Integrated Solutions for Electrophysiology, Biophysics, Cellular Imaging and Electrochemistry

AAxxoonn IInnssttrruummeennttss,, IInncc.. May 1996

The NEW Axopatch 200B is the Coolest: LLoowweerr SSiinnggllee--CChhaannnneell NNooiissee,, GGrreeaatteerr OOppeerraattiinngg RRaannggee

Axon Instruments is proud to announce the Axopatch 200B,the latest advance in patch-clamp amplifier technology. Wehave incorporated many improvements in this, the latest inthe Axopatch 200 series of amplifiers. Most notably, usinginternal cooling of critical electronic circuitry, we havereduced to unprecedented levels the open circuit noise inpatch mode (low-pass filtered with an 8-pole Bessel filter):

< 15 fArms below 1 kHz bandwidth< 60 fArms below 5 kHz bandwidth< 130 fArms below 10 kHz bandwidth.

Even with a pipette holder attached, noise is still low (145 fArmsbelow 10 kHz bandwidth). This translates intolower noise during actual recordings. James Rae, of the MayoClinic, reports that noise is lower than he has ever observed in arecording situation.

Better noise performance is only part of the story. We haveredesigned the headstage so that it is long and thin, makingit easier to fit under your microscope. Also, we nowinclude BOTH whole-cell current ranges (previously avail-able only in two separate headstages) in one headstage.

The Axopatch 200B includes all of the features of theAxopatch 200A, plus a few more. Command and bandwidthranges are larger. With its 1 V maximum (instead of 200 mV)holding and voltage command ranges, it is now ready forelectrochemists without modification. The maximum pass-band of the low-pass filter is now 100 kHz twice that ofthe Axopatch 200A. Now with built-in CapacitanceDithering, the Axopatch 200B comes with a SeriesResistance Dithering unit to make it ideal for sinusoidalmembrane capacitance measurements. Series Resistancecompensation is now active in current clamp as well as involtage clamp mode to enable bridge balance.

Axon is proud of its success with the Axopatch 200B. Visitour World Wide Web site (http://www.axonet.com) or call(415) 571-9400 for more information.

AIW 2 SUPPORTS PHOTOMETRICS CAMERAS

Axon Imaging Workbench (AIW) version 2 isAxon’s highly functional and modestly priced software

for ratiometric and nonratiometric ion imaging. Version 2 nowworks with Photometrics cameras that are supported by thePVCAM interface software developed by Photometrics, Ltd.The models supported include the premier PXL series, thenew, moderately priced SenSys series, and the Image Pointcamera. On applicable models, the user may select a Regionof Interest, choose readout speed and number of bits of digi-tization, and define pixel binning parameters. If you wouldlike to try AIW version 2 with your Photometrics camera,you can use the new Demo mode feature; this is automaticallyselected if the computer does not have the AIW parallel portkey (a security device). When in Demo mode, AIW workswith any supported camera, frame grabber and wavelengthswitcher, in all operating modes. You can even do a real

In This Issue

New Product News . . . . . . . . . . . . . . . . . . . . . . . . .1

The New Axopatch 200B is the Coolest . . . . . . . . . . . .1AIW 2 Supports Photometrics Cameras . . . . . . . . . . . .1Need to Buy a Computer for Imaging? . . . . . . . . . . . . . .2New Features in AxoScope 1.1 . . . . . . . . . . . . . . . . . . . .2AxoScope Junction Potential Calculator . . . . . . . . . . . . .3AxoGraph’s Synaptic and Quantal Analysis . . . . . . . . . .4

AxoGraph e-mail, FTP & Web Site Update . . . . . .5

Restoring the Sensitivity of Used ProCFE Electrodes with a Simple Cutter . . . . . . . . . . . . . . .5

Notes on Hardware Compatibility for the Digidata 1200A . . . . . . . . . . . . . . . . . . . . .6

Axon Replies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

Employment Opportunities at Axon . . . . . . . . . . . .8

Third-Party Product Information fromBMT Research Services . . . . . . . . . . . . . . . . . . . .9

Focus on MethodsCV-4-1/100 Headstage Adapted for Scanning Tunneling Microscopy . . . . . . . . . . . . . . . .10

The Sales Corner . . . . . . . . . . . . . . . . . . . . . . . . . .11

New Product News

NEW!

experiment; the only function missing is the ability to writeimage or data files to disk. This is a risk-free and effectiveway of trying our ion imaging software with yourPhotometrics camera and hardware. You can download AIWversion 2 from our Web site at http://www.axonet.com; orget it by anonymous ftp from ftp.axonet.comin the directory\pub\demos; or request it from the Sales Department via e-mail (sales@axonet.com), telephone (415-571-9400) orfax (415-571-9500).

