts from the DAC many volts from the amplifier equals 1 unit at the cell equals one unit fom the cell Vclamp Vclamp Cho 005 Cho Chi 005 Ch1 EM Ch2 Ch3 Ch3 Iclamp Iclamp Cho Cho oo ESAE Ch2 Ch 3 th2 i E Ch3 fi EC Ch1 Chi 70 0 C m m Extra Gain and 4 D units for ChO 3 check to use in lieu of above cho ji funit Cha ho wt thi i ut chs h funit C cha fh dut che Mo funit Rn Jw Ch dunt audi 550 Com Scl Factor 2 1 12 15 12 14 36 p The configuration settings must be set correctly for each AD board CED boards The Power 1401 from CED 16bit in out works in WIndows 95 98 NT 2000 XP Pro Follow the setup described by CED Digital in 0 Event Input 0 is used for triggering pulse to low for trigger Trigger BNC is not used Digital out 0 hi byte 0 on front panel is scope trigger Other digital outs are low byte on expansion connector Fastest clock supported is 3 usec for synchronous in out CED command files must be placed in the jclamp32 CED directory They are ADCMEM MEMDAC and MEMDAD Analog output on channel 2 and 3 is controlled with DAX window CC monitoring for a 3rd and 4th jClamp32 Whole cell voltage and current clamp 25 5 0 Manual amplifier uses channels 2 2 and 3 3 in out
20. 1 etc If you select a channel not supported by your board then a pop up will indicate that Alternatively you can simultaneously synchronize multichannel collections with the Sync drop down box Selecting 0 will set up a single output input channel 0 0 Selecting 1 will setup a dual output input collection 0 0 1 1 And so on Each parameter file contains information for separate and distinct command waveforms for each output channel The number of input channels is limited to eight 0 7 Mouse movement over the InChan area opens a box indicating the number of channels selected their associated programmable input and the physical channel associated with the software channels Double clicking on the channel number in red increments jClamp32 Whole cell voltage and current clamp 25 5 0 Manual through the available channels 0 7 Physical channels are sampled in descending order 1 e in the software channel order Double clicking on the gain number for each channel in blue increments through the available gain settings 1 2 4 8 In addition the auto check box allows the users to obtain input at an optimal gain setting for all channels This works by setting the gain to maximum 8 initially Each episode is run and the data is checked for clipping If the data is clipped then the gain is lowered to the next gain setting and the episode is repeated This continues until the data are unclipped or the gain is unity This process will maximize
21. 11878 14847 167 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 8908 11878 14847 The response from dual sine stimulation is analyzed with FFT to obtain the real and imaginary components at each stimulus frequency These data are used to determine the parameters of a simple model of the recorded cell a series resistance in series with a parallel combination of a membrane resistance and membrane capacitance The equations are from Pusch and Neher 1988 I outlined the application of their equations to the novel 2 sine technique in one of my grant proposals in 1992 which is photocopied in the appendix below along with the final progress report of the grant It makes for interesting reading Single sine stimulation can also be used to measure membrane capacitance with the same cell model See jClamp s help file for details Each technique requires that you obtain a calibration file that is used to correct for the frequency response of the patch clamp system electrode amplifier filters Correcting for the system response is important since the parameter solutions are model dependent and assume that the system response is flat It is the job of the calibration file to flatten the unflat system response It is important for correct measurement that the electrode s stray capacitance which is not included in the model is balanced out during pipette membrane seal formation Another important step to take for proper paramet
22. 3 Exit Cancel Copy to folder New Folder lees Open ABF Search engine window from the Data load window Ability to search ABF files in multiple subdirectories for particular characteristics of the command protocol or clamp characteristics Shift right click on list to copy to clipboard e g to paste into Excel Copy all listed files to a new directory to do group file analysis Double click on a file to plot data in Analysis window Episode Math Episode Math 138 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 139 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Bran TR gota YT 149971 10 abt vom Amd mis eR ai uas a LEE LL l s dee r3 1 da xm 140 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Bn rae actas Vs TEC aM Ivano rom MW Aca a a es Za m L Ep tS 1 280 mt Y 7 709 0A Fat 7 Commang 29 000 mv Re anf 1 Ed CC Data Plot CC Data Plot 141 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Plot 1 x Du L fect 2 ch gt Tine ardi S le F a Fil Um lt 0283 lh O69 Cm 3535 Vm Moi E ua 14 44 281 41 7 55 4 58 1 82 8 96 5 110 1 123 8 Hour 01 23 12 X 5347 Y 35 022 Time s Display channel is selectable Choose Cell number and CC x and y data to plot If parameter data files and comments were collected w
23. 5 0 Manual Script File Script file Commands jjclamp scp semicolon precedes a comment p n true false p n leakage subtraction enable automatic file naming and saving is toggled on off in Cell Censor Window or thru ini file script filename call a script filename scp don t use extension from within a script cc save true false save CC data to a file auto file name true false enable auto filenameing auto file save true false normally set in ini enable disable automatic file saving if disabled user will be queried to save avg on true false enable averagi clk 1 20 set command protocolclock in microseconds play sound filename play a sound file filename wav don t use extension from within a script this is a Microsoft wav file that can be made with MS sound recorder and must be placed in the jclamp lt sounds gt subfolder jClamp now has a stand alone version of lt jc_audio exe gt which can 109 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual be used to record or play wav audio files since Windows Vista does not have a sound recorder that does this It is accessible in the jClamp directory or the jClamp start menu pass hold true false enable disable passing of CC hold to next parameter file that is run pass clk true false enable disable passing of CC clock to next parameter file that is run disable file true false enable disable file save box in CC to enable disable automat
24. 562 90 402 90 402 4 52 4 52 28 87247 19 1 47 595 57 346 57 346 2 868 2 868 29 71508 20 1 48 628 9 223 9 223 0 462 0 462 28 4931 21 T 49 661 41 323 41 323 2 065 2 065 14 39729 22 1 50 695 81 923 81 923 4 096 4 096 0 415068 23 1 51 728 99 574 99 574 4 98 4 98 0 332444 24 1 52 761 91 063 91 063 4 554 4 554 0 371365 25 d 53 794 58 626 58 626 2 932 2 932 0 260019 26 I 54 827 10 786 10 786 0 542 0 542 0 239274 27 1 55 860 39 888 39 888 1 993 1 993 0 262699 28 1 56 894 81 012 81 012 4 049 4 049 0 187586 29 T F 927 99 417 99 417 4 969 4 969 17 64089 30 1 58 960 91 7 91 7 4 585 4 585 29 01914 31 1 59 993 59 891 59 891 2 994 2 994 29 83155 32 1 60 1026 12 346 12 346 0 617 0 617 28 71466 33 1 61 1059 38 442 38 442 1 922 1 922 14 24534 34 J 62 1093 80 081 80 081 4 004 4 004 0 408446 35 1 63 1126 99 235 99 235 4 962 4 962 0 335673 36 1 64 1159 92 316 92 316 4 618 4 618 0 304436 37 1 65 1192 61 142 61 142 3 059 3 059 0 312708 22 1 RR 1995 12 QAN 12 QAN N 607 n AQA7 N 72n22 as ds jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 150 100 JA HL 0 TT TTTT TTTTT TTT TT u V mV a 4 16 1 MS 28 3 7 404 49 52 5 58 6d4 64 R 100 150 Look32x Ini File these data can be set upon starting Look32X TOOKS sc abii sem
25. Amplifier number 1 with 10 kHz filter collected with lt accalib prm gt set to 5 usec clock Amp2_10us_10kHz cal Amplifier number 2 with 10 kHz filter collected with lt accalib prm gt set to 10 usec clock Ampl 10us 3kHz cal Amplifier number 1 with 3 kHz filter collected with lt accalib prm gt set to 10 usec clock For calibration if you want to make altereations say to voltage the altered file lt accalib prm gt or lt accalibi prm gt must be saved in order to use the changes 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 may 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 fi e 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 on startup 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
26. Axon no longer supports with their new software See supported hardware for more information Support for the IOtech PersonalDaq It is a great board 1 400 with many capabilities close to the Axon 1440 series Detached Telegraph window with only the enabled channels from 1 4 for each of the jClamp32 Whole cell voltage and current clamp 25 5 0 Manual two supported amplifiers displayed Vista and Win7 support Detached PDF Help file in addtion to Windows Help file You can search and print out the help file Separate Audio program jc audio exe for recording playing MS wav audio files which can be played during script execution now since Windows Vista does not include one Multiple instances of jClamp can be run each controlling a different A D D A board Or use another instance to analyze data while the first instance is actively collecting data Frequency analysis can successfully monitor bilayer prep capacitance making jClamp a good choice for these types of studies Cell Censor window provides full cell parameter analysis either in Time or Frequency domain Axon Multiclamp telegraph support User selectable channel input for telegraphs Real time scalable plotting of Cm or Im vs time or voltage collected with protocols Support for Axon 1440 series Support for National Instruments M series boards NI 6251 USB and NI PCI 6221 in particular NI boards that use Ni daqmx driver M series and also the
27. C Time C Dual Single Her 5 12 f peg Cem Y MD f 1953 EI Car f1 17 Anak F2 17 A30 F2 17 21 To switch between VC and IC in CC when in ext mode in multiclamp select VO or 10 Channel 0 amplifier Rapid Parameter Directory Rapid Parameter Directory jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Allows instant data collection using the selected parameter file or script file Can be used during either Time or Frequency domain monitoring of cell parameters in the Cell Censor window Parameter file text appears in black Script file text appears in red Press the key or click the mouse Pressing Ins key opens and closes the window The keys F1 thru F9 can also be pressed to initiate data collection without the RPD window being open Keys 1 thru 9 must have the RPD window open Data are saved automatically if auto save and auto filename are selected After the data collection is complete the holding potential is maintained at the final potential in the protocol usually the command holding potential however if CC Censor is enabled the holding potential is set to the monitoring holding potential Script file execution can be cancelled using the mouse to click on the cancel button or by pressing the lt esc gt key A sample script file is illustrated below in the Script section all commands are shown jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Maintain View RPD File Aut U Santos Sacchi
28. Phase tracking Very detailed information for all files in data directory in data load window when info button is pressed IV plot window calculates reversal potential and slope resistance multiple I v plots for comparison Quick notes window allows button click insertion of pre assembled often used notes into the log file Conditional scripting with parameters from Cell Censor For example wait for membrane resistance to reach a certain value before running a protocol DCM files are only saved when enabled in ini file under History tab CED Power 1401 support Four DAC outputs control four patch clamp amplifiers Channel watch channels moved from 2 5 to 4 7 for all boards Shell out from Data Load window to Adobe Acrobat or reader to look at lab notebooks I suggest that you scan in your hand written notebooks as an image and place a PDF conversion into each day s data directory for viewing NT and 2000 support for Labmaster DMA and Daqp 308 boards j Clamp for the old Labmaster DMA board is FREE No license needed Great for student labs Break out those old boards and put them in some new fast and cheap computers Multiple stopwatches Streaming video capture and playback record video during data collections control through script or via Look video window Screen shots available from scripts CM calibration changed to provide more resolutions accalib prm and accalibi prm are revised Please recalib
29. Synchronous digital out issupported Other models may be supported Axon A D D A boards The 1200A 12 bit in out and 1320 1440 series 16 bit in out boards are useable in WIndows 95 98 NT4 2000 XP Pro Follow the setup described by Axon For both boards digital in 0 is used for triggering pulse to high for trigger Start trigger is not used because it is not directly accessible to third party software except for the 1440 series in this case start BNC is used to collect data Digital out 7 is scope trigger out The 1200 Axon boards are configured by supplied Axon software e g Axoscope and the axdd1200 ini file located in the Windows Win 95 amp Win 98 directory is used by jClamp If there are problems check the hardware address interrupts and DMA channels in Axoscope Get that to work The 1200 board may be functional only in Win 95 98 Unlike the other boards the 1320 series boards treat the synchronous digital output as another channel therefore when using these boards the clock will change in the command utility when synchronous digital output is enabled The 1440 series supports synchronous digital output and multiplexing is not used so clock remains the same regardless of input channel number The DD1550 board is supported but sets clock increment to 2 us because that is required by Molecular Devices There is still a jitter that I detect that MD cannot recapitulate I think it is present not just in jClamp but also in C
30. Whole cell voltage and current clamp 25 5 0 Manual AxDD1400 dll wdapil 140 dll wdapi1011 dll 1550 boards AxDD1550 dll DD1550fpga bin wdapi1140 dll wdapi1011 dll You may want to copy all regardless of board you have The the required files may change so email me for information The software version of the Axon Multiclamp Telegraph system is supported see telegraphs Labmaster and Axon 1200 boards jClamp for the Labmaster DMA and for the Axon 1200A and B boards is free no license required However it is not sure whether they will work with the newest versions of jClamp since I don t have the boards anymore to check The Labmaster 12 bit in out must have the Port address set correctly in the jClamp ini file Remember the free license is only available when the ini file is set to use the Labmaster board or axdd1200 board under gain tab Set the proper address for the Labmaster The Labmaster board 1s controlled exclusively through Port I O witht the jClamp VXD or SYS driver The only differences with the hardware setup required by pClamp is that the ports A0 A7 should all be configured for output A7 is scope trigger See the hardware manusal BO is trigger input C channels are not used Cm tracking and gap free are functional only in Win 95 98 althought 2 sine command protocols are supported Win 2000 XP Pro performance is superior The Axon 1200 board needs the DriverLinx installation NT version for Win2000 an
31. are written to the directory where the data file resides Series resistance correction of voltage is done when the Rs checkbox is checked The value of the resistance is entered into the pop up text field Additionally the traces can be filtered 142 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Plot 1 36 77 x Dut L cet 11 Ch x Time s On y Eme gt Plots 36 17 35 87 35 57 35 26 34 96 34 36 14 4 281 41 7 55 4 691 82 8 96 5 110 1 123 8 Hour 01 23 12 x 53 47 Y 35 022 Time s Plot 2 X Dut L cell 16 lt UTE On y Cm oF lt Ca Fix Scales 30 52 7 5 97 11 8 142 164 187 20 9 Hour 01 22 35 X 16543 Y 36 221 Time s Analysis Script Analysis Script Commands should be placed in a text file with extension lt ana gt Then place the file name in the User Analysis directory e g my analysis directory uad which is set in the inifile No spaces allowed except in the text of a note wait 1000 ms beep 1 2 3 note text as in script analysis do as in script loop 143 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual choose files true opens window to choose files to analyze all commands which follow apply to each file filename filename choose specific file from script do iv avg peak tpeak integ rms as in episode math episode all 75 show all episodes start_point show
32. both 16 bit in out PCI MIO 16E 1 1 usec support 12 bit in out and PCI MIO 16E 4 4 usec support 12 bit in out should be set up with version 6 7 of NI DAQ which comes with the boards Inputs should be single ended Triggering is done on PF10 TRIG 1 Scope trigger uses digital out 7 Both are setup to have an ADC range of 10 V Programmable gain is available 1 2 4 10 for PCI 6052E and PCI MIO 16E 1 and 1 2 5 10 for PCI MIO 16XE 10 The PCI 6035E 16 bit in 12 bit out and PCI 6024E 12 bit in out are boards that have 5 us analog in but only 100 us analog out They are supported but they are functionally limited Support is for 100 us in and out so that synchronous in out can be performed Gain is limited to 1 or 2 with 20 supported in CC manual gain mode The slow speed limits CC parameter estimation since only slow time constant current responses can be fit accurately The M series NI 6251 USB is supported 16 bit in out with synchronous digital output Clocks down jClamp32 Whole cell voltage and current clamp 25 5 0 Manual to 1 usec for a single channel The NI PCI 6221 board is also supported with synch digital and clock to 4 usec Programmable gain option is employed with both These boards will be the preferred boards for jClamp starting 6 2007 The NI PCI 6221 board is used by Neobiosystems to control their PatchMA X 100Aand jClamp will work with their system to provide user friendly patch clamp and two electrode voltage
33. can be subsequently run in the script could be an Iclamp parameter file that has its holding current passed to it from CC if passing hold check box is enabled and increments test pulses upon that holding current screen shot name name is a screen shot file name scn used without the extension note This a test This note can be used to indicate actions which the user needs to make before continuing with Script execution A note to the user will pop up and the program is suspended until the okay button is pressed run new1 run a parameter file jclamp_beep 1 2 3 make sound N number of times do begin ado loop all commands within the loop will be repeated loop N times loop 2 end a do loop While a loop is running hitting the L key will cancel the loop and continue with the following script commands More than one loop can be placed in a script Variables can now be embedded in a do loop The variables are vara varb varf and can be used to represent any number in a script Before the loop the starting variable value e g vara0 should be defined vara0 5 and the increment varainc should be defined upon encountering vara within the loop vara will become by a multiple of the loop number If the variable e g vara0 or varainc is set to a question mark lt gt then the user will be prompted during the script for an input value For example 112 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual va
34. can load the scale for the image if already set in FastLook by running fl set scale ps al FastLook jc Prosilica GE680 S N 02 2020C 06588 Ext View Live on Capture Controls About Frame ait C m select vda file vda_to_avi 30 fps vda to Image 159 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 4 Image eis 23 File Edit Image Process Analyze Plugins Window Help dB cale RIN A SOIE SI 0 415 gt Scrolling tool or press space bar and drag a L d 02 27 2010 1128 24 vda Les te jes Shortcut to 1 15 320x240 pixels 16 bit 2 2MB jclamp32 exe el Image now n New Folder 4 160 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual H Image File Edit Image Process Analyze Window Help B ojejo RN AIS wars Shortcuts Utilities no ten eclipse Shortcut to Compile and Run jclamp32 exe Magnifying glass or use and keys 3D Analyze Examples Filters Graphics ra 9 Biorad Reader Macros DM3 Reader Scripts gt Deltavision Opener n stacks LICUIT redta Tools FastLook loader IPLab Reader K LSM Reader ZVI Reader stretch FastLook Ini file The ini file sets startup conditions for FastLook These can be changed in a text editor such as Notepad Here is the default fastlook ini file content Commands set checkboxes in the Controls form fastlook ini semicolon is a comment in jClamp these ini comm
35. cell characteristics Analyze opens a window to permit some analyses Communications GPIB under construction and serial communications are available to control peripherals Control via scripts is possible Clipbrd opens the Windows clipboard to view data which has been captured for pasting into other programs Quick Shell Move mouse over free space indicator to open quick shell Add useful program links to run without leaving jClamp See the quick shell txt file in the jClamp directory jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Quick Shell EJ A list of programs to execute located in quick shell txt Links ass TE 12 40 AM Cell Censor Cell Censor D Cell Censor vClamp with model Labmaster DMA selected SS CE 1004003 ab 16 Vhold mV Ca cik us i JE E CCl Save 21 mq Pass hold S a mm Pass Clamp Type gt ix v0 x 1 ix V2 x v3 i wien t 12 T 13 ul mV x weOiX wel 49 g x we2 x wc3 x Model J E b T TeleShow i Dis dual e ter ms 12 095 Jo Ih nA NN 0 065 Offset mT Vm nm To cen Oo JE NN CC Domain Freq options Fi2 0 256 y CM Calib LR Dith r Time Dual Single Res 2565 E a er on ES m C Freq eCm C MD 5 396 EI gt mmi c 100K oE Thetop left comer of the window shows a graphic in this case a photograph of a spiral ganglion cell T
36. clamp solutions When controlling their system you must switch back to math model in jClamp accessable in the pop up window when the mouse goes over the user graphic in CC After cell encounters press their release button and turn off math model in jclamp to start clamping Digital out 1 2 and 3 are used exclusively by their system and are set high and disabled in jClamp Digital 0 is available for synchronous output with analog out Out 4 5 and 6 are useable for non shynchronous use 7 is scope output trigger and digital in 6 is for episode or start triggering To enable use with their system you must check the NeoBiosystems box under the History tab in the jClamp ini file In jClamp ini editor select lt nidaq gt for E series boards and lt nidaq_mx gt for M series boards The PCI 6052E can also be used with the lt nidaq_mx gt drivers Support for new board NI USB X Series Amazing 1 megaHz 16 bit sampling on all 8 channels without multiplexing Synchronous digital output About 3K Could not ask for more The new NI DAQmx 9 3 or higher drivers need to be installed Trigger to start is PFI6 Visit NI at http joule ni com nidu cds view p 1id 2337 lang en Synchronous digital out with analog is not supported by the E series boards so DAX is not supported although jClamp can provide non synchronous digital out Board number eg 1 obtained from NI Explorer NiDaq 6 7 drivers for E series NiDaq mx ver 8 5 for M series
37. directory that was previously set params to fit filename parameters for fit passed to matlab environment uses script data directory that was previously set rs cor True false turn on off rs correction rs val override value replace rs value to use for rs correction script data directory path eg Cscript data data files will be saved as first five leters of directory and filenumber scrip001 abf scrip002 abf in that directory 147 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual FastLook overview SciSoft s FastLook Copyright 2010 d Santos Sacchi Loading Ini file Hardware Fa Fad D SS lL Ea Supported Hardware Prosilica cameras www prosilica com that are supported by their SDK are required Andor iXon X3 860 EMCCD Camera 148 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual XXX s i 200 300 400 500 600 700 800 900 1000 Wavelength nm The cameras are interfaced with jClamp patch clamp program to externally trigger the camera using a variety of A D D A boards See jClamp website www scisofico com for more information main FastLook Main Window 149 EZ FastLook jc Prosilica GE680 S N 02 2020C 06588 fe Exit View Live on Capture Player Options l Image Control Cntrst Brite hl Rotation 1 fo d Pixel X 8 13 2010 1 18 06 p Free Memory 1 X 451 Y 7 Mag 1781 X055 l
38. from this point end_point end at this point epi_avg true commands below as in episode math epi rcfilter Hz epi gaussfilter Hz epi offset epi sub episode number epi int true epi diff true epi _zero epi_set_pts epi_mult epi_add iv_print true print the i v plot trace_print true print the analysis plot export asc mat abf export file files are put in subdirectory export cursor1 set cursors cursor2 cursor3 cursor4 cursors cursor6 144 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual partition highlight given partition with cursors at start and stop sp do transform filename xfm run transform in SigmaPlot transforms must be in the Transforms subdirectory of SigmaPlot ml do command matlab command run MATLAB command org do command Origin LabTalk command run Origin command raw data leakage subtraction sub trace episode to use as scaled subtraction trace volt_partition partition number for voltage range leak sub part partition number to be leak subtracted use lt h gt for hold region automation export to external applications set SigmaPlot notebook template else selection window opens if set to auto then new notebooks are created templates located in sigmaplot subdirectory of jclamp32 sp template templatename jnt export data cc matlab sigmaplot origin or excel export data abf matlab sigmaplot origin or excel export data iv
39. gt 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 fi e 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 lt abf gt data files are saved raw and analyzed each time the file is opened but lt Cm gt files recorded in the Cell Censor Cm track section cannot be reanalyzed This means that for lt abf gt files different calibration files can be used to analyze