Need to Buy a Computer for Imaging?

Are you facing the purchase of a computer to be used withAxon Instruments' Cellular Imaging software or hardware?If so, there are several issues that affect the installation anduse of the computer, such as processor and speed, memory,interrupt (IRQ) lines, PCI Bus support chipsets, displaycards, device control methods and image file backup.

Axon Instruments has prepared a Guide to PurchasingComputers for Imagingthat summarizes our experience andrecommendations. You may request this document fromAxon Instruments Technical Support or directly from ourweb site on the Technical Support page. Go to the Technical

Notes section and under Technical Tips and Hintsclick on“imagcomp.txt”. We also invite you to fax your computervendor’s quotation to us; we will evaluate it and give youfeedback on price, performance and technical issues.

New Features in AxoScope 1.1

The latest release of Axon’s powerful oscilloscope and chart/tape recorder program for Windows 95 and Windows 3.x isshipping as part of the DigiPack 1200 system bundle. In theversion 1.1 release you will find a number of new features aswell as a small number of bug fixes. A free upgrade willautomatically be mailed to all registered users. Or, you candownload the full production release of AxoScope 1.1 fromAxon’s Web site at http://www.axonet.comto demo it or toupgrade from AxoScope 1.0. If you are unable to get accessto our Web or ftp sites, please contact Sales and a free set ofdisks will be sent to you. You must purchase a DigiPack 1200system bundle to perform real data acquisition with hardware,but a demo driver in AxoScope can be used to simulate acqui-sition. Please note that if you currently have a Digidata 1200,you must upgrade it to a DigiPack 1200 in order to acquiredata with AxoScope.

New in AxoScope 1.1Voice Tags− In addition to comment and time tags, you cannow record and replay voice tags. This new feature requiresthat you have a microphone, speakers and a sound card. Youmust set up your sound card and microphone in theWindows Control Panel before attempting to use Voice Tagsin AxoScope.

Low-Pass and High-Pass Filters− Low-pass and high-passRC (single pole) filters have been added in software thatallow you to filter incoming data. The filter cutoff frequen-cies can be set individually for each channel.

New Product News . . .

COPYRIGHT 1996, AXON INSTRUMENTS, INC.

Editor . . . . . . . . . . . . . . . Jay Kurtz

Axon Contributors. . . . . . Brian Carter. . . . . . . . . . . . . . . . . . . . Michael Delay, Ph.D.. . . . . . . . . . . . . . . . . . . . James Fox, Ph.D.. . . . . . . . . . . . . . . . . . . . Burt Maertz. . . . . . . . . . . . . . . . . . . . Pam Middaugh. . . . . . . . . . . . . . . . . . . . Michael Wu, Ph.D.

Additional Contributors . . Peter H. Barry, Ph.D.. . . . . . . . . . . . . . . . . . . . David Dunlap, Ph.D.. . . . . . . . . . . . . . . . . . . . Zhuan Zhou, Ph.D.

2

D I P S I I N D U S T R I E An Approved Reseller for Ion Imaging Products

Axon Instruments has formed relationships with companies thatnow offer Axon Instruments’ Cellular Imaging products. TheseApproved Resellers (ARs) of Axon Instruments offer significantexpertise in cellular imaging and/or offer their own line of imaging-related products.