40. in the IV window by clicking on the parameter column Fitted parameters original traces and fits can also be exported in tab delimited ASCII format for importing into spreadsheets Shift click on Fit button will automatically copy data to clipboard for pasting If the Man button is checked then you may choose the filename to save as or accept a default If auto is checked then all subsequent fits are saved to the default filenames based on data filename If group is selected then all data is save into a common group filename If param is selected then only the parameters are saved Sdev is the SD between real and fitted traces The user equations are placed in the user fits directory under the jClamp32 directory Use the open and save buttons to access the files You can also simply write the equation in an ASCII editor and place in the user fits directory Each equation is housed in an ASCII file with the extension fit An example of a double exponential fit is given below The format must be followed The word xdata represents the x values in mV or ms and the word Yfit must finally equate all previous equations A semicolon must end all lines and the headings equations and initial must be used Most math functions are available They are ABS ACOS ARCCOSINE ACOSH ATN ATANH SIN SINH COS COSINE COSH TAN TANH EXP LOG SQR LOG10 Make sure that non parameter names are unique since they will be used to place their equivalents
41. 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 Ampl 10us l10kHz cal Amplifier number 1 with 10 kHz filter collected with lt accalib prm gt set to 10 usec clock Ampl 5us IO0kHz cal Amplifier number 1 with 10 kHz filter collected with lt accalib prm gt set to 5 usec clock 165 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Amp2_10us_10kHz cal Amplifier number 2 with 10 kHz filter collected with lt accalib prm gt set to 10 usec clock Ampl 10us 3kHz cal Amplifier number 1 with 3 kHz filter collected with lt accalib prm
42. jClamp32 Whole cell voltage and current clamp 25 5 0 Manual charge transfer Below I stimulate with ramps with the two sine technique of differing rates using jClamp s incrementing time feature Parameter File c Njcdata M5 0320653206033 abf S cm ramp 2 6 03 Partition Design gt abf template private K T si Boe comment x password Properties Partition 2 Start 150 Cm 1 2 sine Analog Channels p Digi 5 Inc s rome C S ut ln TF Pr ps c abf P Tw i En PES ms c us C user 1 N 1 R s Part Op T fx T Pre T PAN Averages p gt Episodes f Points Cik us Hold um a 512 xp 42 9 1fioo Cfo mv 2 o xf T Dh rr rH La rep Ve Command Pat 2 Epr 8 x 432 Below is the capacitance plot derived from the incrementing ramp stimulations 169 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual In the analysis window I plot all episodes It is clear that the magnitude of the peaks of capacitance depend on ramp rate the faster the ramp rate the larger the peak even though the model parameters are the same for each ramp data collection The I V plot window clearly shows the relationship Several years ago I implemented detrending in jClamp because of this potential problem If you use only step stimuli then these is no rate problem except at onset Below is the response when the detrending
43. level Additionally toggleable timing increments by a factor of 2 Automatic saving of individual runs during averaging Protocols can be run at the press of a key or click of a button External file based user stimulus incorporation Arbitrary number of input channels and order of input channels in parameter files Analog output is through channel 0 or channel 1 or both simultaneously Each parameter file contains information for separate and distinct command waveforms for each output channel Random episode delivery auto supplemental A D gain 1 8X Axon 1200 DAQP 308 and daq2000 boards dual telegraph inputs to control gain and clamp type and monitor capacitance and filter frequency Digital output during before after or synchronous depends on board with analog output TTL or key triggerable starts full run or individual episodes Text audio and video time lapse grabs or linked to data collections after each average or episode clips can be saved and easily retrieved and played during analysis Any Windows compatible cards with wav audio drivers and video for Windows drivers will work Script files can run individual protocols or loops can be programmed wait periods are allowed between protocols and during these periods cell and clamp characteristics are continuously monitored and saved to disk Control holding potentials main board and DAX output serial port commands e g to control external hardware GPIB under con
44. levels can be instantly set by checking the corresponding selector For example before establishing whole cell mode an appropriate holding potential can be set The scroll bar can also be used to set the potential or a value can be entered directed by overwriting the indicated potential blue numbers The holding potential is set for the selected channel 0 being the Cell Censor channel and 1 being analog channel 0 If PASS is enabled then the CC holding potential is used for any subsequent data collection via parameter files i e the parameter file holding potential is overridden Sbutton Movement over the S button will open a window to allow setting of holding potential stepping information Clicking on the S button will step the holding potential to the various potentials for the set time interval Check T to enable saving CC trace at the end of each step and V to enable saving a video clip at the end of each step Executable in a user script see Script File Clk sets the clock for the CC stimulus Since the stimulus length is fixed the clock is used to change the length of time of the stimulus For proper CC evaluation the response must reach steady state and this can be achieved either by setting the clock manually in the text box or by clicking on the transient trace icon which sets the clock automatically so that the stimulus delivery time is 10 times the response time constant Alternatively AUTO can be enabled which will per
45. make backups The jClamp32 lic is not replaced If your hardware license your_name lic is in the jClamp directory it will not be touched and it can be copied and renamed anytime to jclamp32 lic You can keep a back up store of the msi versions for safety as the automatic installation does in jclamp32 jc_msi special files for installation on old Win95 machines may be obtained by email to me w95 bat oleaut32 dll regsvr32 exe SCRRUN DLL JC readme utility Jcreadme txt This is a new method for communicating with a jClamper during experiments without interference during recording A text file named jcreadme txt can be placed in the jClamp32 directory at any time e g remotely via network This note will pop up at the start of jClamp or if jClamp is running jClamp32 Whole cell voltage and current clamp 25 5 0 Manual when the Cell Censor is turned off The note can be printed prior to exiting the window Upon exiting the file is deleted but the information is stored in the file jcreadme bak Additionally if a text message is present the audio file named lt jcreadme wav gt located in the jClamp sounds directory will be played You can record your own audio message using Windows sound recorder for example along with the text message or just play a notification sound when the text message opens Just rename your recorded wav file to lt jcreadme wav gt and place in the sounds directory A separate Audio program j
46. matlab sigmaplot origin or excel analyse cm abf file and plot previously loaded data file must be a two sine or phase track Cm collection select raw true select cm true select rm true select rs true select 1m true select vm true 145 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual select time x true select cm x true select rm x true select rs x true select im x true select vm x true set scales for plot of loaded data file fix yscale true or false y max value y min value fix xscale true or false x max value x min value set fit parameters fit on true open fit window fit now true do fit now fit target analysis iv cc fit expl true fit exp2 true fit cap true fit boltl true fit bolt2 true fit line true fit user equation filename fit replace with any user equation filename that is present in the user fits directory 146 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual auto export true false part partition partition to plot in Result Analysis Window 0 for all group true false check box in fit form param true false check box in fit form choose files fromz filename list of files to analyze rather than choose from dialog uses script data directory that was previously set x data to fit filename x data for fit uses script data directory that was previously set y data to fit lt filename gt y data for fit must be same length as x data uses script data
47. of data trace with automatic zero padding Support for the National Instruments PCI MIO 16EX 10 10 usec and PCI 6052E 3 usec boards 16 bit A D D A boards with breakout about 2K Support for the IOtech daq2000 PCI board 16 bit A D D A 100kHz board is 500 Cabling and breakout extra Important contact me before getting one there is a glitch on analog out High time and magnitude resolution capacitance measures during episodic protocols Measure capacitance Rs Rm simultaneous with your favorite step ramp or AC protocols Automatic analysis Episode length up to 1 million pts for Axon boards and jClamp32 Whole cell voltage and current clamp 25 5 0 Manual IOtech daq2000 measure voltage induced capacitance changes up to 10 seconds per episode 10 us clock 10 million pts for National Instruments boards Real time monitoring of cell and clamp characteristics in Cell Censor Window Full control over two patch clamps Channel 0 0 A D D A patch clamp 1 channel 1 1 A D D A patch clamp 2 both channels or junctional coupling possible Pseudo simultaneous measures under current and voltage clamp on channel 0 and 1 in Cell Censor Automatic pulse polarity flip flop for charge buildup control Four additional channels can sample peripherals e g pressure monitor at user selected rates Works with either the DigiData 1200 1200A or 1320 series boards from Axon Instruments Labmaster DMA IOtech da
48. or Origin and save what you like Matlab will ask before closing Xcel will ask to save data before closing SigmaPlot will not close Origin Selection of DD1550 board sets clock increment to 2 us because that is required by MD There is still a jitter that I detect that MD cannot recapitulate I think it is present not just in jClamp but also in Clampex I will fix when they acknowledge and fix In order to use MD Axon boards you will need to copy some files to the jClamp32 directory The required files change so email me for information FFT and PSD analysis bugs fixed A Matlab script to do PSD analysis on j Clamp files can be provided 12 15 14 JClamp now supports Molecular Devices DigiData 1550 there is an intermittent one point jitter in their driver so that high frequency Cm measures are not stable Molecular Devices MD is working to fix the jitter and I will update when this happens Lower frequency Cm measures are less susceptible to the jitter Update SEE ABOVE MD has just released a new version of pClamp v 10 5 0 9 which apparently fixes the jitter issue in pClamp Clampex However clock rates are limited to multiples of 2us Thus older versions of Clampex collected data that had the potential for inaccurate timing when using the 1550 jClamp does not currenly support the 2us increment limitataion on sampling and in any case the new drivers still produce jitter in jClamp even at 2us sampling I m trying to figure thi
49. order If averaging is on each set of episodes will be delivered in the same random order Check boxes in the episode and average interval frames allow the capture of video frames after each episode or average Move the mouse cursor over them to identify them with the jClamp info panel Smart Averaging jClamp32 Whole cell voltage and current clamp 25 5 0 Manual piss D Start d c C1 Tea nga Time e a Sync 0 zl bee we a Digi Dp g Part Pre Bun Epi P Averages Smart Avg ict Number part N H 512X There are an additional two check boxes in the episode and average interval frames which control averaging In the episode frame the left box is checked by default to average after completing all episodes first If unchecked the number of averages per episode are completed first before moving on to the next episode In this case you may enable the Smart Averaging checkbox left in the Average frame and the Smart Avg window appears Normally when you supply a number of averages the episode is run until the total number of averages is complete Now you can stop averaging when a particular signal to noise ratio S N is reached as entered in the S N text box The signal RMS value from the chosen signal partition part S is compared to the RMS value in the noise partition part N So if you select a S N ratio of 10 the response will be averaged until the ratio of RMS values reaches 10 if this is after 10 aver
50. program In the properties window target line you should append the word skip reg For example C Program Files jclamp32 JCLAMP32 EXE skip reg is taken from the target line of a shortcut It will run jclamp but not display the registration window You could also specify a particular ini file other than the default jclamp32 ini by doing the following lt C Program Files jclamp32 JCLAMP32 EXE my ini file ini skip reg Note there is a space after the executable name and between the ini filename and skip reg Gain setup in ini file First check that the output is working Set the gain for DA to 0 001 in the textbox Save the ini file Restart jClamp or reinitialize Set Vpulse to zero Set the cc hold to 1000 mV and the meter connected to your output channel should read close to that Verify that the output corresponds to changes in cc hold jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Now set the DA gain to that required for your amplifier For example if you need 10 times the voltage set the gain to 0 01 Save the ini file Restart Clamp or reinitialize Verify that the output corresponds to 10 times the changes in cc hold Now that the DA is set correctly you can set the AD scale Remember to always set the DA scale first Use a model cell to check everything before doing real experiments Supported Hardware X Axon Instruments Inc Supported Hardware The program is written to work with the Tecella Pic
51. re entry to analysis mode rather than checking for connections each time No need to double click on the SigmaPlot Matlab Excel or Origin icons in Analysis mode If programs are available but need to be set to check for in in file then icons will display and drag drop icons to transfer data will be immediately available In order to use SigmaPlot j Clamp templates available in jClamp SigmaPlot directory you must load them into the template jnt file according to Sigmaplot instructions You can add templates for access in jClamp by creating your own in SigmaPlot and putting the names of the template in the jClamp SigmaPlot directory e g make a file name my new template jnt in the directory it can be an empty file only the file name is necessary and must match the template name my new template in the SigmaPlot template jnt file Check that the new template is available in sigmaplot It is required that you reload SigmaPlot to install new templates that are added to template jnt Sigmaplot transfers will now increment notebook numbers so that now saving with new names is not required between jClamp gt SigmaPlot transfers JClamp data file names are inserted in notebook sections jClamp32 Whole cell voltage and current clamp 25 5 0 Manual All transfers of data to other program via automation will leave the automation program files open after jClamp closes You can continue work on the data in those programs SigmaPlot Matlab Excel
52. resistance obtained from CC or entered manually The 129 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual open file button in the iv plot window will allow selection of multiple files of similar protocols to compare plots Order of plots are RGB then dark RGB then all red The text button will show all the x y data that can be copied to the clipboard and pasted into any other program For i v plots the reversal potential and resistance at zero volts are calculated for each data set plotted Pressing the Erev button will copy the results to the clipboard jClamp for Windows RE Analysis File e vr2002 VR02314 R2314014 abf vClamp 10l xj Avg 1 Epi 10 Epi ali zC sei Channel vg pk 10 Epi all C sel 0 0 Fix Scales ymax 5 81471 136 97 77 58 38 Task an r Estee eatem 3j Lar ee s Js S D gi ne cursor sj PERE H 3 17 2002 10 13 32 a Free Memory 321 764K CM Cei O CC Celi 130 File Math File Math jClamp32 Whole cell voltage and current clamp 25 5 0 Manual X s gem Papp al cAiosepFAisS7n28 Vs 7028004 abf Subtract Files C Average s n28001 ab Subtract X Group Group scere CAM 57n28003 abf s7n28004 abf szn28004abt Directory Gac joseph E c MAI All these manipulations can be done in an external package after export of the data This window is exposed for those who wish to inve
53. run sequentially by renaming first file to jclamp test test1 test2 prm synchronous iun ese main eor orc orba pol he main protocol tt can start and end before during jClamp32 Whole cell voltage and current clamp 25 5 0 Manual MultiClamp IC lt gt VC switching Current and Voltage Clamp switching for the MultiClamp 700 jClamp can do real time switching between current clamp and voltage clamp in protocol files when an Axon 700B Multiclamp amplifier is connected The amp must be in external mode Channel 1 Channel 2 V mV p V mV p 0 5 16 9 0 2 0 0 Resistance Resistance dms Mode vc 20 sic Ext D mv 10 mv 100 Hz 3 02 mv L Fino 3463pF 1 00ys Q00pF s 100ys p IS 12 93 pF 9 992MQ 1 02 kHz 0 D r Membrane Current 0 5 n4 10 kHz DC T 10 00 MQ Membrane Potential 10 mV mV 1 10 kHz 10 mv 10 ms 500 us 500 MQ You will need to setup current and voltage clamp in CC window to make sure things are working first in each mode separately To measure Cm you will need calibration files for IC and VC and the file names need to be placed in the ini file Everything talk about here was done with an electrical model at the headstage Real cells require careful interpretations due to nonlinearities To balance out stray capacitance use the MD feature under Frequency domain IC Cm is not as good as good as VC because stray capacitanc
54. shift gt lt X gt toggles between the two Plots can have axes or a scale bar The scale bar will indicate magnitude for the plotted data The scale bar can be dragged to any location in the plot window Select the corner of the scale and a finger pointer 122 ESISTESESESES IS 2 gape es v2 223 AME us 7 5 6 El sl i um X1 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual appears drag very slowly with the mouse down to a new location In the scale bar mode voltage protocol 1s displayed as well In addition if the digital partition output is enabled in the Command window then digital output is plotted In either plot mode a colored marker up to 3 can be placed on the graph by clicking the right mouse button over the data plot Left clicking on them will erase them The click clip check box if enabled will display a magnifying glass cursor which can be used to capture X y points from the plot to the clipboard Points are appended to the clipboard with each left click of the mouse You can paste the data into any spreadsheet Unchecking the check box clears the clipboard See info panel at bottom of main jClamp window for button info when mouse moves over each button They are enabled after a data plot is created If a ramp command stimulus was employed to collect any part of the data a panel is displayed to allow i v plot generation CC Data Plot Window button use to view saved Cell Censor data cc c
55. stray capacitance is balanced out but see MD Cs method below because the evaluation of the cell s characteristics is model dependent and does not include a pipette capacitance 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 In cell stray capacitance neutralization with software assistance Although you may have made efforts to remove stray electrode capacitance prior to popping into a cell things may change during the course of an experiment For example fluid level may rise or fall around the electrode and additional stray capacitance may interfere with Cm measures I now provide simultaneous multi dual MD sine wave analysis to handle this problem In CC select the MD checkbox and 5 dual f1 f2 frequency analyses will provide a plot of Cm versus frequency index low f to high f Simply flatten the line with electrode
56. 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 cell s physiological response Let s 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 cell s 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 cell s 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 o
57. the desktop and select properties ro Shortcut to jclamp32 exe Properties Ea Shortcut to jclamp32 exe CA UsersNoseph santos sacchi Desktop 2 88 KB 2 959 bytes 4 00 KB 4 096 bytes Saturday January 10 2015 6 45 04 PM Monday January 12 2015 2 36 34 PM Monday January 12 2015 2 36 34 PM Readoniy C Hidden Go to Compatibility jClamp32 Whole cell voltage and current clamp 25 5 0 Manual If this program isnt working correctly on this version of Windows try running the compatibility troubleshooter Run compatibility troubleshooter How do choose compatibility settings manually Compatibility mode L Run this program in compatibility mode for Windows 8 v Settings L Reduced color mode 8 it 256 color v C Run in 640 x 480 screen resolution C Disable display scaling on high DPI settings C Run this program as an administrator C Enable this program to work with OneDrive files ji Change settings for all users Change settings for all users jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Compatibility for all users If you have problems with this program and it worked correctly on an earlier version of Windows select the compatibility mode that matches that earlier version Compatibility mode Run this program in compatibility mode for Windows 8 v Settings Reduced color mode 16 bit 65536 color v C Run in 640 x 480 screen resolution Disable displa
58. the resolution of the AD conversion but will potentially deliver more voltage or current stimuli to the cell than the parameter file calls for Consider the benefits and disadvantages before using auto gain The number of averages can be set as well as the time between averages Each set of episodes 1s performed and the set 1s repeated for averaging In the average frame a check box allows the saving of individual data collections in a separate file in addition to the averaged abf data file The filenames for individual traces transformed into episodes for display purposes are derived from the averaged abf filename For example the file lt average abf gt which contains averaged data from three episodes will be associated with other files average AVG_1 abf gt average AVG 2 abf gt average AVG 3 abf one file for each of the averaged episodes This works with leakage subtraction on as well and is useful for noise analysis where variance needs to evaluated The number of episodes can be set with an inter episode wait time The wait times in msec in jClamp are approximate since they are produced by the Windows environment Data collection however is exact since DMA transfer is used by the Axon acquisition board In the episode frame a check box allows delivery of episodes randomly For example if the command protocol has incrementing voltage pulses of 10 20 30 and 40 mV enabling random delivery will deliver the pulses in random
59. yovi Bru x N 2E22 A o is SME gt IT cn Sigmaplot transfers will now increment notebook numbers so that now saving with new names is not required between jClamp gt SigmaPlot transfers jClamp data file names are inserted in notebook sections All transfers of data to other program via automation will leave the automation program files open after jClamp closes You can continue work on the data in those programs SigmaPlot Matlab Excel or Origin and save what you like Matlab will ask before closing Xcel will ask to save data before closing SigmaPlot will not close Analysis Window A large window displays the data file traces and permits certain types of data manipulation Descriptions of the button functions are provided in the lower main window panel by moving the mouse over the buttons Data is loaded with the file open button in the task section Other options include plot printing I V analysis zoom viewing Cell Censor data viewing toggling of leakage subtraction exporting files into abf format Matlab format or ASCII format clipboard capture of the displayed traces data or graphic plot file math capabilities and episode math capabilities 121 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Avg 1 Epi 15 Part 2 Part all sel Epi allC sel le SSS 1 72 0 102 204 307 Epi 1 27 ms y 2 107 nA Part 1 Command 120 99 4 mV Rs 9 8 Mohm 3 r
60. 0 Manual 3 Freq 2 n 195 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual B Command Utility vClamp with lOtech PersonalDaq3000 x tech v 247 cicdata v5 1402754027036 abf A mode 7008 tesa 10727714 Parton Design T gt abf template Author N Select Copy J Santos Sacchi private 5 3 ABI 3 gt gt 1 comment password Properties Partition Elem 1 2 MD Analog Chan YC clamp Start 1 nA m 0 zi eCm Chirp Out In Eun 2 ast PLS al ms SA k ns Sync 0 re N 1 E step user auy o DERE T T T lenge e EUN da T Ie Epi Points Cik us Hold Number PA HN ERT sys ho 0 mv 1 custom chan no TT Dh Inter ms Inter ms 50588 rp 0 rp 0 B fa arai Vrange Plot Results Trigger ET Epi mx Init f s Ky Di Epi u e mij epi non I Ky Di Run Pat 5 Epi 1 x 4323 y 0 0 Building a command starts with a voltage clamp protocol Select Axon 700 mode D3 and Part in the Digital section Digital output 3 is used to switch between IC off and VC on Trace 3 of the digital protocol is red By setting digital 3 for any partition you can make that partition VC or IC You can select a partition from the drop down box or move the mouse onto red vertical line partition demarcations in the command plot When a partition is in VC mV is displayed and nA is displayed for IC Values are in n
61. 21 Offset mV Vm mV on 3 4 Arnt 40 TEN Freq 2 n 195 Hz CC Domain i options EE S z Am t lib l Dith Time Dual C Single Hn ian d Freq C etm MD 11953 F P paak EE Cm f1 17 14485 f2 17 11503 f3 17 11957 f4 17 15355 f5 17 10505 15 Capacitance Compensation C30 zero guess cate 0 6219513 pF 8 596919 Mohn D pF fo Mohr Rcs Es1 Model Cell Model Cell jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Model lonic Extra JSS Cell2 Ch1 Cell3 Ch2 Celia Ch3 Simulink via m file Edit Script Sim choose a model lt Edit Up MList Parameters Vesicle mI Rs py 10 Gp nS fea Rm MQ 100 amp v nS es Cm pF 30 00 Cv fF 1000 00 Vh mV 30 Qmax pC 0 00 Param change Vall 0 E Va2l0 Noiz 0 001 PH D z jo RN pus my o oM Cpip pF 0 0100 Rpip MQ 10000 0 RC Gauss filter Hz lo 0 1 2 3 Tele0 An Tn 5 Vm mV Cpip is pipette stray capacitance and Rpip is the resistance in series with Cpip Values of 5 each produce a transient current with tau of RC riding on top of the cell currents jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Cv If Qmax gt 0 then a two state Boltzmann is used to simulate a nonlinear capacitance in parallel with Cm Checking Vesicle switches to the model shown above Gp is 1 Rp and Gv is 1 Rv The sta
62. A for IC and in mV for VC partitions Since the command plot shows both nA and mV the view may appear odd Select a partition to view the magnitude of that partition command jClamp32 Whole cell voltage and current clamp 25 5 0 Manual B Command Utility vClamp with lOtech PersonalDaq3000 eS wi pie v DAE c jedata J 14028 none A mode 7008 vi pulse 10 24 14 Porn Design gt abf mid AUS Select J Santos Sacchi private um z Jf BIS ai PS E alf E B comment password Ei Properties Partition 2 a Chan E VC I clamp m 5 mV 2 E a na Inc 0 e C On sa Fl isa Time 100 ac stair CO nChan m ing jo ms ramp abf Sync 0 gt N 1 step user Dig T Axon 700 mode D3 Dp T TT Ix eno 3 Pre T PAN Bio T HE Se Clk us Hold Averages j Episodes Number Number 512 X 139 sys 10 o mV Li 1 E custom chan 10 T T Dh Inter ms Inter ms 71168 ry o Tp 0 dcs B fa eng We Plot Results Tri gger Epi mx 5 cum aw o Ky C Di Epi r au e mn epi non ee Run Command Pat 7 Epi 1 x 608 8 y 0 0 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual A jClamp for Windows v 24 7 0 Cell Censor iClamp with lOtec 4027036 abf 1355 IX CCl Save Oms oie Yo oe vO x Vi Ix v2 x V3 x10 H i E x weO X wel x wc2 X wc3 Model x TeleShow Dis dual Vh Trk peg options F2 00
63. Capture VDA files or AVI files VDA is much faster Grab a bitmap during viewing or during playback see viewing VDA files elsewhere in this help file FastLook jc Prosilica GE680 S N 02 2020C 06588 w T dt View Live on Bees Player Options p E ames EE T de S l f Image di TIFF Contro Entrst Bril TT A box may be draw to select a region of interest ROT that will be captured when saving video Frame rates can be much faster when small ROIs are selected After a box is drawn and the mouse button is released the Check max fps checkbox is automatically set and a one second simulated video collection is made with the maximum rate and frame time period displayed Check the box again to see how fast the capture rate is for any already drawn box Simply press the left mouse button down on the screen to erase the box and return to full screen collection mode The max fps will be displayed for full screen You can reduce or increase the viewed image by changing the scale Video collection is always done with display disabled By using binning set in the control Panel and small ROI fast video collections can be made jClamp32 Whole cell voltage and current clamp 25 5 0 Manual EZ FastLook jc Prosilica GE6BO S N 02 2020C 06588 PEE Exit View Live on Capture Player Options Image Control Entrst Brite I Rotation 0 T PielxX Control Control Panel Auto save is default If unchecked you wi
64. PCI 6052E and Axon s 1320 and 1440 series now can have dual analog output and up to 8 input channels jClamp32 Whole cell voltage and current clamp 25 5 0 Manual The NI PCI 6221 board is used by Neobiosystems to control their PatchMAX 100A and jClamp will work with their system to provide user friendly patch clamp and two electrode voltage clamp solutions User choice of Dual frequency Cm calulation from f1 f2 or aveage of both Phase Sensitive Detector PSD exposed for phase analysis Automatic fusion event analysis Easy capacitance measures new method based on actual angle of dY dCm perfect at last Average after all episodes are run or average each episode before continuing to the next episode Smart averaging only average as many runs as required to reach a desired signal to noise ratio More flexible delivery of frequency stimuli Axon 1322A support Storage view in Analysis mode view two data file plots simultaneously previously opened and current Independent analysis and data collection CM calibrations file loading Streamlined generation of command protocols from ABF data files User choice of detrending of raw data prior to CM analysis Technical Note released NI PCI MIO 16E 1 NI PCI MIO 16E 4 support jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Fitting with user supplied equations Now measure capacitance with either 2 Sine analysis or
65. Watch jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Seal Display input resistance during seal formation Zap is a parameter file which can be modified by the user The scroll bar dynamically increases or decreases the amplitude of the signal Pressing the cartoon button delivers the zap stimulus which hopefully will establish whole cell configuration If not suck One zap can be delivered automatically when a given seal value is reached if auto is enabled Settings in the ini file Command Utility the Command utility is used to develop voltage or current protocols Quick Notes open window to insert preassembled often used notes into the log file Rapid Parameter Directory toggle open Edit Script opens Notepad to edit script files with the extension lt scp gt An example script is shown below with all commands Scripts can be edited with any text editor Impedance analysis open impedance window C mcealibrate ieon capaeitance traeking ealibration and ealibratien filelead Calibration accessible from CC window now About info on the jClamp and computer The Help file can be accessed from here as well LOG open log window only works in analysis mode Model the program can be run without hardware if model is selected from the Cell Censor window move mouse over the user graphic in the Cell Censor open selection window containing model toggle Clicking this button opens a window to adjust model
66. aPlot and Origin are available Attention parents Get Merlin Programmer for Kids an absolutely FREE fun program I wrote to help my kids now 18 and yours ages 5 8 learn how to program within Windows should work even in Win7 and hopefully ultimately help support their higher education but more importantly help them to think logically Nanion Port o Patch support is here jClamp32 Whole cell voltage and current clamp 25 5 0 Manual New Feature Update e 3 12 15 Ability to search ABF files in multiple subdirectories for particular characteristics of the command protocol or clamp characteristics Shift right click on displayed file list to copy list to clipboard e g to paste into Excel Copy all listed files to a new directory to do group file analysis e g to analyze all data collected with a particular protocol file Double click on a file to plot data in Analysis window 1 19 15 Command protocol window shows actual max min including 2 sine stimuli During build if magnitude is over board settings then a notification is given If such a protocol is run a choice is given to cancel or proceed Parameter directory fixed at jClamp diretory subfolder param with subfolders below automatically entered into search path from RPD and scripts Crash detection implemented in jClamp registry key Logs crashes for debugging purposes 12 30 14 Automation links remain in effect when exiting Analysis mode This allows quick
67. ages averaging will be stopped even though the number of averages was set to 100 If the S N 1s not reached the total number of averages will be performed The reject box is an RMS value of either the signal or noise partition that will be rejected from inclusion in averaging since it is deemed to be an artifact If the RMS value is negative then the whole episode all partitions is checked for potential rejection The combination of rejection and selected S N requirements provides for powerful smart averaging The Partition Design function produces the voltage or current waveforms Holding potential is used at the initial 6 partitions points and for a variable number of points at the end of the command at least 6 partitions points to make a total command length which is a multiple of 512 points Clicking on mV in the Properties Section changes the command window to current clamp mode and clicking on nA reverts back to voltage clamp mode A partition is a number of points set to a particular type of waveform In designing a protocol one should first determine the number of partitions desired Partitions can be inserted before or after the currently selected partition arrow down buttons or deleted X button The parameters of one partition can also be copied to one or all other partitions Setting the partition number to 0 erases all partition data For a simple voltage step protocol one partition can be used Partitions on the command plot are se
68. ained if CC digital is not enabled otherwise CC digital controls output after the protocol is run jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 1 FU RRR ste Pat Cs 2 pean cho 3 Pre esis Digital FlipFlop p fus 5 iv p 1 ms ys 10 ms lm E ms an 10 0 p2h ms ew Comman zs f HT PrePulse Trigger n E Epi m RCo me EE U Z The prepulse function allows delivery of a voltage stimulus prior to delivery of the command waveforms Itis the same as Axon s prepulse protocol See there manual for details The currents produced by prepulse are not saved however average steady state values are stored to enable series resistance corrections to the prepulse voltages when using the built in I V plot window When using prepulse there is a machine dependent time delay between the end of the prepulse and the beginning of the command waveform The time is somewhat variable and represents the time needed to fill the command buffer with the episode voltage after the prepulse is delivered The time on your machine should be checked with an oscilloscope it is about 10ms on a 90 MHz pentium To help with this potential problem the INI file has a setting called prepulse hold If the value is set true in the IniEditor then the final prepulse point is set to the protocol parameter holding level filling the variable time with the holding potential If set false then the prepulse time itself will include t
69. an_f a SOT gap 5 5 Acre jClamp32 Whole cell voltage and current clamp 25 5 0 Manual confined to aimited array size n the case of the Axon 1200A boeard denger times are allewed than with the other beards Utility Files scp these are ascii script files that can essentially run an experiment by sequentially performing a host of commands listed in the help file rpd these are ascii files that contain a list up to 20 of the names of protocol prm script scp and capacitance tracking or gap free cmt files In the RPD window these can be executed by a button or key press scn automatically generated screen arrangement file types Created and utilized with Screen Shot utility ana analysis script file in ascii Commands are listed in the help file uad user analysis directory of analysis script file names in ascii This directory is used in the Result Window to assign buttons to the individual listed scripts The buttons are accessed by arrowing down on the up down control at the lower left in the Results Window fit ASCII text files of user equations for fitting data traces See Fitting section Data Files abf data files saved after running a protocol prm file The file types are Axon compatible and loadable in their programs as long as Axon maintains compatibility with their earlier ABF version This filetype is also used to save gap free data when gap free saving checkbox is enabled
70. ands can be run from a script as well Set camera types here if supported by FastLook value of 0 will be default camera cameral start 0 serto emo So mo camera as onwien progran starto routs ideor jClamp set to 1 for Ando to start 2 for Prosilica 7camera krnd andor Tt pneamemnasmdpmnoseea27 161 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual camera kwund spob 3 ij path 0 imageJ set ImageJ path here capture length 20 set the capture Meng th prosilica poll on set polling mienie emere control capture with a particular camera spot_poll on andor poll on auto gain threshold 700 set auto gain to obtain this average random pixel intensity of camera image not screen intensity capture frames set capture length to equal frames capture time set capture length to equal time in seconds time out 10 set time out to wait for hardware trigger if exceeded capture is halted but file is saved frame rate 25 set fixed frame rate frame mark on enable frame marking during recording frame mark off audio mark on enable audio frame marking audio mark off audio threshold 50 set audio threshold for frame marker 1 127 disk stream on when capturing video stream directly to disk may be slower than to memory disk stream off when capturing video stream directly to memory before writing to disk autogain push autogain button to automaically set
71. ant to inset an LED into the periphery of the camera light path in the microscope in order to get fiduciary marks on video captures 102 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Home Insert Page Layout Formulas Data Review View Acrobat e 4 cut Calibri Ju X S E wrap tet General i3 Co MIT 2 ee EE E EE TOS irene B iy JE I A E mS EH Merge amp Center 7 La VIE Clipboard fa Font Ta Alignment ta Number Q7 f T H mami mna lmai Damn mnis mma ma Sn omui Hla mm mm m mama 1 Epi frame T ms V mV V mV I nA I nA R G B Y 2 1 30 31 0 0 0 0 2 24392 3 1 31 64 45 567 45 567 2 277 2 277 2 249892 4 1 32 97 83 684 83 684 4 182 4 182 0 489875 3 1 33 131 99 898 99 898 4 992 4 992 17 77801 6 1 34 164 89 015 89 015 4 45 4 45 28 93131 7 1 35 197 54 745 54 745 2 737 2 737 29 88543 B 1 36 230 6 091 6 091 0 305 0 305 28 63121 9 1 37 263 44 163 44 163 2 208 2 208 14 07409 10 1 38 296 82 814 82 814 4 14 4 14 0 461233 11 1 39 330 99 814 99 814 4 991 4 991 0 361273 12 d 40 363 89 72 89 72 4 486 4 486 0 331703 13 1 41 396 56 052 56 052 2 803 2 803 0 26205 14 1 42 429 7 658 7 658 0 385 0 385 0 237391 15 1 43 462 42 748 42 748 2 137 2 137 0 231173 16 1 44 495 81 923 81 923 4 095 4 095 0 149744 17 1 45 529 99 706 99 706 4 984 4 984 17 50016 18 1 46
72. apacitance tracking data cmt and capacitance data dcm obtained from protocol files prm that incorporate two sine voltage stimuli t 1 JE Mum Two data files can be displayed simultaneously by turning on storage The previously loaded data plot is retained in the color green and its filename is displayed in green in the Analysis window Cm Analysis in the Data Plot Window It is now possible to derive Cm Rs Rm and Im from the single dual or eCm method abf files in the Analysis window Most analysis features that are used for currents exponential fitting etc are then available to use on the extracted data When a file that has employed 1 or 2 sine voltage stimulation is viewed in the Open data file window see that help section you can enable a checkbox to load the embedded calibration file or if unchecked use one already loaded or load a new one for analysis from the Analysis window A calibration file opened while in Analysis mode will only be used for analysis the data collection file remains the same and can be changed only when Analysis mode it exited Upon plotting the abf file in the data analysis window a panel is displayed at the lower right that allows choosing which data to plot Raw Cm Rs Rm Im or Vm You may also choose whether to analyze with single sine dual sine or the new eCm Method and correct for series resistance effects on voltage The green scale icon can be used to load calibration f
73. apture to occur at fixed frame rates This will be limited by Windows fixed rate off no fixed frame rate speed will occur at maximum rate permited by software in Windows set frame sec set fixed frame rate in terms of time duration of frame set frame fps set fixed frame rate in terms of FPS frames per second The default directory for VDA files is c Mastlook data plus the day s date e g see below The directory can be changed here in FastLook or in the ini file at program startup see ini file section in this help file Thus each user can set his her own user directory When FastLook is used in conjunction with jClamp jClamp control the data directory path directly Communication with jClamp is via background polling 152 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual EZ Controls FastLook jc Prosilica GE680 S N 02 2020C E BS Stats Be Cooler e ath c Mastlook dataX08 13 2010 Calibrate check box will show two flashing white lines 100 pixels apart Determine the actual size and place distance and units to be saved with VDA files Simple measurements to plot line or box profiles or average of marked areas RGB plot Export VDA video files to ImageJ to do sophisticated image analysis Delta length and save data are jClamp only features During image capture you can enable viewing of live images at entered frame rates Uncheck box for best high speed capture results
74. arameters set independently The stimulus may be either continuous burst with a linear on off ramp or burst with a Hamming window The duration of the on off ramp can be set to 1 8 or 1 4 of the stimulus duration Other AC stimuli i e chirp logarithmically ramped frequencies white noise random noise or ternary noise TN can be selected Data obtained with AC stimulation can be analyzed with the built in FFT analysis or analyzed after export to another software package It is possible to perform automatic capacitance measures see Cm Analysis section using the command utility Protocols are built as usual with step ramp user or ac stimuli in each partition If the check box 1 2 or eCm is on either a dual or single sinusoid is superimposed upon the command in that partition Fi2 checkbox enables one of two sine stimulus protocols see Cm Analysis for details From the recorded currents capacitance membrane resistance etc are derived automatically It is possible to run dual simultaneous capacitance protocols i e ChO out in and Chl out in Build the command as usual for each channel separately A special protocol file accalib prm 1s used to calibrate the system prior to measurements It is run automatically when the system is calibrated or can be run from the command utility window and a calibration file is saved with a selected name e g test cal The calibration file contains data for two channels When one channel is calib
75. ard type to the board that was used to save the data when viewing the data You don t need the board present but can work in math model mode Actually I have tried to make the aforementioned not a requirement For example the board type is saved with a data file and I use it to select the board type when analysis mode is in effect so that data files are matched for board characteristics In the command utility the board that made the protocol is now exposed and jClamp32 Whole cell voltage and current clamp 25 5 0 Manual when loading data files the board that was used is exposed in the window Nevertheless it is safest to select the board on startup to look at data generated with a particular board Gain setup in ini file 1 First check that the output is working 2 Set the gain for D A to 0 001 in the textbox Save the ini file Restart jClamp or reinitialize Set Vpulse to zero Set the cc hold to 1000 mV and the meter connected to your output channel should read close to that Verify that the output corresponds to changes in cc hold 3 Now set the D A gain to that required for your amplifier For example if you need 10 times the voltage set the gain to 0 01 Save the ini file Restart jClamp or reinitialize Verify that the output corresponds to 10 times the changes in cc hold 4 Now that the D A is set correctly you can set the A D scale Remember to always set the D A scale first jClamp32 Whole cell voltage and current clamp 25 5
76. ates of junctional conductance Gj0 is the conductance between the cell recorded with amplifier 1 and that recorded with amplifier 2 and visa versa Series resistance and membrane capacitance are measured with the technique of Santos Sacchi J Neuroscience 13 3599 3611 by default which is designed for simple fairly small single cells and is robust despite filter settings There are two check boxes in the Rs Frame If the top check box is checked the series resistance 1s and capacitance is obtained by Rs corrected steady state command voltage divided by maximum instantaneous electrode current estimated at zero time and total integrated charge divided by Rs corrected steady state command voltage If the bottom box is chaeked Rs is ignored and input resistance is only displayed Under this condition Auto clock is disabled since tau is not measured Check boxes in the plot window permit various options including automatic software selectable hardware gain if supported by the board if disabled clicking on the gain number can scroll through hardware gain Zero current trace fix plot gain normally it is automatically scaled to fit within the window if enabled a spin button is used to manually changed plot gain store function to maintain previous traces FFT to look at no signal frequency response clock initially set to observe 60 Hz noise clock can be changed to change frequency range Beware of aliasing jClamp32 Whole cell vo
77. b a 9 BTT Rm MQ p 313 30 Cm pF Inter ms 18 345 0 th n r 0 121 Offset mV Vm mV i on 3 4 40 L Freq 2 n 195 Hz 4 5813 n Cm pF CC Domain Freq options Fi2 f S E m Z lib Dith f Time C Dual Single 2 al ither on G Freq C em MD nasa Fh v pde sd i a Cm f 18 33020 f2 18 13252 f3 17 35681 f4 14 46723 f5 3 05423 8 Capacitance Compensation Do Cs0 zero guess fit 0 pF bes 0 Mohr Cs1 pF jClamp32 Whole cell voltage and current clamp 25 5 0 Manual D Cell Censor vClamp with Axon Digidata 1440A none 10456 Vhold mV B clk us C Sp x EF Pass hold S Pass amp x ae nave ns x Auto ocara E 10 s mI CCinter Disable File 2 ge Cmdhid Chan Watch L EN 3 N tau ms Vpul mV 0 1781 1 10 Rs MQ aia 10 6 Rm MQ ows 316 13 ms Cm pF nter ms 17 334 JU Ih nh r 0 122 ffset mV 3 4 Vm mV on s Freq 2 n 195 Hz Zero x ER 8 0 01 ms 4 5813 n Cm pF CC Domain Bs options Fiz T s E oa a Dither on Time Dual Single x Freq C etm MD f1 195 3 S pee ADOKHZ domi Hei Cm f 17 33305 f2 17 26488 f3 17 13551 f4 16 59281 f5 14 85998 15 Capacitance Compensation 3 Cs0 guess fit D pF lo Mohr ResO Cs1 j pF D Mohr bes jClamp32 Whole cell voltage and current clamp 25 5 0 Manual D Cell Censor vClamp
78. below to allow entry of the exact value of the resistor and the sample clock rate The protocol file accalib prm accalibi prm for current clamp is used for calibration and is run automatically during calibration It can also be run manually from the command utility in which case it will simply open up the calibration window The voltage magnitude in the protocol can be increased as long as saturation is not evidenced in the current response The protocol generates a correction file for the real and imaginary components of the system for 5 time resolutions which correspond to different frequencies in the frequency domain The resultant calibration file has calibration data for a given clock and filter cutoff if either are changed another calibration file is necessary Remember that when used to measure cell capacitance the magnitude should be as small as possible I use 10 mV peak See Takahashi S Santos Sacchi J Distortion component analysis of outer hair cell motility related gating charge J MEMBRANE BIOL 169 3 199 207 1999 for potential problems with the technique The non harmonic protocol now implemented in jClamp can jClamp32 Whole cell voltage and current clamp 25 5 0 Manual overcome these distortion problems if you have significant distortion See description of the non harmonic technique above A new approach I have devised a new way to get around some problems with stray capacitance which heretofore had to be removed by c
79. c audio exe for recording playing MS wav audio files which can be played during script execution now since Windows Vista does not include one Located in the jClamp directory Itis a good way to remind yourself or another jClamper of something important jClamp32 Whole cell voltage and current clamp 25 5 0 Manual jClamp for Windows Details Data Collection StartUp Start Up E S First Joseph Pos L Last Santos Sacchi A EM 1 Settings Choose startup ini file Ini File model ini model Data Path c jcdata js15118 Free Space 150 189 Mb Board Type IOTech board License Licenced to 1 Joseph Santos Sacchi 05 18 13 Serial Pioneer r Current Version D osci oFT GO V 29 5 0 software tools z suiesstists Upon double clicking the jClamp icon a window is displayed requesting user identification First and last names are required and may be specified in the INI file The initials of the first and last name are used to name files and data directories automatically see below The current INI file is indicated jclamp32 ini being the default A scroll box displays other INI files available in the JCLAMP directory Clicking on the INI filename loads the file and also displays a unique user graphic set in the INI file An image of the user is an ideal graphic for identification The data path and the drive s free space is also shown INI files are setup using the separate program iniedit32 exe A us
80. capacitance compensation circuitry on the patch amplifier to correct for stray capacitance at all frequencies Color of the line changes towards green as the correct compensation is reached An accurate calibration file is essential to use this feature With this technique you need not worry if you pop into a cell before adequately compensating stray capacitance It must be emphasized that this procedure demands that cell capacitance must be linear i e surface area dependent only If a nonlinear capacitance exists this must be performed in a state where it is absent A Capacitance Compensation window is now available to aid in stray capacitance Cs compensation It is opened by checking the Cs checkbox in the CC window in Calibrate sub frame Some amplifiers e g Axon Multiclamp may not have enough compensation to flatten out the Cm vs F curve in MD Additional compensation is provided by the sliders Start with the Cs0 Res0 slider first One could do all compensation with the software controls but it is better to balance fully with hardware if possible This software compensation works only under Frequency domain but hardware compensation works under all stimulation conditions since it is at the amplifier One could compensate Cs 1n Freq domain and switch back to Time domain knowing that the compensation remains in effect It is really impossible to balance out stray capacitance under the Time domain when inside the cell thus the MD Cs method is ver