In this issue we introduce DIPSI Industrie. Based in Asniéres,France, near Paris, DIPSI has been active in supplying Axon’selectrophysiology equipment to European scientists, and is nowalso an AR for Axon’s ion imaging products. DIPSI is a supportand service company that represents a few carefully selectedleading manufacturers and provides equipment ranging fromcomponents to complete custom-made systems. Their customersinclude laboratories, research centers and R&D departments withneeds for scientific instrumentation in biomedical, electrophysio-logical and other fields. DIPSI offers (1) sales and service ofintegrated systems for signal conditioning and data acquisitionand analysis on PC and Macintosh computers; (2) sales and support of systems for electrophysiology; (3) sales and service ofintegrated systems for ion imaging, including monochromators,optical fibers, cameras, plug-in boards and software; and (4) peripheral components such as fast data streaming devices,and mass storage and computer networks. DIPSI has the capability to sell to all European countries, and, since its establishment in 1991, has had significant experience in France,Belgium and Switzerland. Their personnel includes one memberwith a Ph.D. in Pharmacology and an electronics engineeringdegree, an engineer specialized in signal processing, and anadministrative and logistics team.

DIPSI Industrie, 19 rue des Parisiens, 92600 Asniéres, France.Telephone 33 1 47 90 21 11; telefax 33 1 40 86 06 61.

. . . New Product News

Store Trace− You can now store traces on the display withthe Store Trace command in the View menu. This featureallows you to freeze a snapshot of a waveform in the ScopeWindow for real-time comparison with incoming data.

New for ElectrophysiologyJunction Potential Calculator- This is an electrophysiologyutility for calculating liquid junction potential corrections invarious electrophysiological situations. It can be used forthe correction of patch-clamp measurements (for whole-cell,perforated, intact and excised patch configurations); intracel-lular, epithelial and bilayer measurements; and for the correction of direct experimental measurements of junctionpotentials.

AxoScope Junction Potential CalculatorPeter H. BarrySchool of Physiology and PharmacologyThe University of New South Wales

A significant source of error in many electrophysiologicalmeasurements is due to the unavoidable development of liquid junction potentials wherever two solutions of different ionic composition come into contact. Although ithas concerned electrophysiologists over the years (e.g., (1,3)),it has become even more important with the advent of thepatch-clamp technique where exotic ions with low mobilities, necessary for the investigation of ion permeationthrough ionic channels, are present in fairly high concentra-tions (2,4,7,8). Errors of 15 mV and more can be readilygenerated and although the researcher may believe that zeroing the patch-clamp amplifier before patching onto acell has compensated for any junction potentials, this is adelusion. For although the original junction potential of thepipette, VL, is normally initially balanced by an offset, VL',within the amplifier, when the pipette seals on to the cell, VLwill be replaced by the membrane potential across the patch,but the offset, VL', will still remain. The only way to correctfor this source of error is either by calculating the liquidjunction potential (4,2) or by measuring it (7,8) and then bymaking the appropriate corrections (with the correct signand magnitude) to any measured membrane potentials.

In order to make it easy for researchers to both calculatesuch junction potentials and apply them correctly, in addition to showing them how and where in the circuitsthese corrections arise, a graphical DOS program, JPCalc,was developed by Peter Barry (2). This program also storesthe valencies and mobilities of a large number of ions, in addition to allowing the user to enter his own values. Theprogram handles all of the different patch-clamp configura-tions, with additional solution changes. It also handles intracellular, bilayer and epithelial measurements, and eventhe small corrections that also apply when attempts aremade to directly measure these junction potentials. Thejunction potentials are calculated using a form of the generalized Henderson Equation (5,6,2).

Working with Peter Barry, programmers at AxonInstruments developed a Microsoft Windows version of theJunction Potential Calculator for inclusion in AxoScope 1.1.For those without AxoScope, a stand-alone version of theJunction Potential Calculator is available for purchase directfrom Peter Barry (9).

A typical layout of a dialog box is shown in Figure 1, wherethe magnitude and direction of the junction potential correc-tion is clearly seen for an intact patch clamp measurement,with the experimental set-up and the involvement of the various potential components illustrated graphically. Theionic composition of the solutions is entered with a simpleAdd/Edit/Delete dialog accessed by the menu buttons shownabove. Users can choose from about 40 common and exoticions already included within the Factory Ion Library andfrom their own User Ion Library. A continuously updatedCharge Balance output aids users as they input the ioniccomposition of each of the relevant solutions. Solutioninformation can be saved, loaded and modified and theresults of calculations printed out.