81. cell with a mechanical stimulus for example while monitoring the current response Labmaster DMA selected lt Vhold igi o Pass hold CINE a clk erm 5 10 a 3 The Offset box allows one to zero amplifier offsets with the pipette in the bath prior to patch clamping The voltage offset correction is added to the command potential but is invisible to the user It can be enabled disabled with the check box but will store the offset value when disabled I prefer to use the offset knob on the amplifier but this will do it in software automatically The CC window can be collapsed by clicking on the left arrow bar on the upper right At the bottom of the window the domain time or frequency can be selected for cell parameter tracking See Cm Tracking for more info CC on checking this box begins monitoring of cell clamp characteristics The text Save of the adjacent check box blinks indicating that it should be enabled to save CC data see below Clamp time constant tau electrode series resistance Rs membrane resistance Rm cell capacitance Cm holding current Ih and series resistance corrected holding potential Vm are displayed If you place the cursor over these values a popup appears which shows the corresponding RMS noise value over a short period of time jClamp32 Whole cell voltage and current clamp 25 5 0 Manual CC save if checked CC data are saved to disk as an ap
82. cess If we check the fusion event FE checkbox and place single or linked cursors at the section before fusion we see that in fact the capacitance jumps to final values instantly the wiggles are due to the ODE solver If we now change the plot to the fusion pore conductance Rp in the dropdown box and switch to a plot of conductance Gp lt shift gt lt Y gt we see that the time course of fusion pore expansion is about 500 ms and the conductance corresponds to the model settings 125 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual pe rad cmm Hem um E d m Umm EL s o i othe ER ACI 126 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 27 8 2 560 202 240 407 040 511 840 816 640 1021 440 Epi 1 x 506 88 ms y 31 072 pF Part 1 Command 0 303 9 mV Rs 9 8 Mohm 127 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 615 936 Epi 1 x 967 68 ms x idi e Part 1 Command 0 10 4 mV Rs 9 8 Mohm 2asainaiaaas EI E Es n Fa Ew 3 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual IEEE ES E en s Berri Eus ES E ER LZ m IV Plot IV Plot Select iv plot button to plot a range of values avg rms peak integral time to peak time to positive 1 2 peak time to negative 1 2 peak versus episode or voltage at particular partitions If Rs check box is checked then the voltages are corrected for series
83. d You must select the Multiclamp telegraph checkbox in the History tab of the jclamp ini file editor and you must select the channels to use although the physical channels are not actually used Also jClamp automatically sets up telegraphs as enabled in the jClamp ini file You need to supply paths to the Multiclamp 700A or 700B executable 700A exe or 700B exe 5 jClamp Ini File Edit 32 bit v 13 0 1 ft Goes User Files Gain TO T1 T2 T3 cc Hist CC Script 2 MultiClamp v Load MC7004 Commander and Telegraph Servers MC7O0Apath j Load MC 00B Commander and Telegraph Servers MC 70068 path C Program Files x86 Molecular Devices MultiClamp 700B C Channel Watch Part Plot skip Enable Interval 1 sec 20 Chan 2 Chan 3 Label T C Label PIPI Fastlook Scale 1 Scale f Neobiosystems X Enable Enable CM Detrend Chand Chan 5 Slow Telegraphs an an t Label voi Label test a CellSeeker Scale 1 Scale 10 MagicSeal Enable X Enable lOTech Diff PujiClamp CC History Resolution 0 025 sec 1 IRs 3 Cm Time 30 sec EN fe Of 4 CCHistory on by defaut 2 Am wi 4 LC for 4 fe ord gaphs 4 Sealtime 0 sec 1 12 15 1 46 44 p In the ini file under History Tab gt you can set some features Checking no tele will disable telegraph jClamp32 Whole cell voltage and current clamp 25 5 0 Manual checking and allow you to enter a gain manually in the tele gain box Chec
84. d above Axon supplies the drivers Email me if you have a problem getting them Axoscope 9 2 is the last version of Axoscope to support the board so you must use that program to verify that everything is working before using jClamp Normally if there is no hardware license the registration window will appear indicating that the license is software only or that so many days are left in the hardware fulldemo license For the free labmaster or Axon 1220 support there is no license so the register window will open If you want to bypass that window you may create a shortcut on the Windows desktop to jclamp32 exe as you would do for any other program In the properties window target line you should append the word skip reg For example C Program Files jclamp32 JCLAMP32 EXE skip reg is taken from the target line of a shortcut It will run jclamp but not display the registration window You could also specify a particular ini file other than the default jclamp32 ini by doing the following lt C Program Files jclamp32 JCLAMP32 EXE my ini file ini skip reg Note there is a space after the executable name and between the ini filename and skip reg jClamp32 Whole cell voltage and current clamp 25 5 0 Manual DAQP board The DAQP 308 16 bit in 12 bit out must have the Port address set correctly in the jClamp ini file The DAQP 308 should be set up according to the manufacturer The board is controlled exclusively through Port I O witht t
85. d current clamp 25 5 0 Manual Drive free 127 611 Mb files 920 E c wW indows8 05 FileName Time Parameter 2sie Epi C Pts ccdataMS13D 5 54 35 PM jclamp false 5 1 204i c NjcdataV513D 5 56 01 PM jclamp false 5 1 204 c jcdata513D 5 5812PM jclamp false 5 1 204 4 4513D07 c cdataJS13D 6 01 43 PM jclamp false 1 1 204 23 4513008 c NedataV513D 6 06 25 PM model full freq false 1 3 125 813009 c cdata JS13D 6 14 08 PM model full freq false 1 3 125 69814307 G Nedata MS 1 3D 6 16 36 PM model ful freq false jS 125 814311 c iedata WS130 6 21 49 PM model full freq false TUER 125 814312 c icdata JS13D 6 27 26 PM model full freq false 1 3 125 94514325 c NjedatasJS 13D 6 27 53 PM model full freq false 1 3 125 mm cNjcdataV513D 6 37 03 PM model full freq false 1 3 125 a JS14326 c icdataW 13D 6 39 59PM modelfullfteq fase 10 3 125 1 J514327 c icdataWJS13D 9 58 44 AM model ul freq fale 10 3 125 Lay 3514402 c jedata JS13D 10 07 22 modelful freq fale 10 3 125 1 4514403 c NcdataMS13D 10 26 24 model cm hy r true 4 4 527 234514404 c NcdataV513D 3 12 58PM model full freq false TU 125 234514406 c NcedataVS13D 0 19 28 AM model full freq false My 3 125 234514407 v cjedataM5S13D 1 55 24 PM model cm hy r true 1 4 527 Command Type Values sep I ramp T Rs s ab T jac Rm gt fim user stair param
86. dows Bime in d Rere leE ROL VidClip Grabbingimages in the Look Window saves image files to the day s data directory bitmap format as a note file not rename it to bmp to open in any image editor or view it in jClamp s VidClip image viewer accessible in the Analysis Window The VidClip viewer can be opened in Analysis mode by pressing the VidClip button Linear measurements can be made with the mouse after calibrating X and Y pixels per unit Always save a calibration image known X and Y magnitudes that can be used to do the calibration Press the Cal button then drag the mouse vertically along the known 135 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual length of the calibration image Upon mouse release a window prompts for Y image size enter and click OK Then drag horizontally over the known width ofthe calibration image Upon mouse release a window prompts for X image size enter and click OK A prompt for units appears enter units and click OK Place the mouse over the Cal button and the calibration information is shown in the information panel at the bottom of the main jClamp window Help for other buttons is available by moving over the other buttons These values can be entered into the ini file for automatic loading This is useful if the scale of the video capture remains the same day to day Otherwise recalibrate daily for proper readings Data Load Window Data Load 136 jClamp32 Whol
87. during the analog stimulus to the cell Triggering is repeated for each episode You must make sure that the rate can be handled by the camera see FastLook help file to determine frame rate of the camera You can run the command by pressing the Init button or by saving the protocol and running from the RPD directory or script as usual 105 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual B Command Utility vClamp with model Parameter File c jedata 10504 none Mene OOOO O Partition Design gt abf template AGO N Select Copy SanosSaces miwat e 1 Seas ipd s CAS Dp x 77 ch0 3 ere com id Points Cik us Hold B s L5 max 21 H sys 10 o0 mV Inter ms Inter ms custom chan HD Xl T Dh ry o 10752 View Command PrePulse Vrange Trigger rr Epi max 50 Ky C Di Epi E T Cron C Ky C Di Run S el _ amp Data and video files are saved to the jClamp data directory The filename of the video clip is a combination of jClamp data file name and current time plus the extension vda In analysis window opening a data file that has an associated video vda file will open FastLook internally within jClamp for viewing of the video see windows below IN the jClamp Data Load window when an abf file is clicked on if an associated FastLook video exists it s filename will be displayed If the checkbox next to lt FastLook Video gt is unchecked the video file will not be loaded when th
88. e abf data file is loaded If it is checked both abf and vda files will be displayed 106 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual v FastLook video C f01 19 2010 11 45 40 vda fizssssPm iko videols Size P Inone associated 240188 Episo a e List type Select param HH r f Home Office If the play button is pressed then the video will play and the jClamp red cursor will jump to each time location where a video frame was captured Pressing up down frame will do the same You can also drag the cursor and the frames will follow the red cursor time 107 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual E Analysis File c jss manuscripts working manuscripts cl_coupling reanalysis LS10225 S0225010 abf vClamp with cam c Avg 1 Epr1 Part 2 Parts all sel Epi all sel G3 e e L a es o 123 904 185 856 247 808 309 760 LANE E EE E 122 etm asii mipx rme Sn Si Cursors E aa SAI cz Sie Select Cip click T a ir v 4 P es fige Bue 52 ms E Fu c l 42 S 14 Jel Frame p25 ds 22 2 28 2010 4 18 20 p gl x 56 Y 0 M Enabling Export data to file will save data read at each cursor position during playing to a file for future analysis in external programs Also video can be exported to ImageJ see FastLook help file 108 jClamp32 Whole cell voltage and current clamp 25
89. e cell voltage and current clamp 25 5 0 Manual 5311002 abt Ss3r1001 abt s5311008 abt 55311009 abf s5311010 abf 55311002 8 05 21 PM cm ramp 55311003 8 08 28 PM cm ramp n 55311004 9 09 57 PM cm ramp 55311005 9 14 19 PM stair cm 55311006 9 16 11 PM cm ramp 55311007 9 32 09 PM cm ramp 55311008 9 32 51 PM cm ramp 55311008 9 53 56 PM cm ramp nm ramn AL oA oA oh oA o 2oo0oooooot Info button toggles information for all files located in the data directory for example time protocol clk Rs Rm etc Select Export to Integer format to convert from floating point to integer data saved in same directory with dat extension Plots data as saving Loadable in Axon Clampfit or Synaptosoft Mini Analysis programs Auto plotting or printing is done for a group of abf files scales can be fixed and set in plot window prior to output if the selected data files are two sine Cm collections the plotted or printed parameter will be the one selected prior to output By default the saved calibration data for 2 sine files are loaded calib checkboc checked Uncheck it to use the calib files already loaded Axon ABF and DAT files can be loaded Extensions can also be PDF and if selected Acrobat or reader will be opened with the selected file displayed Use this to looked at scanned lab notebooks for each day Press search button to open ABF search engine ABF Search 137 jClamp32 Whole cell voltage an
90. e cell voltage and current clamp 25 5 0 Manual jClamp for Windows Introduction Overview jClamp for Windows Fantastic patch clamping C l amp for Window Sis a user friendly whole cell voltage clamp data acquisition program which features real time monitoring of cell and voltage clamp characteristics The program has no frustrating menu labyrinth Instead all choices are displayed on each main window or in some cases mouse movements over an area on the window pops up additional choices At all times the patch clamper can determine cell status Data collection is initiated through a rapid parameter directory RPD which maintains a list of 18 parameter filenames or script filenames Either a key press or mouse click begins the predefined voltage or current delivery protocols Scripts are text files that allow groups of parameter files to be called and run Most tasks are automated Some data analysis is available However data is saved in axon binary format abf and can be loaded into programs like Clampfit and Axoscope Axon Instruments CA Additionally data can be exported in ASCII format or via automation to MATLAB Origin Sigmaplot and Excel for further analysis When the mouse icon is placed over a particular item in a jClamp window be sure to check for quick information on that item in the left lower panel of the main jClamp window Copyright 1996 2015 J Santos Sacchi Last modified Jan 1 2015 ty sy Home Send jCla
91. e comparable In fact I suggest that you read the pClamp Clampex manual The currently selected parameter filename is displayed along with its date of creation The author field can contain the designer s name and if so desired the file can be marked as private so that no other users can view the protocol A password is then needed to open the protocol A comment can be appended to the protocol Check the box and mouse movement over the box will open up the comment window For example the solutions used for the experiments could be detailed Upon loading the protocol the user knows that a comment exists if the box is checked WREST e eedem cs 25000 Patton De 2 N Selec res private Ie Ji noi comment x password Properties Analog Channels gt Start 30 in EH ines ae Figs Time 10 24 RE Inc fo fous L s pU ER ium o JI on T Plot Results T Tagger Kof ma Ps Analog output depends on the board that you have Version v 22 0 0 and later supports 4 DAC outputs for the Axon DigiData 1440A and IOTech Personal Daq CED Power will follow If you collect data or make protocols with this version then you will not be able to view them on the previous 2 DAC versions You may select any channel out and the associated input channel will be selected a pop up window Gain will show this e g selecting Chan 0 will select input channel 0 selecting Chan 1 will select input channel
92. e date The directory name for the information given above would be C TJ95913 If auto file save is disabled the user is prompted for a filename Auto FileSave This entry is now only in the INI file file are saved after collection If disabled the user is prompted to save file Disable File if enabled no data is saved after collections and no prompts are displayed This is useful when equipment is being checked Chan Watch if enabled movement of mouse on this item will open a new window Four channels 1 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 4 in addition to CC Censor input Ch 0 are allowed at a sampling rate defined by the user Labels and scales can be entered These data are saved to disk with CC Censor data and can be viewed with CC History Digital Out mouse movement over the icon of the amplifier will open a window which can set or clear TTL level output on digital channels 0 7 channel 7 is reserved for oscilloscope triggering First select the desired channel or channels and then press the Set button The On column will indicate which channels are on high or 5V The Clear button sets all channels off low or OV The digital out set here is steady state Digital out can also be set in the Command Utility to provide transient or synchronous output with the analog output signal Following any parameter run the digital channels are reset to the values set here in Cell Censor if CC on is enabled
93. e is balanced out with positive feedback and is achieved when the system is near oscillation Of course you should back off compensation a bit so as not to kill cells with these oscillations You should have multiclamp telegraphs working in jClamp so gains and mode can be jClamp32 Whole cell voltage and current clamp 25 5 0 Manual registered by jClamp H AS jClamp Ini File Edit 32 bit zu Q pe User Files Gain T0 T1 T2 T3 CC Hist CC Script i Files Data Drive 3c wWindows8_05 Auto 5 x FileSave Data Directory auto x FileName Data Path jcdata x DataDirectory Param Directory c program files 86 Njclamp32 param b H J icdata US Eo v B U Parameter jclamp prm RPD jclamp rpd X UAD iclamp usd k CMT jclamp cmt S Screen Shot 1 CM track Calibration V mclp v ch 02 apr 14 10us cal L CM track Calibration osc2 12 oct 14 10us cal 10 24 14 10 56 55 p jClamp32 Whole cell voltage and current clamp 25 5 0 Manual L jClamp Ini File Edit 32 bit v Ta oe E CC History Resolution 0 025 sec 1 IRs l 3j Ccm Time 30 sec fe of 1 efor 1 T CC History on by defaut 2 Rm lt 4 Ji i User Fies Gain TO T1 T2 T3 CC Hist CC Scit MultiClamp V Load MC700A Commander and Telegraph Servers ile L MC 700A path S iv Load MC 700B Commander and Telegraph Servers J MC 700B path C Program Files x86 Molecular Devices MultiC
94. e new j Clamp version or a quickie update which only replaces the lt jclamp32 exe gt executable file with other files as needed The update files are now named with version number the quickie file will have the form jc exe v 21 10 12 exe and should placed in the jClamp32 directory for extraction and the full MSI file with the form lt jClamp32_v21 10 2 msi gt should be saved to the jc msi directory under the jClamp32 directory If you update automatically from the web the files are downloaded in the appropriate directories and run automatically You may turn off checking for updates in the ini file under the History tab but will still be offered the opportunity to check for updates in the Identity Window when jClamp starts or in the About windows accessible from the popup Bar menu at extreme left movement of the mouse Manual updating Upgrading to a new version is simple with the Microsoft installer Download the msi file into a directory on your hard drive say jclamp32 jc_msi jJClamp installation file double clicking on this file will install or upgrade or repair jClamp jClamp32 msi To install a new version double click on the jclamp32 msi file and remove the old version first Then double click on it again and install the new version All files that are initially delivered with j Clamp e g jclamp ini and parameter files are replaced so if you use those which you shouldn t make your own with new names
95. e sure that CC holding potentials are the same as the protocol holding potentials for all channels that are active Prior Update Midnight issue fixed At midnight a new directory will be made for data collections If CC save is on it will turn off and restart for saving in new directory Inter episode times are now saved with data file View by moving mouse cursor over inter episode time in command protocol window in Analysis mode Prior Update Sister parameter files individual files can be linked to follow each other simple alternative to script files For example jclamp prm test prm test1 prm and test2 prm can be run sequentially by renaming first file to jclamp test test1 test2 prm New box in Parameter load window lists files by date or by today s use This supplements typing names which lists by matching ABF fileload window info reports if math model was used If Matlab model was used script is appended to abf file and then shift right click on filename opens script file in text box Cm calibration file is correctly listed when mouse is over calibration button in Analysis window The file has always been appended to abf files for Cm corrections jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Works in Win 8 Set run as administrator in shortcut And of course bug fixes Full installation to use 7Zip compression for quickie updates Bugs fixed as well Updated MD in cell stray capacitance compensation w
96. e the parameter estimation method of Pusch and Neher 1988 A poster on the subject was presented at the Yale Neuroscience Retreat in 1992 The following description is taken verbatim from by funded NIH grant proposal R01 DC02003 submitted in 1992 Also see the Detrending technical note supplied with jClamp accessible from Windows Start menu It is very important to detrend the data when sinusoidal stimulation is superimposed on rapidly changing voltage commands as would be the case for investigation of voltage dependent capacitance Starting in version 17 11 a new method of detrending is used which does not add any additional noise to the analyzed data When you check the detrend button during Cm analysis a widow pops up below the checkbox The choices are auto and a range of numbers detrending iterations from 1 20 The auto feature chooses the correct number of detrend iterations but there may be a number below that which has less noise in the Cm trace so you can choose the number of iterations manually Detrending removes rapidly changing currents without filtering the 2 sine response prior to FFT analysis See the Detrending technical note supplied with jClamp In the present experiment a discrete frequency analysis will be attempted Pusch and Neher 1988 have used a single tone sine wave actually technique to determine cell characteristics under whole cell voltage clamp By injecting a sinusoidal voltage into a cell and mea
97. econds video grab now true grab a video clip bitmap not extension to disk 113 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual video auto on off turns on or off time lapse video collection video on off turns video on or off video save V on off enable saving of an ascii file same name as the avi filename but with extension asc which holds info on frame number and pt within command protocol which corresponds to frame episode time into command and time after video start load_cmt filename without extension cmt assumed commands below still work but this does it all cm_trk start enable record stop start record or stop cm tracking FastLook with Prosilica GigE fastlook capture frames set to capture frames fastlook capture time set to capture time fastlook capture_length in time or number of frames depending on setting above fastlook fixed rate on enable capture at fixed rate useful for time lapse or fixed sampling fastlook fixed rate off disable capture at fixed rate maximum rate will be used fastlook set frame sec set frame rate in seconds fastlook set frame fps set frame rate in frames per second fps fastlook frame_rate frame rate time in seconds if lt set frame time is set number of frames if lt set frame fps is set fastlook disk stream on set saving direct to disk may be slow fastlook disk stream off fastlook aut
98. ector PSD is exposed aS Admittance Viewer rx R dither PSD Angle 0 1376 step 2 4 42 4993 nS 1 708 rad C dither T dY das 309 vid 0137 Set step 2 dY dCm 04079 Set Tk J Zeol LE 100 nS Export Azero lm When single sine analysis is selected a check box is displayed which allows you to use the phase sensitive detector The method is detailed by Gillis 1995 but instead of integrating over a full cycle I use the FFT to get at the real and imaginay component at any selected angle Any angle can be chosen by moving the slider Right clicking increments by 1 57 radians The angle of OY OCm derived from two sinusoids see Santos Sacchi 2004 or the angle OY OGs 90 can be set or toggled on for tracking Zeroing and scale selection of the in phase Y1 and quadrature 90 Y2 response is possible Pressing the left and right arrow keys will increment or decrement the PSD angle You can use this see what angle is returned by dithering either the Rs or Cm compensation controls The buttons on the right side implement angle estimation from dithering either the resistance or capacitance Step one button is pressed to begin the process then either RS compensation or Cm compensation controls need to be changed a small amount no need to modulate the changes just change a steady amount then press step 2 button There is now a dither feature in the main CC window which lets yo
99. en plots have selectable interplot periods and a pause feature Each data file has appended the parameter file in ASCII format allowing the display of all information in the build utility during data loading Key ASCII information can be searched for and files save to a new directory for group analysis Logging implemented Recall and play abf cc cm not audio video text cc clip files from log window with button click Search logs for information Show all logs on disk Edit them Log is run from Main menu bar in jClamp when in Analysis mode or can be run as an independent program jc log exe Capacitance tracking allows monitoring of cell characteristics using two sine FFT based analysis with analog and or digital stimulation prepulse incrementing pulse ramp at 2 5 or 5ms resolution and noise of 10fF model cell with 200 MHz Pentium Additionally capacitance Rs Rm can be measured simultaneously with your favorite step ramp or AC protocols Automatic analysis Very powerful feature Analysis scripts under construction Binary save of Cell Censor and CM tracking data files Faster save and load and smaller size Ability to save in ASCII format set in INI file Screen Shots to save jClamp window arrangements for instant retrieval Collapsible Cell Censor window Sizable plotting window during data collection drag to size Unobtrusive RPD window updated searchable help file including help on the new features Sc
100. er INI file can be loaded in place of the default jclamp32 ini file if the file is appended to the command line for example in a shortcut icon c clamp iclamp exe myfile ini Clicking the EXIT button unconditionally closes the program After clicking the OK button the Cell Censor window appears and the Menu Bar is accessible by moving the mouse cursor to the extreme left If data had been previously saved to the data directory of the current day a flashing window indicates the number of old files and the next file will be automatically named with a higher number no fear of overwriting Often times jClamp windows will be arranged to suit a particular working condition It is possible to save the arrangement information for quick retrieval rather than repeatedly aligning windows manually Clicking jClamp32 Whole cell voltage and current clamp 25 5 0 Manual command scn on the main screen with the right button opens a small Screen Shot window Clicking again on another area of the screen moves the small window to that area To save a face jClamp window arrangement type in a file name do not add an extension the default is scn and click the disk icon To retrieve arrangements double click on the filename in the screen file list box Clicking on the screen with the left mouse button simply closes the Screen Shot window jClamp was designed to use a screen resolution of 1024 x 768 16 bit color and small fonts all set in t
101. er estimation is detrending of the data FFT measures of selected frequencies in our case the frequencies of the stimulating sine waves are best evaluated if other slow time dependent changes in the response are removed In jClamp if you superimpose the stimulating sine waves on a time varying stimulus e g a ramp or slow sine wave then it is best to remove this slowly changing response from the data prior to FFTing I use a linear approach to detrending but it is on a very small relative time scale so it avoids problems with whole dataset nonlinearity An example Given a 10us clock and a 128 ms ramp with 2 sine stimuli superimposed at a resolution of 1 28 ms these are all set in the command utility there are 100 repetitions of a unit stimulus consisting of one period of the low frequency f1 component and 2 periods of the high frequency f2 component the ratio of f2 to fl is 2 I detrend on the scale of the unit stimulus which for the whole stimulus 100 times larger proves to be much better than full linear detrending and I think on par with a nonlinear spline approach The success of this piece wise linear approach is shown below Here I have used the math model in jClamp to generate nonlinear responses owing to a voltage dependent capacitance A difference equation technique is used in the model to obtain solutions The nonlinear capacitance derives from the first derivative re voltage of a simple two state Boltzmann 168
102. f 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 166 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Detrending for Cm Analysis Technical Note 1 SciSoft Company www SciSoftCo com Detrending of dual and single sine data in jClamp detrending now exposed in jClamp Cell membrane capacitance can be measured with the 2 sine or phase track technique in jClamp Either a 2 sine or single sine stimulus can be superimposed on any waveform created in the Command Utility 94 0 20 4 16 4 90 0 0 Command Pat 2 Epi 1 x 115 v 500 Single Sine 90 0 0 Command Pat 2 Epi 1 x 932 y 500 The response from a simple cell model is shown below 2969 5939 8908
103. g the original implementation where f2 2 f1 index into the FFT array being 1 and 2 and the other f2 lt gt 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 fl of 390Hz and f2 of 781 Hz could generate distortion components at f2 f1 which would sum with the fl 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 lt accalib prm gt
104. gain for proper viewing See camera instructions autoexposure push autoexposure button to automaically set exposure time for proper viewing see camera instructions savebox on save only that area of screen within a drawn box savebox off save whole screen shutter open open camera internal shutter if present pshucter Close close camera wnternal shutter af present showbox on show only that portion of the screen that is within the box showbox off show whole screen movie trig on set whole movie capture possibly consisting of many frames to occur when hardware trigger is sent to camera trigger input 162 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual movie trig off disable movie trigger trame ELL VOM see sangile frame capelure oou with cach trigger sputum number of desired frames is acheived Eeamey UGiG oTt See frame trigger ot PELXeC EacelOM Sel Caprure Eo Occur az fixed frame races Phils wall beim ted by Windows fixed rate off no fixed frame rate speed will occur at maximum rate permited by software in Windows gsermsamegsecon Seba eeciiteamesarseimars embriones amete set frame fps set fixed frame rate in terms of FPS frames per second data pathid testl gt set data path to ds restl Che current date is automatically added as a subdirectory e g d test1 02 28 2010 in jClamp directory is auto set to jClamp data directory The default ini file can be re
105. h a space in between the two To access the properties of a desktop shortcut right click and select properties jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Support for both Axon Multiclamp 700A and 700B telegraphs Previous version only supported the 700B See the jClamp help file under Telegraph Support for new board NI USB X Series Amazing 1 megaHz 16 bit sampling on all 8 channels without multiplexing Synchronous digital output About 3K Could not ask for more The new NI DAQmx 9 3 drivers need to be installed Visit NI at http joule ni com nidu cds view p id 2337 lang en User info License owner board operating system disk size and memory collected when check for updates occurs Useful for me to identify user issues and aid in debugging Automatic or manual check for new version of jClamp on the web set choice in ini file under Script Tab Impedance analysis with Nyquist Bode Plots and model fitting Additional script commands to control CC window parameters jClamp Features Vista and Win7 users must disable User Control Account in Users Dialogue In cell stray capacitance compensation for Cm measures Digital flip flop overrides partition digital out in command protocol Look32X video capture with cameras and devices with DirectShow or VFW drivers Synchronized video capture with electrophysiological data as best as Windows will allow TIFF and other image file support FastLook Andor www a
106. he Cell Censor vClamp with r 9824014 abt Ix CO on Sav x VO x V1 10 H rt x wc UX wel hul ms x Model 10 d X TeleShow I Dis dual kvg gain tab or eid x Inter mun check the model checkbox while jclamp is running Anytime you make changes to the ini file you must save the file and restart j Clamp or press the lt re ini gt button on the Bar Menu It is probably better to restart jClamp The settings open in a pop up window when you move the mouse over the graphic default is a spiral ganglion cell replace that graphic with a picture of individual jclamp users set in ini file in the upper left region of the Cell Censor window jClamp32 Whole cell voltage and current clamp 25 5 0 Manual You must make sure all paths in the ini file match your machine s paths For example in the ini file default the directories are C program file jclamp32 but yours may be different Make sure everything is set right in the ini file and then save it as a different name like your name ini You can specify your ini file rather than the default jclamp32 ini by doing the following C WProgram Files jclamp32JCLAMP32 EXE my ini file ini gt If you find that the file lt scrrun dll gt is giving problems then it may be necessary to register it Run the following on the command line REGSVR32 EXE C WINNT system32 scrrun dll Make sure your windows directories are correct For win98 it may be
107. he Windows Display property settings Menu Bar Menu Bar Accessed by moving mouse to extreme left of jClamp main window If desired the bar can remain visible by moving it move mouse after mouse cursor is down between icon and X button Closing the MenuBar window with the MenuBar s X button sets it back to the hidden position If the MenuBar becomes somehow inaccessible simply click on the main window s X button in the upper right corner It should reappear jClamp32 Whole cell voltage and current clamp 25 5 0 Manual _ jClamp for Wind Exit exits the program after verification If data were saved to the current day s data directory the user is notified that data files exist If no data has been saved then the directory is removed Edit ini opens the separate program Iniedit exe with the current INI file This file contains information which should be set for the program to run correctly An example INI file is shown below with all possible settings The file can be edited with Iniedit exe outside of jClamp as well Re ini if changes to the ini file were made while jClamp is running clicking this button will incorporate the changes without having to exit the program CC history Displays a plot of cell and clamp characteristics during the past N seconds N and the sampling resolution are defined in the ini file Three plots are available but each can be used to display any of the Cell Censor data items or data from Chan
108. he extra variable time before the command voltage If the process being studied can recover during this extra holding time then use the command protocol to deliver prepulses because the time between prepulse and subsequent pulses can be controlled precisely in an episode The AXDD132x series systems 1200 board and IOtech daq2000 do not have a delay between prepulse and command for prepulse lengths less than or equal to 1 sec Above one second a delay of about 40 80 ms occurs which may be machine dependent The National Instruments boards function the same except there is no delay up to 10 sec Check on an oscilloscope The P N protocol is used to subtract leakage currents as described by Bezanilla and Armstrong The setup is the same as Axon s except that subtraction currents are always saved so that the user can toggle between raw data and subtracted data traces If you enable the Size checkbox a fixed size percentage of commands is given instead of P N size The number N of pulses at the subtraction hold potential is still delivered but appropriate scaling 1s made to subtract the linear currents You can open the jClamp32 Whole cell voltage and current clamp 25 5 0 Manual subtracted files from the data load window choose sub extension Currents are unit size before scaling you can confirm that subtracted currents are linear If the currents are not linear the data file can be un subtracted in the analysis window to get the raw data to
109. he jClamp VXD or SYS driver The port address is found in the windows Control Panel That address should be set in the jClamp ini file Only four 0 3 digital in and out channels are available Scope trigger in on Ch3 ChO is trigger input Cm tracking and gap free are functional only in Win 95 98 although 2 sine command protocols are supported Win 2000 XP Pro performance is superior IOTech boards The IOtech daq2000 XP Pro PCI board 16 bit in out 100kHz should be set up according to the manufacturer It will work in Win NT and 95 98 It is inexpensive 500 and quite nice It has features not present in the other boards Unfortunately currently it has a noise problem on the DACs which may be fixed in the second version The IOTech Personal daq 3000 2 or 4 DAC is a great board at about 1500 There is a 4DAC version supported by jCamp Clock down to lusec Trigger in to start episodes is via digital port B7 AND Trig input which should be coupled together a high TTL triggers start Nice Tecella Pico patch clamp system Support for the Tecella Pico www tecella com USB patch clamp amplifier Fully contained in a very small enclosure Clock down to 25us in 25us steps Synchronous digital output with analog out in Four individual units supported simultaneously Waiting for company to fix some bugs in their driver National Instruments boards National Instruments E series PCI MIO 16XE 10 10 usec PCI 6052E 3 usec boards
110. hile CC data Save was enabled the CC data plot can be used to rapidly access and display the data files and comments Place cursor over the green comment or red data file tags and the file names for either are displayed By clicking the file names the comments audio video text or cc clip are displayed played or the data files are loaded and plotted As the cursor is moved data values for all CC data points are displayed in the text box on the right Channel identification is also indicated in the data text box Data plots can be scaled or manipulated as for the Analysis plot see Analyze The buttons allow file operations printing including automatic printing of all CC data for a given cell trace fitting and toggling between points or lines Opening the second display channel reveals more options such as linking the x or y scales in both plots exporting to ascii and copying to clipboard When both plots are displayed both can be printed together At the bottom is listed the rms value of the data that is displayed in the plot window Clip click see Analysis window is available If the x axis is time then all data parameters are sent to the clipboard at the selected time point The export button if clicked with the left mouse button will export the data from a single plot to an ascii file If it is clicked with the right button all data for each cell are sent to seperate ascii files CM and CC data can be extracted this way Data
111. his picture can be any bitmap jpg that you desire and can be loaded in the ini file Movement of the mouse over the picture opens a window that allows you to choose voltage clamp or current clamp mode The Cell Censor channels are either O for output O for input or 1 for output 1 for input VO for channels 0 0 voltage clamp 10 for channels 0 0 current clamp V1 for channels 1 1 voltage clamp and 11 for channels 1 1 current clamp The check boxes we0 and wc1 control whether clamping polarity is in whole cell mode checked or inside out mode unchecked for channel 0 or 1 This is equivalent to but more accessible than inverting the polarity of input and output gains in the ini file A math model can be toggled on off If a board is present and properly set up in the inifile the name of the board will appear in the caption of the Cell Censor Window otherwise model will be displayed The display of telegraph jClamp32 Whole cell voltage and current clamp 25 5 0 Manual inputs from a patch clamp amplifier is also toggled on or off showing an additional separate window See Telegraphs Enabling Dis dual uncouples the default dual patch clamping setup channels in out 0 0 and 1 1 If checked you may deliver a separate command out of channel 1 by selecting chan 1 in the Vhold box see below while monitoring channel 0 in CC When in effect pulse stimulation out of channel 0 is stopped and is replaced by channel 1 In this way you can stimulate the
112. ic saving of data no user query will be made ec inter true false enable disable cc collections during wait intervals of protocols inter cmd hold true false enable disable use of protocol holding potential during wait intervals else cc hold is used cc alt polarity true false enable disable alternate polarity flip of vpulse CC fix scale value fix CC scale value height of plot window channel 0 value shown in tooltip text on mouse movement over Fix checkbox cc fix scale 1 value fix CC scale value height of plot window channel 1 cc fix O true false fix CC scale enable disable channel 0 cc fix 1 true false fix CC scale enable disable channel 1 cc pulse value pulse size in displayed units channel 0 cc pulse 1 value pulse size in displayed units channel 1 cc navg 1 49 number ofcc pulse responses to average for parameter calculations cc ignore rs O true false just get dc resistance and put into Rm parameter cc ignore rs l true false mark it true put a time mark in cc data file cc zero O true false set togglable or settable options in Cell Censor cc zero l true false cc fix 0 true false cc fix 1 true false cc elk 0 value channel 0 CC clock cc elk 1 value channel 1 CC clock 110 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual auto clk O true false auto clk 1 true false flip trk true false jss specific for Vpkcm tracking cc ho
113. icolon is a comment capture length 15 set the capture length to equal time in seconds Gapture vimed om Set capture length On OEE audio on set capture audio on off Erame rate 20 7 set fixed trame rate video codec DV Video Encoder Video codec no compression audio codec PCM codec quality 100 time lapse 2 dara Pathe look32x dabas ser datd path Bg ce looks2x cata X the current date is automatically added as a subdirectory e g c Alpok32x data 02 23 2010 104 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual auto save on auto or manual save manual asks for directory and filename FastLook Video Capture FastLook Video capture linked with jClamp In order to use FastLook the fastlook checkbox must be set in the jClamp ini file If it is not the FastLook FL camera icon in the Command Utility window will have a big red X over it If the ini file is set FastLook will open as an external instance independent of jClamp but linked to it via background polling push Channels Out TRI cn gas ft Sa LE C nhan Co aisle a xe Averages o If enabled moving the mouse cursor over the camera image will open a pop up window Check the FastLook Linker check box to allow digital out channel 0 to trigger the Prosilica video camera The rate at which trigger is performed is entered into the Rate text box Digital markers are plotted where trigger events will occur in time
114. id Chan Watch tau ms 0 1732 Rs MQ Rm HQ S3540 1 Cm pF 17 879 Ihin DT L in Freq options Fi2 a Dual C Single C etm C MD f1 195 3 EI b S Fas m Res 5 12 lt I Ps e zm Starting in jClamp version 17 0 Cm analysis is more easily available from the CC window now it is also referred to as Frequency domain cell parameter estimation You may toggle between Time and Frequency mode on the fly All selections in CC are enabled during CC data collection for example in Frequency mode you may change analysis type Single Dual eCm or Sample Resolution or holding potential or Vpul size on the fly 3 sine analysis 1s something in progress Place the mouse pointer over the values of Rs Rm and Cm and a pop up shows the RMS value Capacitance measures can also be made within ordinary protocols see Command Utility Now CC Frequency domain analysis and protocol analysis work together to get clean Cm measurements Below is a description of sine wave analysis in jClamp and more details about the new features For all methods except dithering it is imperative that you calibrate your system Things will not work otherwise See below jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Cm Rm Patch cell model Single sine analysis The current that is measured under voltage clamp is the current through Rs namely Irs In the single sine
115. iles and view current settings in the jClamp info panel at the bottom of the full screen You may also choose to detrend data or not You have to read the detrending note if you superimpose sinusoidal stimuli on ramps or other fast time varying commands See the Technical Note in the Technical Note subdirectory under the jClamp directory for information on detrending Detrending now corrects without adding any noise to the data If you set the angle to measure single sine in CC it is available here in the PSD box PSD must be selected to use the angle to correct the magnitude of current at that angle and 90 degrees away which 123 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual can be viewed when accessed under the X Y drop down boxes Also Real and Imaginary components of the raw current can be viewed If you used the Cs feature in CC prior to collecting sinusoidal protocol data you will be able to correct for the electrode stray capacitance by checking the Cs box which will only be enabled if you performed Cs correction The corrections are saved with the data file In the Cm analysis pane the dropdown box f1 f2 selection 1 2 or 3 permits the extraction of Cm based solely on 1 2orthe average of the two respectively While using a math model produces equivalent results real world conditions e g different noise levels at each frequency changes in stray capacitance nonlinear distortion may produce differen
116. iles jclamp32 gt In that directory there is a default software only license named lt jclamp32 lic gt You should rename that to lt jclamp32_soft lic gt copy your license file into the jClamp32 directory and rename it lt jClamp32 lic gt It should not be write protected When you run jClamp you should be able to turn off model mode in the ini file on startup or in the pop up under the user graphic see image below to access hardware You must make and load an ini file to access your hardware See help file accessible from Windows Start button or About button in menu bar in jClamp opens on extreme left mouse movement see second image below for further info on getting started jClamp32 Whole cell voltage and current clamp 25 5 0 Manual D Cell Censor vClamp with model IOTech board selected Vhold mV S1516005 b 0 1 CC on Save SP Pass Ei Ma P ix VO x vi 4 ke ag n C inter f Di There is a new method for communicating with a jClamper during experiments without interference during jClamp32 Whole cell voltage and current clamp 25 5 0 Manual recording A text file named jcreadme txt can be placed in the jClamp32 directory at any time e g remotely via network This note will pop up at the start of jClamp or if JClamp is running when the Cell Censor is turned off The note can be printed prior to exiting the window Upon exiting the file is deleted but the info
117. iodically check for changes in response jClamp32 Whole cell voltage and current clamp 25 5 0 Manual time constant actually clamp tau and adjust the clock appropriately If PASS is enabled then the CC clock value will be is used for any subsequent data collection via parameter files i e the parameter file clock is overridden NB notes can be written to disk in either text format click pencil icon or audio format click speaker icon if a sound card is present This is most useful when CC Save is enabled since links to the comments are available when viewing the CC data file under the Analysis section In addition if a video board is present with Video for Windows drivers clicking on the camera icon will open a window that allows viewing and saving camera output in a scaleable window see Look Section of Help TeleShow Toggle telegraphs display for amplifier program interaction Channel Selection Only channel 0 and 1 are available in Frequency domain mode Choosing 0 channels 0 0 selects output channel 0 and input channel 0 e g connected to patch clamp 1 choosing 1 channels 1 1 selects output channel 1 and input channel 1 e g connected to patch clamp 2 choosing B both channels alternates between channels 0 0 and 1 1 pseudo simultaneous recording choosing C both channels also alternates between channels 0 0 and 1 1 but measures single cell information and responses in the adjacent cell enabling estim
118. iods of time Video size W and H can be set in the Controls window or under Options tab in Look32X All settings are dictated by the camera driver supplied by the camera manufacturer Multiple devices cameras can be chosen from if attached to the system Preview must be set to capture video Overlay can be used to simply monitor in near real time for example to get patches You can zoom in if Zoom is selected in the Controls window by pressing left mouse button down while dragging a box You can crop if Crop is selected in the Controls window by doing the same With cropping only that portion of the image is saved to disk during video capture Bitmaps of video images either jpeg or BMP formats can be made during preview or playback jClamp32 Whole cell voltage and current clamp 25 5 0 Manual jClamp for Windows with IOtech PersonalDaq3000 E Cell Censor vClamp with IOtech PersonalDaq3000 EF Command Utility Parameter File w iotech v 19 7 avi test4 T Vhold mV 50519007 abf 0 Pass si 10 ir Pass NB B CC on Save C 80 X Auto e fe b v SEM C OC IC BC CT Dex 16 e 40 fo MAE CC inter Disable File M rwm Santo Saah Cmd hld Chan Watch 1 Hz 80 TRR o g 0 f 1 Gea comment x pa tau ms Vpul mV ER CRANE 2 10 r Si X Esa FL asma aT T 9 a CRB l D nChan Rm MQ ng Br m 1 Averages mI Cm pF Inter ms Number jo RR Ih
119. ith software assistance including automatic compensation see Cm Analysis in Help file Digital trigger now has minimal delay between triggering and data collection except for protocols with P N prepulse or CC inter use Cs stray capacitance compensation prior to cell pop in disabled Replaced by robust in cell stray capacitance compensation using MD multi dual sinusoidal stimulation see help file Molecular Devices releases new Axoscope pClamp 10 3 1 4 with new DLL to fix spurious voltage output and allow digital output during 4 DAC output j Clamp v22 7 0 and above uses this fix Support for the Tecella Pico USB patch clamp amplifier www tecella com Up to four units running simultaneously Support for 4 DAC outputs for those boards that have four DACS including the Axon DigiData 1440A IOTech Personal Daq and soon the CED Power The previous full version was the last one that supports only 2 DAC channels Future versions coming soon will support 4 DAC outputs for those boards that have four DACS including the Axon DigiData 1440A IOTech Personal Daq and CED Power jClamp will now automatically startup with the last ini file selected in the Identity window An edited shortcut will override last selection if different Remember to make an edited shortcut linking directly to your ini file edit a newly created shortcut Target property by placing your ini file e g my ini file ini at the end of the target command wit
120. king slow tele will allow checking telegraphs every so often as a Windows clock allows about a few hundred milliseconds else every time a CC data point is collected If you check slow then CC collections could be faster You may need to check fast so you can switch between current and voltage clamp without a glitch e g possibly giving a larger stimulus than wanted imagine in voltage clamp you give 10 mV steps in CC then you switch to current clamp If jClamp doesn t respond between the delivery of stimuli it could be possible to inject a 10 nA signal because internal scale factors would not have been changed to current clamp in time For this reason default is fast check Chan Watch Channel Watch D Cell Censor vClamp with model Labmaster DMA selected Vhold mV clk us T T none 2035 Pass s C Pass J R CCon Save L X Auto I HT 1 Bt ET Das 40 z CC j C CC inter Disable File mM 4 mi Cmd hid X Chan Watch 1 Hz 80 mE i m 0 63 Chan tau ms name scale X AutoGain 4 Zero X Fix Z Store BkG FFT 0058 ak fic h Rs M 5I PKP 11 4 84 6 vok M Rm MQ 7 ix 10 489 63 X itest ho Cm pF Every 1 sec 12 086 Ih n r 0 035 ffset mV Vm mV moy uon 40 2 zz o CC Domain Freg options Fi2 7 512 Calibrate Dith P Time C Dual Single Res 5 12 anr e TA m ither on rea DP sm AMD ASIAN xi a Rs Hzfi 0 35 ms 3 1923 n jClamp32 Whole cell voltage and c