Extensive On-Line Help is available regarding all aspects of theJunction Potential Calculator and its use. The On-Line Helpalso includes details of the junction potential corrections for allthe different experimental configurations and conditions,

make sure you don’tmiss an issue! Toreceive AxoBits andother mailings fromAxon Instruments, fillout the registration formon the back of thisAxoBits and return it toAxon or register via ourworld wide web site athttp://www.axonet.com

If you enjoy AxoBits, but thisissue was not addressed to you . . .

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Figure 1. The main dialog box of the Junction PotentialCalculator in AxoScope 1.1.

Assorted Bits . . .

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similar to those found within the 25-page manual availablewith the JPCalc program.

The Junction Potential Calculator should be a welcomeaddition to the useful tools provided in AxoScope 1.1. Theprogram should also help to further highlight the importantrole and significance of junction potential corrections inelectrophysiology and make it much easier for researchers tomake and apply those corrections properly.

1. Barry, P.H. Permeation mechanisms in epithelia: Biionic poten-tials, dilution potentials, conductances and streaming potentials.In: Methods in Enzymology, Biological Membranes, Part M:Biological transport, 171, 678-715, 1989.

2. Barry, P.H. JPCalc, a software package for calculating liquidjunction potential corrections in patch-clamp, intracellular,epithelial and bilayer measurements and for correcting junctionpotential measurements. J. Neurosci. Meth., 5:107-116, 1994.

3. Barry, P.H. and Diamond, J.M. Junction potentials, electrodestandard potentials, and other problems in interpreting electricalproperties in membranes.J. Membrane. Biol., 3:93-122, 1970.

4. Barry, P.H. and Lynch, J.W. Topical Review. Liquid junctionpotentials and small cell effects in patch clamp analysis. J. Membrane. Biol., 12:101-117, 1991.

5. MacInnes, D.A. The Principles of Electrochemistry. Dover,New York, 1961.

6. Morf, W.E. The Principles of Ion-Selective Electrodes and ofMembrane Transport, Elsevier, Amsterdam, New York, 1981.

7. Neher, E. Correction for liquid junction potentials in patch-clamp experiments. In: Ion Channels, Meth. Enzym., 207:123-131, 1992.

8. Neher, E. Voltage offsets in patch clamp experiments. In: SingleChannel Recording, B. Sakmann and E. Neher, eds., 2ndEdition, pp. 147-153, Plenum, New York, 1994.

9. To purchase stand-alone versions of JPCalc, contact Peter H.Barry, Professor of Physiology, School of Physiology andPharmacology, The University of New South Wales, Sydney2052, Australia Tel: +61-2-385 1101; Fax: +61-2-385 1099; e-mail:P.Barry@unsw.edu.au

AxoGraphs Synaptic (and Other) Analyses for PC and Macintosh Data

Did you know that custom analysis modules for PC andMacintosh data are included with AxoGraph 3? You can useAxoGraph’s many analyses for flexible data analysis andtechnical graphing much more easily than you thought. LetAxoGraph put its advantages to work for you!

Advantages: Synaptic Analysesn LTP: Population spike analysis of extracellular field recordingsn Minis: mPSC detection and measurementn Quantal analysis of evoked synaptic events

Advantages: Presentation Graphicsn Error bars n Interval barsn Scale bars n Symbol legendsn Text notes and more…

Here's why:

a) All Power Macintosh computers and most Macintoshcomputers can read 3.5" PC diskettes.

b) Networked computers easily transfer large data filesbetween PC and Macintosh systems.

c) Data from other non-IBM-compatible computers based on theIntel 80x86 CPUs can be read similarly on Apple computers.

d) Data from Atari, DEC and VAX systems based on theMotorola 68xxx CPUs are already in the correct byteorder for reading by Apple computers.