121. l 17 14263 2 17 11517 f3 17 084856 f4 17 04954 f5 17 10370 2 Capacitance Compensation Cs0 guess fit 0 pF Res Cs1 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Cell Censor vClamp with Axon Digidata 1440A none 10504 Vhold mv clk us N BET BENI ims Pass hold s T x free IN 10 fe 0 1 CC C Bes fo orn CCinter Disable File Cmdhid Chan Watch tau ms Vpul mV 0 1800 7 10 Asal si f 9 10 9 MV Rm MQ rmm 311 94 Yerum Cm pF nter ms 17 132 0 Ih n 0 121 Offset mV Vm mV T on 3 4 40 oO Freq 2 n 1 35 Hz CC Domain ose options Ip S z CM Calib Dith B f Time Dual Single ts Io ILLA d aa Y Freq C etm MD ft 1953 F fc Al xj Hef Cm f1 17 13586 f2 17 11029 f3 17 09146 f4 16 99514 f5 16 69996 15 Capacitance Compensation C0 2ero EXT f 0 6079242 pF ResO 0 Mohr Cs1 pF Res jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Cell Censor vClamp with Axon Digidata 1440A none 10630 Vhold mV clk us 10 N x CCI ae 4ms L Pass hold s n Pass LN Z R a x Auto corie p 10 N s s fo of I m CCinter Disable File ES T0031 Cmdhid Chan Watch s So S tau ms Vpul mV Zero x Fix Z 0 1802 Fn ditis Rs M mi d mi l 21 6799 n 10 9 Rm MQ owe 309 82 Cm pF ded ms 17 148 0 Ih n r 0 1
122. lamp 7008 Ci E b Channel Watch Part Plot skip Enable Interval 1 sec 20 1 Label TIE Label P kP Fastlook Scale 1 Scale 1 Neobiosystems P X Enable Enable CM Detrend p Slow Telegraphs no telegraphs Label volt Label test CeliGeeker a Scale 1 Scale 10 MagicSeal Zz Enable X Enable IOTech Diff 4 PujiClamp 3 EE zen P EN qure Wgaphs 4 v Sealtime 0 sec l 10 28 14 12 02 06 a Chin Ch11 Multiclamp must be selected in the jClamp ini file jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Cell Censor iClamp with lOtech PersonalDaq3000 4027036 abf 49 Ihold nA clk us i C 0 z NB t Pass hold S Pass CCT Savel Oms Somme ll foric ag T C 21 a MEE Cocacoog 0 eR Pe CC inter Disable File 9g e Cmdhld Chan Watch n agn m gs tau ms Ipul nA 5 0308 rior rsa 104 C 15 3351 mV Rm MQ nAvg 288 20 8 Inter ms Cm pF 17 456 io Ih nA 0 000 Offset nA 0 1 2 3 4 Vm mV m on 5 E Freq 2 n 195 Hz CC Domain Freg options Fi2 I Bel AE CM C lib z Dith E C Time C Dual Single 51 n E e Freq C elm MD fi 1953 EI lt PLA EN Cm fl 17 42173 f2 17 38139 f3 17 38682 f4 17 30658 f5 17 02276 Telegraphs vClamp se Tele 0 x nis Ch14 Lins Gain ICIp X Mode Tele1 x jClamp32 Whole cell voltage and current clamp 25 5
123. lamp Analysis Window that has been collected with an impedance protocol the Impedance window is opened data plotted and can be fitted Check the Show box and the data are listed hit lt esc gt and the listing is aborted it takes a long time to list the Chirp data at 32K length Right click on table to copy any selected data to clipboard See jClamp Analysis Window help to see how to copy data to SigmaPlot Excel MATLAB or Origin by dragging the icons within the Impedance window onto plots You can enter a surface area to divide currents by surface area with resultant changes displayed check box enabling Reanliz button will reanalyze data for example after loading a new calibration file jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Real kohm 0 27 E Z Calibration iar sis C1C2 3 Cap TOY ka Decifactn mla msan d Rs 0 0075 Ra 10 535t Ca 54285 ed Brers T 2 35 DU tm GP Parts aM sel Epics saiC sei Channel ow LE si Model to N Cab Load 253 CART On kn Peoaos s Cav i1 oi a2 Paneles h OF V guess As 0007S alio 35 Ca 54265 i hw 180 d A Baln 755 Raline as v Puig x Mada 3 50000000 C adir eZ wth nofancl jClamp32 Whole cell voltage and current clamp 25 5 0 Manual D Command Utility lame Pas ameter T de wi itech v 21 8 y jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Look32X Look32x Video cap
124. lampex I will fix when they acknowledge and fix Under NT or Win2000 XP Pro the 1200A board does not work well with jClamp the drivers supplied by Axon from SSI are dysfunctional Cm tracking does not work but the 2 sine command protocol it works The drivers for all Axon boards are installed with the manufacturer s software program Axoscope See the instructions supplied with the boards Get Axoscope to work For the 1320 series 1320A 1321A 1322A Axoscope version 9 or above but see their website to make sure which Axoscope to use must be installed since the new drivers that are released with this version are used by jClamp versions greater than 10 9 0 The boards should be occasionally calibrated with the Axon software Axoscope see the Axon documentation jClamp uses the board s hardware stored calibration data The file lt AxDD132x dll ver 1 1 0 7 gt found within the Axoscope 9 directory must be copied into the jclamp32 directory for the board to work with jClamp For the 1440 series AXDD1440 dll must be copied into the jclamp32 directory However if you have a new Axoscope version that works get the files from that version s directory see below In order to use MD Axon boards you will need to copy some files to the jClamp32 directory You must get Axoscope to work with their boards before running jClamp If Axoscope identifies the boards then jClamp will work 1322 boards AxDD132x dll 1440 boards jClamp32
125. ld 0 90 set holding potential for Cell Censor analysis scaling set in INI file cc hold 1 100 set steady state potential for analog output channel 1 scaling set in INI file dax hold 2 set steady state potential for DAX analog output channel 2 scaling set in INI file gain tab DAX gain text box for CED Power use to control analog channel out 2 dax hold 3 set steady state potential for DAX analog output channel 3 scaling set in INI file gain tab DAX gain text box for CED Power use to control analog channel out 3 dax hold 4 set steady state potential for DAX analog output channel 4 scaling set in INI file gain tab DAX gain text box dax hold 5 set steady state potential for DAX analog output channel 5 scaling set in INI file gain tab DAX gain text box wait 10 wait for 10 seconds before continuing during this time if Cell Censor is enabled cell and clamp characteristics are continuously monitored waitfor wait for the following conditions to be met before continuing Cell Censor must be running cell and clamp characteristics are continuously monitored hit esc key to exit waiting switching between clamp types while under Cell Censor clamp type to 1 continue with script after switching to current clamp e g waitfor clamp type to i clamp type to v continue with script after switching to voltage clamp e g waitfor clamp type to v monitoring Channel Watch va
126. ll be asked for a filename to save to In the Main Tab you can Set the capture length Set capture length to equal frames Set capture length to equal time in seconds Set time out to wait for hardware trigger If exceeded capture is halted but file is saved 151 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Set fixed frame rate disk stream on when capturing video stream directly to disk May be slower than to memory disk stream off when capturing video stream directly to memory before writing to disk autogain push autogain button to automaically set gain for proper viewing see camera instructions autoexposure push autoexposure button to automatically set exposure time for proper viewing see camera instructions bitdepth8 set bitdepth to 8 bit denoted by 0 in file header bitdepth12 set bitdepth to 12 bit denoted by 1 in file header savebox on save only that area of screen within a drawn box savebox off save whole screen showbox on show only that portion of the screen that is within the box showbox off show whole screen movie trig on set whole movie capture possibly consisting of many frames to occur when hardware trigger is sent to camera trigger input movie trig off disable movie trigger frame trig on set single frame capture to occur with each trigger input until number of desired frames is acheived frame trig off set frame trigger off fixed rate on set c
127. ltage and current clamp 25 5 0 Manual 3 Cell Censor vClamp with model Labmaster DMA selected ins S1004003 abf 228 Vhold mV clk us xus 10 z N NB ense f Passhold S Pass cog ow Oum Sip iiw 40 ie a FI em 20 amp amm eo mi 5 md EC inter T Disable File m d gt Cg fe roe T Cmd hid Chan Watch go y scu tau ms tau ms Vpul mV ChO X AutoGain 4 Zero X Fis E 0 058 0 265 4 10 246 Rs MQ room mir m wa 48 9 5 Fos Rm MQ Rm M SI 233 995 53 100 41 a 00 4 08 1215 C F C F nter ms C 2 008 AE jo Chl X AutoGain 8 Zero X Fis Z Ih nA Ih nA m 0 065 0 182 Offset mV 0 18 Vm mV Vm mV Emo jon Pss Ata 39 9 zio 20750 an 44 A amp TRO CC Domain Freq options Fi2 z 256 wj m z lib m Dith B Y Time rc Dual C Single Bes 256 x S Ss Flag Cem CMD f 3906 FN wj pa ll BE Quick Notes extra_solutions txt great cell intra_solutions txt lost cell methods txt Na80 soultion started pipette plugged rationale txt started video tape x new new note note The Quick Note window allows you to make quick note entries into the log file and if CC save is on have graphical displays green bars of the note in the CC data window which will open up to show the text when the mouse is moved over the bars see CC window below Quick Note files are stored in the application subdirectory lt quicknotes gt The file quick notes txt
128. ltage connected to the MODE telegraph input is undefined e g if the amplifier is turned off then an asterisk 1s displayed next to the mode which is set to the selected checked mode setting for the affected channel It is useful to use the VOLT function while altering amplifier gain or mode in order to determine whether voltage levels are as specified in the amplifier manuals If not actual values can be used in the INI file Evaluations of telegraph input are made with an error window defined in the INI file For the DAQP 308 board only Gain and Mode are available for the two CC Censor channels since only a total of 8 input channels are available lt l Q ea User Fies Gan TO T1 T2 T3 CC Hist CC Script 2 Telegraph 0 x Enable Telegraph Channels must be sequential and increment left to right Chans Ch13 X Chi4 NC x gain X mode volt vol Clamp V Clamp Clamp Mode Voltage Gain Voltage IClamp E zero n VClamp 712 Emrs 1 Filter Frequency Voltage j n G EJ EROR T a PEP ET ERN NER T ia a a Error filt err Capacitance Scale x ho Error Error amp 1 c ce e c c e 1 12 15 1 45 51 p The telegraph inputs on the AXDD132x systems are not used The above details apply instead jClamp32 Whole cell voltage and current clamp 25 5 0 Manual The Axon Multiclamp Telegraph system is supported Support for the 700A and 700B is provide
129. lues chan watch chan gt test_value wait for channel chan s value to be greater than test value before continuing script channel number can be 2 3 4 or 5 e g waitfor chan watch 2 4 when Channel Watch channel number 2 is greater than 4 scripting will continue chan watch chantt value wait for channel chant s value to be less than test value before continuing script channel number can be 2 3 4 or 5 e g waitfor chan watch 3 6 when 111 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Channel Watch channel number 3 is less than 6 scripting will continue CC parameter dependent switch rs gt value rs lt value waitfor rs gt 10 continue with scripting after success other clamp parameters can be used as well rm cm tau hold 1i and vm timeout 25 if condition does not happen within timeout seconds then continue must be set before conditional statements default time out is 10000 seconds Here is an example of what can be done Monitor a cell under voltage clamp Track its membrane current at a particular voltage Run a script that collects some data in voltage clamp then pauses to wait for the switch to current clamp With the passing of holding current to current clamp mode checkbox enabled in CC switch to current clamp at the amplifier With telegraphs enabled CC will sense the mode switch and change into current clamp mode which will cause scripting to continue The protocol that
130. mp Features jClamp Features Copyright 1996 2015 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual e a papa iil SciSoft al L ay Featurey Voltage amp Current Clamp Text audio and video clips User friendly analysis Rapid Parameter Directory fast protocol deployment Script files experiments with a button press ENS Linkautomatically with Excel MATLAB Origin SigmaPlot Tecella Pico High Res capacitance measures with concurrent voltage stimulation CED Power Micro Fast synced Video capture with Andor and Prosilicacams Natl Inst E M X Series Quatech PCMCIA Simultaneous quad patch clamping with quad telegraphs IOTech Cell Censor constantly monitor cell characteristics Labmaster DMA ANON AUD eel EI ent email joseph santos sacchi yale edu jClamp for Windows v 25 5 0 is a user friendly whole cell voltage clamp data acquisition program which features real time monitoring of cell and voltage clamp characteristics At all times the patch clamper can determine cell status Data collection is initiated through a rapid parameter directory RPD which maintains a list of 18 parameter filenames or script filenames Either a key press or mouse click begins the predefined voltage or current delivery protocols Scripts are text files which allow groups of parameter files to be called and run Most tasks are automated Some data analysis is available In addition automation links with MATLAB Excel Sigm
131. n the MD Cs method should work here as well To confirm things I have made a simple cell model in which I can manually disconnect the series resistance from the cell parallel resistance and capacitance Balance out the series resistance stray capacitance when it is disconnected into the air as you do during calibration Then connect the rest of the model and the measured values will be quite accurate and should match those you obtain with the MD Cs method 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 5 useable noted above The calibration file that is loaded will be used and automatically chooses the appropriate resolution correction factors for the jClamp32 Whole cell voltage and current clamp 25 5 0 Manual 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 Ampl 10us IO0kHz cal Amplifier number 1 with 10 kHz filter collected with lt accalib prm gt set to 10 usec clock Ampl 5us 1O0kHz cal
132. n files Each channel must be calibrated if you want to use them together or individually You can calibrate channel 0 first and save the data file then calibrate channel 1 and resave the same file The channel is selected in the calibration window When you calibrate channel 0 is defined as channel 0 out with channel 0 in and channel 1 is defined as channel 1 out with channel 1 in Check the goodness of the calibration with an electrical model You will need to balance out the model series resistance s stray capacitance first which you can now do under whole cell conditions using the MD Cs method see below You can look at the calibration file in a text editor The calibration values are listed for each channel If a channel is not calibrated the list contains all zeros At the end is a comment section where comments on filter frequency etc can be added from the calibration window text box or in a text editor The default capacitance tracking calibration files vClamp and iClamp are set in the ini file Here is another point to remember The Cm determination is model dependent and assumes no stray pipette capacitance This is also true when you use a model cell If you have a commercial cell model typically in the past you cannot really balance out the stray capacitance of the series resistance under whole cell conditions Now this can be done suing the MD Cs method below Additonally you could not rely on balancing out in the patch conditio
133. n loaded with associated data files are synchronized to the data If you drag the data cursor in the Analysis Window the video will step through frames captured at the time denoted by the cursor Alternately you can step through frames in the Look32X Player and the cursor will move to the corresponding time On either side of the stop button in the Player you can automatically step forward or backward The rightmost button simply plays the video in real time Synchronization can be turned on and off with the LX checkbox under Cursors area of the Analysis Window If turned off the cursor no longer moves with the video and all frames can be inspected if synchronized only frames within the data time frame are accessable Rectangles can be drawn on the video image to monitor average gray level within the rectangle Simply hold down the right mouse button and drag to size and release Five areas can be set red green blue yellow orange Click once one the rectangle then move mouse to reposition click again to dock Double click on any rectangle to delete The data are monitored in the X Y Levels window Position of upper left corner and value 0 255 are given for each rectangle and for mouse PERSEO umm calibrate f distance delta edge Lines to average E Box Plot Check box to view fiduciary T avg box lines vertically marks Enter distance and units y between the two blinking white avg box lines horizontally lines Uncheck box
134. n the Main Menu Bar or by simply running the lt accalib prm gt protocol from the command window In order to make accurate AC measures the frequency response of the system needs to be corrected for When the calibrate button ruler icon is pressed a window appears and allows you to either load an existing calibration file or run a calibration Upon calibration the red icon turns green Any changes in the stimulus settings which require a new calibration will require a new calibration file The user must also recalibrate after changing the clock or external filters Filters should be set to a cutoff frequency fc less than that indicated Calibrations can be saved to or loaded from disk ascii file named filename cal using the file command buttons Calibration depends on the pure resistivity of the calibration resistor and in the case of the dither technique the pure resistivity of the whole dithering unit Unfortunately stray capacitance and inductance accompanies these components I find it best to work with low frequency stimulation sine 1 at 390 Hz and sine 2 781 Hz the default setting at a clock of 10 us At these relatively low frequencies reactance and inductance errors are not so great Nevertheless you should always check the reliability of the measures with an electrical cell model The method for calibration is to use a resistor between headstage and ground A value of 10 Mohm is typical During calibration a window opens see
135. nA 1 000 k Offset mV re in Vm mV Ln fon 0 o v 19 7 1 Plot Res Init cum i i i C epi CC Domain Freq a Fia l librate Dith E Time Dual Single Res 256 Cs TUA ither on C Freg etm fo H 3906 FN mmc 100kHz in Eluzi E File none Avg 0 Epi 1 407 55 1222 65 1630 20 2037 75 T Fix Scales me NU FlFlansed Time tst 0 vst Fl Pat All T jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Fac EJ icdata 50519001 abf 051 9002 abf 051 9003 abt 051 9004 abf S051 9007 abt I25143aM M Look l i Pira mjecwen sg 2 m Iv G y 505 S005 ab _ D 505193006 abf When loading data files if a file has an associated video file it is listed FastLook Vda files are collected with Prosilica cameras see FastLook Help If the check box near the video filename is unchecked then the video file is not loaded when the data file is loaded Otherwise data and video are loaded simultaneously see below jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Analysis File c jcdata J 10519 S0519007 abf vClamp with avi_test4 prm 3 08 39 PM Avg 1 Epi Part 2 Parts all sel Epi all sel f 407 550 815 100 y 1 141 nA Part 2 Command 22 825 22 8 mV RS ms Ls Cursors 2 H E as ESSE Fd Rab Tres ama 1 s ZE L jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Video files whe
136. naptic currents recorded under voltage clamp can be fed back to cells under current clamp In addition to using any of the build command functions on the abf partition abf data can be modified via a box that opens when the mouse moves on the abf button Scaling and zeroing relative to a point in that partition can be done While moving the mouse over these function controls see the information panel at the bottom of the jClamp main window for info on settings These features apply to user stimuli as well Digital output can be presented before the whole run pre before each episode epi during the whole run run or synchronous with the analog wave form partitions part Selecting any of the first three opens up a window allowing the user to choose which digital output channels should be used The time on and the time before starting the command delivery can be set Exact timing may depend on computer hardware and should be checked with an oscilloscope Partition part selection displays a plot ofthe digital output from channels 0 to 3 and the level of these outputs can be set independently for each partition Dp check boxes set for each partition and holding level Dh check boxes Additionally upon moving the mouse over Dp a pop up window appears allowing flip flop of digital output that overrides partition settings Set duration of pulses for whole duration of command Negative duration will invert pulses The holding digital level is maint
137. ndard model is shown in the Cm Tracking section If Param change is enabled then any one of the possible parameters is changed from Vall at Pt1 to VaD at Pt2 The value is ramped linearly from Vall to Val2 The point values are the point numbers in the episode of a command protocol So it is possible to simulate vesicle fusion during the measurement of capacitance using eCm by changing Gp between points Vall and Val2 An example of this is shown in the Analysis section Impedance analysis Impedance Analysis Analyze button will open a pop up window to choose a protocol to run Under voltage clamp nyq chirp v_ prm gt or nyq sine v_ prm gt are available with different voltage Under current clamp lt nyq_chirp_i_ prm gt or lt nyq_sine_i_ prm gt are available You can modify and save Place a resister or empty filter holder for epithelial measures and enter resistance determined from CC and voltage or current to stimulate with then run calibrate to obtain corrections for system characteristics You can enable or disable its use once collected Calibrations are saved and automatically appended to each data collection if the check box is checked After data is collected a fit to the chosen model can be performed For chirp signal you may need to limit the points used for the fit to get better results set the deci factor above to decimate a value of 2 extracts an octave spread When you open a data file in the jC
138. ndor com camera support and Prosilica Gige www prosilica com vision camera support Link video frame capture to patch clamp data collection Synchronized data for each frame Export automatically to ImageJ for video analysis Combined with j Clamp or standalone program available jClamp32 Whole cell voltage and current clamp 25 5 0 Manual CellSeeker and MagicSeal coming soon Automatic offset current cancellation User control over audio feedback during seal formation Enhanced P N subtraction Switch polarity for whole cell or inverted patch in CC window Disable dual patch mode in CC for stimulation with channel 0 or 1 D A while recording from channel 0 Removal of stray capacitance effects on calibration and during cell recording for Cm analysis Plots of Real and Imaginary components of raw currents Simple way to set angle of dRs dY 90 or angle of dC dY in Phase detector Admitance Window and angle will be used by single sine protocol to get Magnitude at 0 and 90 degree phase for C and R analysis Note that using Rs compensation or an Rs dither and C compensation or a C dither for dithering is a poor method for dithering actual Rm and Cm if Rm is not much greater than Rs or if Rm or Cm changes substantially during your experimental measurements the limitations of single sine analysis Non harmoninc two sine stimuli to avoid nonlinear distortion interference with Cm measures Free support for the Axon 1200A and B boards that
139. ne click on them to move another click to set Shift down left mouse button down and drag to draw line thickness set in Control Window Control Window has plotting and disk output options OH FastLook Player a Sven es i sed gt ini w Pe NIZ C Mastlook_data 03 01 2011 03 01 2011_12 2 iv Length 1720 frames W 640 H 480 ee X Y Levels With Prosiica GE680 S N 02 2020CO6675 T muere s 646 274 172 124 317 4185 m 539 278 3097 Image Cm Control ZH FastLook d no camera attached Crirst Brite 86 165 1010 6 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Simple left mouse button down and drag to draw box Scale video by dragging window to larger size imageJ ImageJ Link ImageJ is a free powerful image analysis program from the US National Institutes of Health http rsbweb nih gov ij download html VDA video files that are being viewed can be loaded from FastLook into ImageJ automatically from the Menu Alternatively VDA files can be loaded directly into ImageJ from its Menu see picture below The following two files which are located in the ImageJ subdirectory of jClamp or FastLook have to be placed in the ImageJ directory C Program Files ImageJ plugins Input Output in order to work with VDA files lt FastLook_gui_loader class gt lt FastLook_loader class gt Also in the macro section of ImageJ you
140. needs to be entered in the jClamp ini file under Gain tab after choosing the NI board Functional in Win 95 98 NT 2000 XP Pro DAX no longer available DAX hardware simply connects to the digital out BNCs The single male BNC connects to ch0 the attached white wire connects to ch1 and the other four BNC assemblies attach to any of the digital out channels ch2 5 A 4 5 5 volt power source central pin positive drives the DAX Output from each channel is 5 volts Episode length capabilities etc Axon 1200 boards can have episodic collections up to 0 5 million pts in W98 and 1 million points in Win NT Axon 1320 boards have episode lengths up to 1 million pts as does the IOtech daq2000 XP Pro Episode time for 1 million pts at 10 us is over 10 sec per episode If longer episode lengths are required this may be possible Contact me Episodic delivery for the National Instruments is up to 10 million pts this should be checked with your hardware before using on cells CED board and Axon 1440 1550 boards is up to 5 million points When in dual voltage output mode the default setting is 2 analog inputs For the NI boards using nidaq mx drivers and for the Axon 1320 and 1440 series boards you may increase the number of inputs jClamp32 Whole cell voltage and current clamp 25 5 0 Manual up to 8 channels Graphics speed jClamp runs fastest when graphic settings in Compatibility mode are set Right click on the jClamp shortcut on
141. nels up to four for Axon and Labmaster boards two for DAQP 308 frrom the DAX outputs This output can be used to drive analog recorders such as strip chart recorders Command Utility jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Command Utility B Command Utility vClamp with model Parameter File lt w axddl44x v 22 c NjcdataJS15112 none S iclamp 9 30 11 CHER Design gt abf rou Author men ee J Santos Sacchi private 2 hse 1 ot il comment password Properties Partition 1 EU EM MU Analog Chan 9 VO clamp Start 30 I Qut nis mV etm Chirp hee E A gs FL gsi Time 1024 pts ac Sr mChan Inc 0 z ramp abf EE jo ALU N N 1 step user r E Axon 700 mode mat Pre PZN fx Pat Pref Epi Run Epi EET Eu Averages EET sux a ii UN ho rio mV Number Number EN B 5 custom J chanjo x Dh Inter ms Inter ms fme J ro IL o0 2 BI e Enda brd mx po Plot Results Trigger Epi m 5 Init ee fom ks C pi pa I a CIE EN C epi non C Ky Di Run Pat 2 Epi 1 x 1108 y 20 0 When the mouse cursor is moved over the vertical partition lines the partition is bounded by thick red lines and properties in the main window are shown jClamp32 Whole cell voltage and current clamp 25 5 0 Manual EL Command Utility vClamp with m
142. ng When you want to record from a cell you will typically try to balance out stray capacitance after you get a gigohm seal but it usually is not fully successful To overcome this problem you may press the Cs button in the CC window after giga seal formation prior to popping into the cell The button will turn green if you click the button again the calibration will be zeroed and the button will turn red If the Cs check box is checked the Cs calibration data will be used to correct sinusoidal response current before Cm analysis if unchecked raw currents will be used for analysis Remember if you made a calibration file for 10 us and are using that your clock in CC Frequency mode must match that calibration clock and furthermore the Cs button can only be used when the CC clock matches the calibration clock Whether or not you use the Cs to help remove electrode stray capacitance the main system calibration file will be used to correct for system response Thus this new technique will remove any uncompensated currents due to stray capacitance so that the calibration file which also is now be made without stray capacitance interference can accurately be used to correct for system response The check box next to Calibrate will allow you to see the calibration data and Cs corrections that are currently being used You can modify the calibration data real time to see effects For current clamp the procedure differs and an alternate window will appea
143. o patch clamp issues remain however see below Power 1401 from CED 1200 1200A 1320 1440 and 1550 series boards from Axon Instruments Molecular Devices the Labmaster DMA from Scientific Solutions the DAQP 308 PCMCIA card from Quatech the IOtech daq2000 XP Pro PCI board the IOTech Personal daq 3000 2 or 4 DAC and the National Instruments E series PCI MIO 16XE 10 PCI 6052E PCI MIO 16E 1 and PCI MIO 16E 4 boards and M series USB 6251 NI PCI 6035E and PCI 6024E boards are supported in a limited way see below jClamp also supports video capture using a Video for Windows compatible board see the Look32 X section Streaming video capture and playback record video during data collections control through script or via Look video window Check script command section and check menu on the Look window I suggest a Hauppauge video see www hauppauge com capture card WinTV PCI you can get them without the TV tuner for less than 100 The default capture driver is number 0 but now you can provide other numbers in the jClamp ini file under the misc tab Setting the driver number to 999 causes jClamp to skip the check for a board and no capture will be available CAVEAT if you have different board types protocols created with one board selected may not be the same if you use them with another board This is because specifications e g minimum clock synchronous digital output voltage out can be different You should set the bo
144. odel Parameter File Su Diodor 177 c Ncdata S 15112 none Jc steaddl 2 23 08 Partition Design Command Cm F2 NR N X Select V Pk 20 mV lJ Santos Sacchi private n h E BE Res 2 56 ms comment x password Properties Partition Analog Chan YC clamp Start 0 mV 7 L N a R Out In Inc 10 P o C1 E Fi ment a S ee nChan L ino E C ramp abf Sync 0 N 1 step user TN mmm e Pat Pre Epi Run Epi Points Clk us Hold Averages r Episodes 7 P Number Number 512X 20 H sys 10 T jo mV 1 L n 1 custom chan10 TI TT Dh Inter ms Inter ms o i 1 0240 Miew Command rp o L PrePulse Vrange Plot Results Tn Epi mx o Init eet UL Ky C Di Epi es Au e mn 0 ee R T een Aun Command Pat 2 Epi 1 x 31108 y 20 0 The Parameter Load window opens up parameters into the command window The parameter directory is param under the jclamp32 directory All subfolders under param are included in a search path when a protocol 1s run from the RPD Directory window or via scripts jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Author Clamp parm file Current Parameter Data Filename fad dual fup 2 prm abf dual fup i prm dual fup prm 06 2671 2 Time dual out 1440 prm dual out 2 prm dual out 3 prm 3 02 44 PM dual out 4in prm z Size m ramp 1 160mV prm 4530 cm z 3sec 5s pm y List type FolderC sPro p32 param
145. ogain set gain automatically fastlook autoexposure set exposure automatically fastlook bitdepth8 pixel depth 8 fastlook bitdepth12 pixel depth 12 114 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual fastlook savebox on capture only within boxed area fastlook savebox off fastlook showbox on show only boxed area fastlook showbox off fastlook movie trig on hardware trigger for whole movie to start fastlook movie trig off fastlook frame trig on hardware trigger for each frame to start fastlook frame trig off fastlook cap vda now run capture to a vda file fast capture to memory slower to disk if set fastlook cap avi now run capture to avi file slow capture fastlook grab bitmap grab a bitmap image CMT files can be run from the RPD or a script file run cmt test run the protocol in a script cm calib file filename load cm calibration file do not include extension default extension is ca S cm trk prepulse true false choose to deliver prepulse cm trk prepulse volt 40 voltage of prepulse cm trk prepulse dur 100 duration in ms of prepulse cm trk clock 20 set cm tracking clock in microseconds cm trk flmult 2 set cm tracking fl frequency as a multiple of fmin cm trk f2mult 2 set cm tracking f2 frequency as a multiple of f1 cm trk time 10 set cm tracking recording time in seconds cm trk w sine true false set true to make capacitance measures during voltage commands 115
146. ompensation at the amplifier Now although it 1s useful to balance out stray capacitance to limit potential saturation of the amplifier jJClamp can automatically remove stray capacitance during calibration and prior to cell recording EX AC Calibration vClamp mm Place a shielded ungrounded resistor about 10 Meg in the headstage Deliver a 10 20 mV pulse in CC Balance out stray capacitance as best you can enter the sampling clock rate in the text box below default 10us then press Cs button Next ground the resistor enter its actual value in the text box and press lt OK gt f Ch Chi fio Mohm you like enter filter cutoff and comments Cancel DK fo Clk us The resistor should first be placed into the headstage connector With the calibration resistor ungrounded and 1f wanted after stray capacitance compensation press the Cs button in the AC calibration window Then after grounding the resistor measure the resistor s value in CC you can check the lower check box in the Rs panel in CC to sum Rs and Rm to get the resistor s real value jClamp tries to separate out Rs and Rm and the sum of them will be the resistor s value remember to uncheck the box when doing experiments and enter the resistor s value into the text box Remember to enter the sample clock you want to use Press OK and the calibration file will be generated and should be saved The calibration is automatically loaded after savi
147. on are set in the ini file Available channels are from 4 to 15 but if 4 7 are used Channel Watch function will simply look at the telegraphs and not be available for monitoring other devices For each telegraph the channel numbers must be consecutive Each channel s can have any of five functions viz NC not connected VOLT simple monitoring of the voltage on that channel GAIN monitoring of gain telegraph output from a patch clamp amplifier FREQ monitoring of the filter frequency telegraph output from a patch clamp amplifier CAP monitoring of the capacitance telegraph output from a patch clamp amplifier MODE monitoring of the mode voltage or current clamp or Izero telegraph output jClamp32 Whole cell voltage and current clamp 25 5 0 Manual from a patch clamp amplifier You should always shift from voltage to current clamp by first changing to Izero Amplifier knobs usually do this For the Multiclamp you should do this by mouse click in their MultiClamp window The GAIN and MODE are special cases where jClamp can take action on the telegraph input Therefore check boxes are also displayed under these conditions If enabled by checking GAIN will automatically rescale the incoming A D data to correct for the new patch clamp gain If enabled by checking MODE will automatically switch jClamp between current and voltage clamp modes The telegraph values are equated with their specific amplifier definitions in the INI file If the vo
148. ording to Sigmaplot instructions The default template jnt is located in the SigmaPlot program directory see below Y ou can add templates for access in jClamp by creating your own in SigmaPlot and putting the names of the template in the jclamp sigmaplot directory e g make a file name my new template jnt in the directory it can be an empty file only the file name is necessary and must match the template name my new template in the sigmaplot template jnt file Check that the new template is available in SigmaPlot It is required that you reload SigmaPlot to install new templates that are added to template jnt 119 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Look in F SPW10 tct as Name Date modified Type Ji FAQs 3 7 20148 03PM File folder Macro Transforms 3 7 2014 amp 03PM File folder Samples 3 7 2014 8 03PM File folder Desktop Submission Profiles 3 7 2014 8 03 PM File folder um Transforms 3 7 2014 8 03 PM File folder S Layout JNT 5 4 2006 11 49 AM SigmaPlot Libraries NorthAm LayoutJNT 5 4 2006 11 49 AM SigmaPlot Northam jnt 5 4 2006 11 49 AM SigmaPlot E Template jnt 12 30 20144 55 PM SigmaPlot s File name Template jnt Files of type Template Notebook jnt 120 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual File Edit View Tools Toolbox Pharmacology Help Da Eli e X Ba A o ola an CE ES a ES Ss A ED soe Arial
149. original implementation where f2 2 f1 index into the FFT array being 1 and 2 the Fi2 checkbox is unchecked and the other f2 lt gt f1 2 index into the FFT being 2 and 3 the Fi2 checkbox is checked 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 fl 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 fl 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 fl 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 mea
150. otocol is built with the data just obtained as a command in one partition So if data file S0728002 abf were just collected a new parameter file 50728002 abf prm will be created As with the user stimuli the stimulus abf file must be copied it is done automatically while making an abf template to the parameter directory where it is read during command build This process can also be accomplished within the parameter load window where any abf file can automatically be recruited as a command protocol In the window change the default file extension in the drop down box to abf select any abf file in any location on the disk and press the ABF TO PRM button This will copy the abf file to the default parameter directory and construct a parameter file named as above In order to use the parameter file simply select and execute it as you would any other parameter file through the command build window script file or via RPD The abf file can be opened in the analysis window as any abf file and modified with epi math functions Upon saving it back to the parameter directory the changes will be evidenced in the command when reloaded More abf data partitions can be added to the template but the partition size can only be as large as the number of abf data points Enabling abf in new partitions will reuse the data from the abf file In this way repeats can be constructed with intervening partitions of arbitrary stimuli For example sy
151. parated by red cursor lines The number of points in each partition or time in ms of each partition can be entered in the text box Changes can also be made by cursor movements via the mouse Clicking on pts in the Properties Section changes the time input to milliseconds ms clicking on ms reverts back to pts Selecting custom points allows the extension of the ending holding potential in increments of 512 points jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Step ramp stair AC abf and user stimuli are available for each partition These are enabled for a given partition by first selecting a partition by entering its number manually or moving the mouse over the right line cursor which demarcates the partition in the command plot When selected the cursors for a given partition are highlighted green Once selected choose the desired protocol Do this for all partitions The AC protocol allows delivery of single dual or three simultaneous sinusoids Maximum and minimum frequencies are determined by the acquisition clock rate on the main command window and are displayed A given frequency is selected by choosing a multiple of the minimum frequency The phase in radians can also be set Frequency increments can occur during multi episode collections by setting the multiple increment to greater than zero If octave increments are desired then set the increment to 1 The second sinusoid stimulus can be enabled if desired and its p
152. path and filename wild cards allowable e g 116 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Ini File Ini File Q ea User Files Gain TO T1 T2 T3 CC Hist CC Script 7 CAProgram 2Xmulticlamp 1550 ini 1 10 2015 8 55 02 PM Name lJ oseph Santos Sacchi Password iss User graphic C jClamp source 2001 sg bmp Plot graphic C iClamp source 2001 boats bmp 1 12 15 12 13 51 p Use the separate IniEditor to set custom settings for individual users 117 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual SJ c wWindows8 05 C Program Files x86 jclamp32 param Sc Program Files x86 Aiclamp32 param breakdown jesper Double clicking allows choosing files Default parameter directory and subfolders for path are shown Analysis Analyze Window Analyze 118 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Automation Automation link with SigmaPlot 2000 Excel 9 Origin 6 or 8 1 or MATLAB gt v 5 3 Program usage must be enabled in the ini file under the Script tab Linking to earlier versions possible if supported by those versions If application icons are present in the upper right of the jClamp main page when in analysis mode then the programs are available for use When active drag icon in upper right of main jClamp window onto a graph and release in Analysis Window IV Plot Window or CC Data Plot window and data is au
153. pendable ascii file readable by any spreadsheet or text editor The file is saved to lt UserlinitialsDate cc gt e g JS96115 cc this file can be viewed graphically when the Analysis window is opened Ifthe check box in the Ih display is checked then upon switching to current clamp from voltage clamp the holding current is transferred to current clamp mode This should allow the cell voltage to remain close to the same during the switch If the check box in the Vm display is checked then upon switching to voltage clamp from current clamp the membrane voltage is transferred to voltage clamp mode This should allow the cell current to remain close to the same during the switch CC inter If checked then CC data collection is continued during protocol episodic and averaging intervals also during trigger waiting periods Cmd hld Enable the use of protocol holding potential in CC during interval data collection returns to CC hold after protocol is finished Auto FileName This entry is now only in the INI file Enables auto file naming for collected data Files are named with the last name initial date and a number ranging from 1 to 999 For example if the user name is Tom Jones the first file of the day on 9 13 95 would be named lt j5913001 abf gt For months Oct Dec the initial are used instead e g lt j5013001 abf gt Note that automatic directories are made for each day and are named with using first and last initials and th
154. placed with any other ini file by appending the ini filename to the Target command line in a shortcut on the desktop Here myini ini will replace fastlook in1 This is useful if more than one person uses the program and needs to set different data directories and camera settings In order to be able to edit the properties of the shortcut you need to go to the executable file fastlook exe in program files directory right click on the file and send shortcut to the desktop Shortcuts made other ways may not allow editing the target 163 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual ei FastLook exe Shortcut 2 Properties FastLook exe Shortcut 2 Target type Application Target location FastLook Target Program Files Fast Look FastLook exe myini ini Start in C Program Files FastLook Shortcut key None Run Comment i 164 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Notes on CM calibration Update Technical Note 3 SciSoft Company www SciSoftCo com Notes on Calibration Running the calibration parameter file lt accalib prm gt 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 lt accalib prm gt will produce corrections for six resolutions jClamp now uses two approaches for 2 sine stimulation one bein
155. q2000 National Instruments PCI MIO 16EX 10 and PCI 6052E boards or Quatech DAQP 308 PCMCIA have Lap can Clamp I suggest a Hauppauge video capture card you can get them without the TV tuner for less than 100 Convert digital outputs to analog outputs with the optional DAX D A eXtra hardware add on Control up to four more amplifiers Monitor seal formation set holding potentials monitors cell characteristics synchronous episodic delivery Four channel graphical history display of selectable cell and clamp characteristics time window user adjustable Graphically view seal formation with frequency varying audio cues Customizable initialization settings for individual users including graphic ID in Cell Censor window Separate initialization file editor Automatic data directory naming file naming and saving Powerful graphical voltage current protocol build utility Episode length up to 10 million for National Instruments boards 1 million pts for Axon boards and IOtech daq2000 128k pts for Labmaster and DAQP 308 Step ramp and AC stimuli one two or three sine chirp Dual command protocols support simultaneous current and or jClamp32 Whole cell voltage and current clamp 25 5 0 Manual voltage clamp Gap free collection is supported Full command waveform scaleable based on user scale factor set in INI file For example with a button press increase or decrease the wave form magnitude by a factor of 2 about the holding
156. r you must compensate jClamp32 Whole cell voltage and current clamp 25 5 0 Manual capacitance The current clamp calibration window will open if CC is set in current clamp mode else it will open for voltage clamp calibration Cm measures under current clamp are very tricky and I am still working on how best to do this EX AC Calibration iClamp xa Place a shielded grounded resistor about 10 Meg in the headstage Deliver a 1 n pulse in CC Adjust capacitance compensation as best you can until a square wave is obtained Enter the sampling clock rate in the text box below default 10us enter the resistor s actual value in the text box and press lt OK gt Y Chet Chi 10 Mohm f you like enter filter cutoff and comments Cancel OK ho Ciklus In current clamp mode the resistor should be grounded and the voltage response to a short current pulse should be squared by using the capacitance compensation Then calibrate by pressing OK button Current clamp calibration and measurement are still under development Beware Save the calibration data file and note that it can be reloaded as default set in the ini file or at any time with the file command buttons or menu bar button and used only when the clock and filter settings in CC and protocols match the original calibration settings Remember voltage and current clamp frequency responses within the same amplifier unit may differ so each may require using unique calibratio
157. rate to use extra resolutions Remember to check proper function jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Channel watch is functional if enabled even when CC is not running Conditional scripting Continue scripting after switch from to current voltage mode or based on values obtained from analog Channel Watch e g based on temperature change reading Ability to transfer voltage clamp holding current to current clamp upon mode switch Also current clamp membrane potential to voltage clamp Ability to set all partition start voltages currents to holding level in Command Build Utility Useful when passing hold from Cell Censor to protocol Better memory management in Win 98 IniEditor can now be opened from within jClamp without out of memory error Tested with 128 Meg memory system Automation link with SigmaPlot 2000 Excel 9 MATLAB 5 3 and Origin 6 Rapid data transfer and plot generation May work with some earlier versions Enable use for each program seperately in jclamp ini file Scripting of application commands in jClamp analysis scripts Two sine FFT based CM tracking now supported under current clamp Ability to use data file abf traces as commands in Command Build utility Automatic generation of command parameter files from abf data files For example immediately stimulate cells in current clamp with currents obtained under voltage clamp Also FFT can now be computed on any portion
158. rated only the data for that channel is written If channel 0 and 1 are calibrated separately then the two data sets can be put together in an ascii editor Alternatively two systems can be calibrated simultaneously It is now possible to derive Cm Rs Rm and Im from the abf file in the Analysis window Most analysis features which are used for currents exponential fitting etc are then available to use When a file that has employed 2 sine voltage stimulation is viewed in the Open data file window you can select to load the embedded calibration file or use one already loaded Upon plotting in the abf file in the data Analysis Window a box is displayed which allows choosing which form of the data to plot all automatically analyzed Raw Cm Rs Rm Im or Vm If a dual sinusoid command is chosen then fusion event analysis can be performed When enabled the user selection allows the incorporation of user defined stimuli into a command waveform Only one partition of an arbitrary number of partitions can be defined as a user partition Only one channel is allowed The user waveform is actually added to an underlying step partition voltage starting level and increments are allowed therefore but could be set to zero to deliver the user stimulus as is but riding on the holding potential or current The number of points should be fixed with no increments this is allowable actually see below First define a command protocol setting up
159. re0 5 vareinc 3 do wait vare loop 3 This loop will do three waits of 5 8 and 11 seconds Another example timeout 50 varb0 10 varbinc 5 do waitfor chan watch 2 varb run temperature loop 5 This loop will watch channel 2 under channel watch and wait until it is greater than 10 15 20 25 and 30 before running the temperature prm protocol If the timeout of 50 seconds expires before hitting the condition the loop will continue and run the protocol This loop could be used to monitor temperature with channel watch also saved in CC and collect data sets as the temperature is changed continuously and automatically from say 5 C to 35 C Imagine you could do multiple conditions e g simultaneous pH tension etc with channel watch digital 01111111 set digital output channels 0 through 7 if a digit is gt 0 then digital out is high else low chan 5 oscilloscope trigger no care e g digital 01234567 digital 10000000 shell path amp filename optional command line argument an executable with or without a command line argument e g shell test exe commandlineargument streaming video capture vidclip filename seconds for example vidclip test 20 will save a 20 second capture to test avi vidclip stop stop capture if running vidframespersec value value is frames at 1 to 30 per second vidcaptureaudio true false enable disable simultaneous audio capture video auto interval video time lapse interval in s
160. reen shots can be loaded at the command line in a shortcut jClamp exe myinifile ini collect scn will bypass the Identity login form and load the saved sreen jClamp32 Whole cell voltage and current clamp 25 5 0 Manual format Getting Started Getting started If you buy jclamp you will need to register After you install and run jClamp on the computer you want to use press the registration button after running jClamp Press the copy info button and paste all the info into an email to me It looks like this To SciSoft Co joseph santos sacchi yale edu http www SciSoftCo com Date 03 17 07 Time 10 55 24 Application jClamp32 exe Version v 17 0 0 System Windows NT 5 1 Build 2600 Application path C jClamp32 Registration code A40079BD 22585A 26A3654 10196A30 5E72F78 I will send you the hardware license file for jClamp or a link to download it Alternatively you can use the fulldemo license on the web for a month to check out the program with hardware or you can use the built in math model to test the program Your hardware license is named lt your_name lic gt It may be zipped and named your name zin and unzipping will extract the license file lt your_name lic gt Download and save the file Do not open it it is simply an encrypted text file The file is necessary for jClamp to work with A D D A hardware Make a backup copy You should have installed jClamp into the default directory C program f