Here's how:

a) AxoGraph v3.0 can analyze and/or graph your data froman expanded list of file formats. The recognized file formats now include:

Program Versions Company UsesCED* 2.1 Cambridge PC electrophysiology data

ElectronicDesign

C Lab* Indec data acquisition development environment

Igor Pro* 1, 2 WaveMetrics Mac graphing/data analysisNeuroCom* 4, 5 NeuroCom posture and movement

EMG dataScope* MacLab Mac episodic data acquisition

pCLAMP 5, 6 Axon Instr. PC/DOS episodic/continuousdata acquisition

AxoData 1 Axon Instr. Mac episodic data acquisition

AxoGraph 1, 2 Axon Instr. Mac graphing/data analysisAxoScope 1.0 Axon Instr. PC/Windows continuous

data acquisitionAxoTape 1, 2 Axon Instr. PC/DOS continuous data

acquisitionChart 3.4 MacLab Mac continuous data

acquisitionCricketGraph III CricketGraph Mac spreadsheet

graphing/data analysisKaleidaGraph 3.0 Synergy Mac spreadsheet

Software graphing/data analysis

b) Multi-column text data can be read in either comma,space(s) or tab delimited formats, and are exported in atab-delimited format.

c) For opening binary digitized data files that are in anunrecognized file format such as AxoBASIC, simply fillout the 'Data Acquisition Parameters' dialog box thatpops up (see next page). Thereafter, these "foreign" datafiles are automatically recognized by AxoGraph.

d) For conveniently opening many foreign binary digitizeddata files in the same file format, binary input/outputfunctions are provided so that you can create your owncustom file import modules.

e) Binary input/output functions are provided so that youcan create your own custom file export modules.Examples are provided for the Igor Pro and AxoGraphfile formats.

If you have a Macintosh or Power Macintosh computer inyour lab, you cantemporarily take advantage ofAxoGraph 3.0 for free! The "demo" version of AxoGraphis actually an evaluation copy of the complete program.This free version will run on your computer for up to 3 months. You can download AxoGraph 3.0 from our website at http://www.axonet.comor call the Sales departmentand ask for the AxoGraph 3.0 demo. Better yet, ask for thespecial introductory price of $295 for AxoGraph 3.0, offergood through June 30, 1996!

*These file formats have been newly added to AxoGraph 3.0 as "Drop-In"modules. By simply placing these modules in the 'Drop-In…' folder, thesefile import formats are incorporated into the AxoGraph 'File Open' menu.

AxoGraph e-mail, FTP & Web Site UpdateThe web site (http://www.axonet.com) and ftp site(ftp.axonet.com) at /PUB/AXOGRAPH have been updatedrecently, as documented in the NOTES.TXTftp file. A second upgrade version of AxoGraph 3 (v3.0.1.02) has alreadybeen posted. To get updates and drop-in modules as fast aspossible, subscribe to the AxoGraph e-mail list by sending ane-mail to burtm@axonet.com. You will then be notified auto-matically whenever new entries are available for downloadingfrom the /UPDATESsub-folder. There is also a new sub-foldercalled /TO_AXON, for submitting your own custom modulesfor distribution through the AxoGraph ftp site.

The AxoGraph Programming Service has a new e-mail address:John.Clements@anu.edu.au. Please send all programming proposals to this e-mail address. See the PROG_SVC.TXTfilein the AxoGraph ftp site for more information.

Restoring the Sensitivity of Used ProCFEElectrodes with a Simple Cutter

Zhuan ZhouDepartment of Physiology, Medical CenterLoyola University Chicago

Carbon-fiber electrode (CFE) sensitivity can be defined bythe electrode’s ability to detect fast amperometric spikes.Low-noise and high-sensitivity ProCFEs (1) can detect ultra-fast single-vesicle release of sub-millisecond spike duration.

However, CFE sensitivity is dependent on the history ofCFE-cell contacts. The electrode’s sensitivity may bereduced by half after 5-8 CFE-cell contacts (2).

The loss of sensitivity after CFE-cell contacts is probablydue to contamination of the CFE sensor tip by organic tissues on the cell’s surface. Fortunately, the CFE sensitivitycan be restored fully after properly recutting the electrode’stip (3). When recutting the electrode, it is critical to avoidany damage of the polypropylene insulation near the sensortip. Consequently, it is best to use microscissors, a 3-dimen-sional manipulator and a microscope to recut CFE tips (4).

Figure 1 is a schematic diagram of a simple cutter used torecut ProCFE tips. Readers can make their own cutters.The microscissors may be purchased from Fine ScienceTools, Inc. for $70 (Cat. No. 15100-09, Tel. 800-521-2109).