161. rmation is stored in the file jcreadme bak Additionally if a text message is present the audio file named lt jcreadme wav gt located in the jClamp lt sounds gt directory will be played You can record your own audio message using Windows sound recorder for example along with the text message or just play a notification sound when the text message opens Just rename your recorded wav file to lt jcreadme wav gt and place in the sounds directory In the sounds directory there is a file no sound wav that can be copied and renamed to lt jcreadme wav gt and or lt giga wav gt so that no sound is played during the execution of these files within jClamp jClamp now has a stand alone version of lt jc_audio exe gt which can be used to record or play wav audio files since Windows Vista does not have a sound recorder that does this It is accessible in the jClamp directory or the jClamp start menu Using the jcreadme feature is a good way to remind yourself or another jClamper of something important When you restart jClamp the second time Windows will attempt to re configure jClamp This normal and should only happen once Because the computer may crash if jclamp is run with the ini file set to a board that does not exist in the computer the default jclamp ini file is initially set to run in a built in math model mode To run with hardware you should first set the board type and address then disable model in the ini file under t
162. ropped 0 missed It received 2390 bitdepth IET exposure 500 gain i10 disk streamed to memory timestampfreq Refresh List Done You can overlay text on the video and save to bitmap Playback frame rate for AVI export is set here s Controls Eeee Config Image Acquisition Exposure Stats GigE Avi Bitmap Main Calibrate Measure File Info On screen text Comment capture date time ic filename Y axis value ie owner Episode Rs MOhm Rm Mohr Cm pF elapsed time filename frame voltage mV Witte evi fps 30 Refresh List Done You can set or view camera properties in the other Tabs of the Control Panel 155 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual DSPSubregionBottom DSPSubregionLeft DSPSubregionRight DSPSubregionTop Exposure uto djustDelay ExposureAutodAdjustTol ExposureAutoAlg Exposure utoMax ExposureAutoMin ExposureAutoOut liers Exposure utoRate Exposure utoTarget ExposureMode ExposureValue ViewVDA View VDA files The default directory is C Mastlook data Select a file to view from the menu and a Open file dialog window will open allowing you to navigate and select a file You can write an already opened VDA file to AVI format at the FPS rate set in the AVI Control panel You can export to ImageJ see ImageJ Section in this help file
163. s attached you may select the LX check box in the Analog Channels area When selected the light will turn green and indicates that synchronous video capture will be made when the protocol is run Video capture will use the settings made in the Controls window acessable from the Options tab in Look32X window Gain exposure can be set here is the video device supports these features Under the Options tab additional controls for video and audio are avaible from the camera manufacturer You can jClamp32 Whole cell voltage and current clamp 25 5 0 Manual preallocate a disk file to save video so that disk access is faster Size needed depends on the codec used and length of recording Play around to see what is best If video is captured simultaneously with a protocol the Capture length value in the text box is ignored and capturing stops after the protocol is run Otherwise the length may be fixed if collection is made directly from the Look32X window or limited by disk space if the fixed length checkbox is unchecked You must then stop capture manusally by clicking Stop Capture in Look32X1 Filename Fn prepend puts those letters at the beginning of the captured video file Filenameing and directory path are set frome jClamp If run alone default data directory is C Look32x_data Manual naming can be enabled The capture rate frames per second fps can be set in Controls or under Options tab Time lapse capture can be done for extended per
164. s out Unfortunately I do not recommend using the 1550 with j Clamp at this time MultiClamp Commander Telegraphs are now supported internal to jClamp no need to run jc Client applications anymore Set MultiClamp on in ini file History tab Provide paths to MultiClamp servers MC700 exe and MC700B exe in the ini file and select the associated check box to run automatically Only one Multiclamp 700A or 700B is supported Multiclamp mode can automatically be controlled by digital out 3 in a protocol file Multiclamp Commander must be set to ext control This means that voltage clamp and current clamp can be switched real time within partitions of a command episode protocol command type must be set to voltage clamp Only channel 0 in out command jClamp32 Whole cell voltage and current clamp 25 5 0 Manual protocol should be used Other channel inputs can be sampled as usual Drop down box in Data Load Window allows quick selections of previously viewed data directories Previously channel protocol holding potentials were set prior to running the protocol 500 ms plus a computer dependent variable time preceded the protocol command This eliminated a transient in the protocol response if the CC holding potential were different Since you should be aware of mismatch in CC hold and protocol hold this feature is now removed Now you will see a transient current response at the onset if CC hold and protocol hold do not agree You must mak
165. stigate what I have under construction Interpol Export Combine m Auto Oma abf Fitting Fitting Fitting of data traces in each of the three graph windows Analysis CCdata and IV in the Fit window choose the desired target can be made with built in and user defined equations using the Levenberg Marquardt method In the Analysis and CCdata window the fitted region is between cursors 1 and 2 In y 3 Exe Analysis View x x prFit T C i C cc Autofx Raw ess ne Epi 0 Iter 0 OEsp2 Bolt OEsp3 Bolt2 Expot OQ tapa O Line Man Group Auto T Param Parameters a0 jo 0075 CO 1 1655 30 2 05 the IV plot all data are fitted but you can remove check box 1s set Uncheck it if you wish to provide your own guesses in the text boxes Upon typing actually upon key release in any of the parameter text boxes a trace in green is displayed to help with initial guesses Remember that initial guesses are crucial for successful fitting All traces displayed will be Expl C User SDev 0 Cap ImetL dicking on the data points in that graph Built in functions will guess at initial parameters if the Auto fitted You should display only the traces that you want to fit 132 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Data from the fits are sent to the Spreadsheet in the IV window see image below You may then plot the parameters vs any of the x values
166. struction set digital out ports trigger video capture or time lapse capture Scripts can be automatically canceled if one or more particular cell or clamp characteristic crosses a predefined threshold e g if resting membrane resistance Rm drops below 50 Mohm or if electrode resistance rises higher than 40 Mohm Analysis features are smooth and graphical Axes or scale bars User text on plot Episode math file math fitting of exponentials Boltzmanns FFT Residual series resistance correction cursor based analysis IV plots togglable leakage subtraction view raw or subtracted traces x axis data shifts Exporting of data to abf atf ascii or matlab Copy plots to clipboard for paste in word processors Trace comparator jClamp32 Whole cell voltage and current clamp 25 5 0 Manual combine traces from Analysis window into new window for comparison across files Data file loading is enhanced by the ability to view key items in the file before loading e g data owner cell and clamp characteristics immediately prior to data collection parameter file name sampling clock number of channels etc All data collected can be summarized in printed form Each protocol and associated data traces are printed along with key protocol parameter information four data files per page All cell and clamp characteristics for each cell studied are plotted versus time Data files can be selected by parameter filename and printed or plotted on scre
167. surement of cell parameters In the method window the dropdown box selection 1 2 or 3 permits the extraction of Cm based solely on 1 2orthe average of the two respectively While using a math model produces equivalent results real world conditions e g different noise levels at each frequency changes in stray capacitance nonlinear distortion may produce different results Using 1 is probably best Averaging was internally set for jClamp versions 12 0 12 5 eCm Analysis This method estimates all parameters based on an exact solution of OY OCm and its angle Though the method uses two sinusoids to get at the angle of OY OCm all parameters are determined from inspection of only the low frequency response The manuscript Santos Sacchi Biophysical Journal 2004 is in the jClamp directory and is accessible from the Windows Start menu jClamp32 Whole cell voltage and current clamp 25 5 0 Manual Single sine analysis is performed on the lower frequency f1 For command protocols the number of frequencies can be set in the command window and is saved with protocols so you can make and run either 2 sine or single sine commands Notes on Calibration read this whole section amp Open CM Data vClamp Collection Calibration file 3 Calibration Files 10k amp0 cal 10k10us10m cal 1320_office_dagan_10k cal 1i cal model 10us wb cal M Calibration can be initiated by clicking the ruler icon Be CC or i
168. suring the current response they calculated from the system s impedance the cell capacitance resistance and electrode resistance R A b 42 B A b R I b A b B A2 Bj 45 b C 1 B 42 B2 45 b 4 b B5 where b I R HR VEI A Re Y B In Y 0 2nzf As can be seen with the use of one tone it is necessary to obtain an independent estimate of the input resistance b However the utilization of a two tone c1 and c2 stimulus allows jClamp32 Whole cell voltage and current clamp 25 5 0 Manual one to determine all parameters given the signal and response That is b need not be determined independently and can be solved for since for example Rs is the same at any two frequencies The evaluations should work under voltage clamp or current clamp The implementation in jClamp allows the use of several different time resolutions which depend on the selected clock It is best to use the default settings since only these have been checked to work effectively but feel free to try different settings and verify with electrical models Real and imaginary components of the current responses are obtained through Fast Fourier Transform Remember the analysis is model dependent see model above although the technique has now been extended to fusion event and junctional coupling analysis see help on Analysis jClamp now uses two approaches for 2 sine stimulation one being the
169. t and a small noise increase is not crucial then use detrending else disable detrending 173 Index J jClamp Features 2 4 10
170. t results Using 1 is probably best Averaging was internally set for jClamp versions 12 0 12 5 8 i 2 560 202 240 407 040 611 840 816 640 1021 440 Epi 1 50688ms v 31 072 pF Part 1 Command 0 303 9 mV Rs 9 8 Mohm Fusion event analysis is also possible The following model is used Basically prior to vesicle fusion Rp is consider to be infinite and the admittance of the cell prior to fusion is assumed to remain constant during the fusion event Thus Rs and cell s admittance is mathematically removed from the circuit leaving only the fusion vesicle for analysis the manuscript detailing the procedure is in review and will be appended when jClamp32 Whole cell voltage and current clamp 25 5 0 Manual published Below a math model simulation is analyzed Cv A command protocol which measures Cm during a 1 second interval 10000 points at 10 us clock 2 56 Cm time resolution with the eCm method will be run with the model setup below With this set up Gp will initially be 1pS 1000G at the start of recording after 20000 points of the protocol have elapsed Gp will change to 200 pS which initiates the fusion event Gp will be ramped linearly between point 20000 and 70000 up to 100 nS then will remain constant Below are screen shots of the analysis If we look at the simple extracted Cm without fusion event analysis the capacitance changes quickly over the course of 100ms This time course is not that of the fusion pro
171. the user defined partition as a step partition After all parameters are set then select user for the desired partition save the parameter file The parameter file e g jclampu prm must be associated with another file with the same name but a usr extension jclampu usr which contains the user stimuli in ascii format The values should be in mV or nA and arranged in one column The number of values must match the number of points u pts in the user partition else an error occurs If the number of episodes is greater than one then the number of values must be u pts episodes all arranged in one continuous column The multi episode stimulus can be a simple repeat of one set of u pt values or any individualized episode stimulus jClamp32 Whole cell voltage and current clamp 25 5 0 Manual The number of episode stimuli in an ascii file can be greater than the true number of episodes in the parameter file This means that the user file which holds 10 episodes of stimuli can be used to deliver up to 10 episodes without error Partition point increments should be allowable if the user ascii file provides incrementing u pts for each episode In this case if an mismatch in number of points occurs the program may crash When enabled the abf selection allows the incorporation of user defined stimuli extracted from an abf file into a command waveform If the check box abf template 1s checked in the Partition Design frame after a collection a new pr
172. tomatically transferred to the application and a plot is generated SigmaPlot templates JNT can be stored in the jClamp32 sigmaplot directory for use of user defined graphs Upon dragging and releasing the SigmaPlot icon a list of template files is displayed and can be chosen Application files should be saved under new names before exiting jClamp or inactivating the application if data loss is to be avoided MATLAB commands can be run from analysis scripts SigmaPlot transforms can be run from analysis scripts Origin LabTalk command can be run from analysis scripts All applications communicate with jClamp via Active X Ole automation except the older Origin program which only supports slow legacy DDE Because of this the full path to the origin executable e g c microcal origin60 exe needs to be entered into the jClamp ini file in the Script tab section Otherwise the application will not be recognized Automation links remain in effect when exiting Analysis mode This allows quick reentry to analysis mode rather than checking for connections each time No need to double clik on the Sigmaplot Matlab Excel or Origin icons in Analysis mode If programs are available but need to be set to check for in in file then icons will display and drag drop icons to transfer data will be immediately available In order to use SigmaPlot jClamp templates available in jClamp SigmaPlot directory you must load them into the template jnt files acc
173. ture with jClamp This is a replacement for Look and now supports VFW as well as DirectShow compatible devices web cams and other cameras with driver support have tested out Logitech C300 web cam and perhaps it can be modified for microscope use The Hauppauge video capture boards continue to work fine cus j Exit P EI If a video device is present clicking on the camera icon in CC will open a window which allows viewing and saving camera output in a scaleable window Streaming video capture and playback record video during data collections control through script or via Look32X video window A stand alone video capture program also called Look32XI is available in the jClamp directory useful to check that cameras are working etc It is accessable in the jClamp start menu Check script command section and check menu on the Look32X Window suggest a Hauppauge video see www hauppauge com capture card WinTV PCI you can get them without the TV tuner for less than 100 The default capture driver is number 0 but you can provide other numbers in the jClamp ini file under the misc tab if you have multiple boards Setting the driver number to 999 causes jClamp to skip the check for a board and no capture will be available As before video can be captured via script files see Script file help for commands In addition video capture can be synced to protocol capture In the command utility if a supported camera i
174. u repetitively dither Cm Rs or Rm when the math model is chosen or dither a digital pulse when hardware is turned on The pulse could be used to dither Cm or Rs altering circuits However in the PSD mode there is a more simple dither technique to get at the right angle for Cm measurement which follows For Rs dithering jClamp will calculate the angle at which currents are unchanged by Rs changes For Cm dithering jClamp will calculate the angle at which currents are unchanged by Cm changes Cm dithering is not really dithering the cell capacitance so it is a rough estimate Rs dithering will correct for changes in Rs but Rm may change Thus obtaining an angle to measure true Cm is not possible with this type of dithering calibration Using two sines changes in all parameters can theoretically be separated In any case this traditional single sine approach can be used and if after obtaining an angle for Cm analysis you collect single sine data using a Command Utility protocol the angle is saved with the data and can be jClamp32 Whole cell voltage and current clamp 25 5 0 Manual used to analyze current magnitude at that angle and 90 degrees from it in the Analysis window You might also use the digital out in a protocol to step a capacitance jump circuit in your patch clamp amplifier to roughly calibrate the Cm Mag0 and Mag90 measurements Dual sine Analysis In 1991 I devised a method to use two voltage sine waves simultaneously to enhanc
175. urrent clamp 25 5 0 Manual Allows user to sample up to four additional channels Enable this selection in the CC window Upon moving the mouse over the enabled selection region a window 1s displayed allowing the user to individually enable channels 4 to 7 in addition to the CC channel 0 Collection is made at the rate entered by the user and the scales and labels for each channel are displayed These scales and labels can be entered here but will be lost upon program shutdown They can be entered permanently in the INI file A Chan Watch window is displayed and shows the results of sampling Data is also logged tot the CC History window and saved in the CC file if saving is enabled Note that if the sampling rate for chan watch is faster than the sampling for CC then no CC collections will be made Remember to always set chan watch interval to a value greater than the CC sampling interval CC History CC History Displays a scrolling plot of cell and clamp characteristics during the past N seconds The length of time N is set in the INI file The sampling rate resolution is also set in the INI file Three plots are available to view simultaneously but each can be used to display any of the Cell Censor data items or data from Chan Watch Display channel is selectable Ifthe DAX hardware is installed and enabled in the INI file then selecting the DAX checkbox will output a voltage representation of the selected CC History chan
176. within the final evaluated equation For example ea and lt eb gt below cannot be confused with any other part of the equation But if you used an da then all the lt da gt s in the equation would be replaced with lt a0 exp xdata tau0 c0 gt when I parse it equations ea a0 exp xdata tau0 c0 eb al exp xdata taul c1 Yfit ea eb initial guesses for parameters a0 1 8549 tau0 0 57 c0 15 3114 al 2 1495 taul 2 7654 c1 15 3114 133 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual E data vh 1 e 3 Beexp 2 38 927 E A qmax 1e 12 2 38 927 H B 1 B 2 Yfits 1e12 clin 134 jClamp32 Whole cell voltage and current clamp 25 5 0 Manual amp jClamp for Windows R BS Analysis File cXicdota 502D28152028001 abf vClamp with Jclamp prm 8 56 44 AM el xl Avg 1 Ept5 Pat 2 Pat allC sel Epi allC sel ar en 76 42 H avg nA 3502028 S2028001 abf x 15 74 10 61ms xj m A Fix Scales n Lm m zm i Target Optic aca panes Cv C ec eof ur 4597 Ee User Ct2 pk 2047 CE ood P fit plot n OOo Ci reke TY T User Equation ex 7 c u sits P Friteatrenp ndatane EN 0 2 mm Ir ek on cay 23 9 aD 2 tau ME 02671128 06532474 53 li 1574 287 21517 EE 0 2089582 06524789 dun SEIEN 5 04437515 2151788 3 06540697 Tsd IO FL V E BE SE CE Odora to Ez win
177. work on P N Leak Subtraction Parameters Fhan m Size 50 Sub Hold E yi E mV ma Order re UN Before After The command protocol plot can be updated after any changes by double clicking on the plot or clicking on the All button Episodes can be displayed singly by clicking on the up and down arrow buttons Prepulse can be seen by enabling prepulse view Clicking on the trace icon button copies the command waveform to the analysis window for detailed viewing or export Data collection can be triggered externally or via keyboard If Init is selected then collection is immediate upon RPD or script action Trigger can be either by episode or by complete run and can be by key press or digital input see supported hardware for input to use Digital trigger now has minimal delay between triggering and data collection except for protocols with P N prepulse or CC inter use Plotting data during collection can be episodic where each episode is plotted alone or cumulative where all episodes are plotted with the most recent episode plotted in red During data collection hitting the lt esc gt key will terminate the run If some data had been collected you will be given the opportunity to save the partial data file Sister parameter files individual files can be linked to follow each other simple alternative to script files For example jclamp prm test prm test1 prm and test2 prm can be
178. y scaling on high DPI settings Run this program as an administrator C Enable this program to work with OneDrive files check the boxes set to 16 bit color then press OK jClamp32 Whole cell voltage and current clamp 25 5 0 Manual If this program isnt working correctly on this version of Windows try running the compatibility troubleshooter Run compatibility troubleshooter How do choose compatibility settings manually Compatibility mode Run this program in compatibility mode for Windows 8 Settings iv Reduced color mode 16 bit 65536 color C Run in 640 x 480 screen resolution Disable display scaling on high DP settings Run this program as an administrator T Enable this program to work with OneDrive files Change settings for all users Boxes are checked and dimmed out Select OK to exit You should have Administrater rights to do this If not change settings for just yourself File Types File types Protocol Files prm these are protocol files that are built and saved in the Build utility window They are the basic file type used to collect episodic data producing abf data files and also to measure membrane capacitance with 2 sine voltage stimuli dcm data files automatically extracted from the abf files emt eapaeitan ee param hat define the y acquisition parameters for atimetimited coHection The amount oftime allewedis no pr gc
179. y useful even if you do not do Cm measurements Under some conditions it may be possible to compensate automatically in software First adjust hardware compensation just below compensation as shown in the red line below Then press the guess button followed by the fit button The success of this depends on many factors including the initial guess result prior to minimizing to a slope of Cm vs F of zero One last note the Cs corrections are saved with the data file and utilized during analysis there is a Cs checkbox in the Analysis window which enables disable the corrections so that you can see the effect of having successful stray Cs compensation Each new cell must be compensated and probably during an experiment with one cell you should check compensation The actual voltage delivered to the cell during MD stimulation is larger than a 2 sine stimulus because 5 sines of equal magnitude are summed Thus you may want to consider the effect of the stimulus on your cell If necessary you could reduce the stimulus size during compensation jClamp32 Whole cell voltage and current clamp 25 5 0 Manual I ms 256 Calibrate 7 Dither on S RE zr EI 2 mai rr o NN D Cell Censor vClamp with Axon Digidata 1440A none 10408 Vhold mV C10 Wi x EE ae 4ms Pass hold s Cfso fe Ot j C e 40 5P e s of CCinter Diable File mdhid Chan Watch tau ms Vpul mV Zero X Fix S 0 1654 830 Remo
180. ye e FIG teen 24 Various plotting manipulations are possible with the mouse Particular episodes and or partitions can be plotted or all data can be plotted Vertical scaling of traces is accomplished by placing the mouse to the left of the y scale where it changes to a magnifying glass image press the left mouse button down and drag it vertically until the desired selection is made indicated by a colored bar along the y scale Upon releasing the button the traces will be magnified Horizontal scaling is similarly done beneath the lower x scale Double clicking on the plot or pressing the reset button will restore full trace scaling if Fixed Scales are not enabled Fixed X and or Y scaling is selectable By pressing the left mouse button down within the graph area which displays a open hand cursor and dragging the traces can be shifted around A small plot in the lower right shows the horizontal position during dragging Movements of the mouse over traces highlights the selected trace and displays information at the bottom of the plot Vertical movements of the mouse to the right of the right y scale displays a horizontal cursor over the plot with a text box indicating magnitude Y scale values are changeable by clicking on them or pressing lt shift gt lt Y gt current scale loops through nA uA mA and pA Default scales are set in the ini file X scale values can be in points pts or time ms Clicking on the x scale label or pressing lt

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