The special advantage of this cutter is that the cuttingprocess is performed directly on the experimental setup;thereby eliminating the need for an additional manipulatorand microscope.

To cut a ProCFE: (i) the cutter is placed on the microscopewith magnification 20 x 10 = 200 (objective’s working distance ≤ 2 mm); (ii) the microscope is focused on the cutting edge of the scissors; (iii) the used ProCFE is placedat the cutting edge by the setup manipulator; (iv) press thescissors’ free handle to safely cut just 3-5 µm of the tip eachtime. Note: > 90% of the iris scissors’ cutting edge is withinthe spring-loaded region. To reduce possible insulationdamage by the scissors’ movement, the cutting point shouldbe within the elastic-force free region,i.e., the very end partof the cutting edge.

Since the original tip length is 15-30 µm, each ProCFE can berecut between three and five times. After each cut, the CFE canwork for 3-5 cell experiments (2). Consequently, if one usesthis cutter, one ProCFE can be used in 10-25 cell experiments.

1. Z. Zhou and S. Misler. J. Biol. Chem. 271:270-277, 1996.

2. Z. Zhou. Biophys. J. 70 (No. 2, Part 2): A403, 1996.

3. Z. Zhou and S. Misler. J. Biol. Chem. 270:3498-3505, 1995.

4. Z. Zhou, S. Misler and R.H. Chow. Biophys. J. 70:1543-1552, 1996.

Figure 1. A simple cutter for re-cutting ProCFE sensor tips.(The scissor’s length is about 8 cm.)

Microscissors

Adapter tohold scissors

Copper or ironstand (≥≥ 200 g)

. . . Assorted Bits

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Notes on Hardware Compatibility for the Digidata 1200A

What Kind of Computer do You Need?Any computer with an 80386 or higher microprocessor(Intel, AMD, Cyrix, etc. CPU) and with the proper resourcesavailable should work properly with the Digidata 1200A.Devices, such as acquisition boards, use various systemresources to communicate with the rest of the computer system. Direct Memory Access (DMA) channels, InterruptRequests (IRQ), and I/O port addresses are the primaryresources that allow devices to communicate with and beuniquely identified by the CPU. These resources are limited,which can lead to conflicts among different devices thatwant to use the same resources.

In order for the Digidata 1200A to work fully, your computer must have at least two free Direct Memory Access(DMA) channels among DMA 5, DMA 6 and DMA 7, andat least one free Interrupt Request (IRQ) among IRQ 10,IRQ 11, IRQ 12 and IRQ 15.

What Brand of Computer Will Meet These Criteria?While most computers have the necessary free resources,these resources are often taken by devices that are added bythe reseller or the customer after the purchase. For example,choosing to configure a sound card one way might leave youwith sufficient resources, while a slightly different configu-ration of the same sound card would prevent you from usingthe Digidata 1200A. Due to the number of different config-urations possible, there is no “list” of compatible computers.We can, however, give you the information you need to evaluate a computer for use with a Digidata 1200A and armyou with the appropriate questions for your supplier.

How Do You Know if the Computer You Want to Buy HasEnough Free Resources?Before purchasing the system, ask your supplier what IRQsand DMAs are free. If he does not know, ask to speak withhis technical support department. Once again, in order forthe Digidata 1200A to work, you must have two free DMAchannels among DMAs 5, 6 and 7, and at least one free IRQamong IRQs 10, 11, 12 and 15.

How Do You Know if Your Current Computer Has Enough Free Resources?While you can call the Technical Support department of themanufacturer or reseller of your current computer, they willonly be able to help you if your configuration has neverbeen changed. In computers without Windows 95, you willhave to search through the documentation of the variousexpansion cards you have. The following is a list of standard assignments for IRQs and DMAs: (“Fixed” meansthey are used by the system and cannot be altered or usedfor other devices).

IRQ Device Comment00 System Timer Fixed.01 Keyboard Fixed.02 Interrupt Controller Fixed.03 Communication Port Alterable, usually used for

COM 2 or COM 4.04 Communication Port Alterable, usually used for

COM 1 or COM 3.05 Free Commonly used for LPT 2,

sound cards and network cards.06 Floppy Controller Fixed.07 Printer Port Alterable, usually used for LPT 1.08 System CMOS Fixed.09 Free Cascades IRQ channels to IRQ2.

Commonly used for SCSI adapters.Some video boards require this IRQ.Some PCI bus systems reserve thisIRQ for PCI devices. On some oldersystems this IRQ can’t be used at allbecause the system will not allow it.

10 Free Commonly used for SCSIadapters, Network InterfaceCards, and sound cards.

11 Free Frequently used by SCSI cards.12 Bus Mouse Fixed on some newer systems

that use a bus mouse. Switchingto a serial mouse may free thisIRQ, but the motherboard usual-ly refuses to release the IRQ.

13 Numeric Coprocessor Fixed.14 Primary IDE Controller Fixed on most systems. Even

systems that do not use IDE orATAPI devices may refuse to releasethis IRQ if the motherboard has anon-board IDE/ATAPI controller.

15 Secondary IDE Controller Fixed on some systems with on-board dual channel IDE/ATAPIcontrollers. On most systemsthis IRQ is free.

DMA Device Comment00 Free01 Free Often used by SoundBlaster

sound card.02 Floppy

Drive Controller Fixed.03 Free04 DMA

Controller Fixed.05 Free06 Free07 Free

What I/O Port Addresses Need to be Available?The Digidata 1200A requires 32 port addresses (or 20h, inhexadecimal notation). The default address range is 320h to33Fh. The lowest number in the range (in this case, 320h) iscalled the base address. Other possible address ranges startat the following base addresses: 100h, 140h, 180h, 1C0h,220h, 300h, 340h. Many SCSI adapters use 340h and 140h

(continued on page 9)

Assorted Bits . . .

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Q: I have no trouble fitting with a Boltzmann functionin Clampfit by setting my cursors and pressing “T” for fiT.However, I want the slope and midpoint. How do I findthem?

A: After fitting your data with a Boltzmann function,look at the Fit (MAIN or X-Y) Results Array to find theSLOPE(the change in amplitude as a function of the independent variable) of the portion of the curve connectingthe plateau portions and the MIDPOINT (point at which thecurve is halfway between its two plateau values). TheBoltzmann equation, which is displayed above the values inthe results array, is

The independent variable is called “t”. Although “t” usuallystands for “time”, it could also be “potential” or any otherX-axis variable. For example, when “t” is potential, as it isfor a steady-state inactivation curve, the slope represents theequivalent voltage-dependence of inactivation. The τ valueaffects the rate at which the exponential changes as a function of “t”, and so gives you the SLOPE.

SLOPE= ττ is written in the results array under its own heading.

To calculate the position of the MIDPOINT, which occurs atthe value of “t” where B(exp(-(t-K)/ τ)) = 1, one must usethe given values for t, start fit K, and shift B in the followingequation:

MIDPOINT = K + τln(B)

Q: I have trouble using pCLAMP 6 to control myCyberAmp 380. When I input my settings inConfigure/CyberAmp using the SetValues menu, they don’tseem to work. How can I use my CyberAmp with Clampexand Fetchex?

A: You are on the right track by using SetValues, but thisis only half of what you need to do. There are two vital stepsin using a CyberAmp (either 320 or 380) with pCLAMP 6.The first step, which you have done, is to control the operationof the CyberAmp processing channels. The second, equallyimportant step, is to define what happens to the CyberAmpOUTPUTS, and to relate them to the inputs to pCLAMP 6.Happily, the second step is no harder than the first. Afterhaving controlled the CyberAmp operations with SetValues,you must use the MapChannels menu to tell pCLAMP 6which CyberAmp channels are connected to which input

channels on the Digidata. This is necessary becausepCLAMP 6 receives all of its input via the Digidata, andcannot directly access the output of the CyberAmp in anyother way (although pCLAMP 6 controls CyberAmp functions via an RS-232 link, the data transfer occurs via theBNC cable connections).

To set up the CyberAmp for use with pCLAMP 6, select“SetValues”:

and enter the desired settings in the table:

to configure the gain, offset, and filtering of each channel ofthe CyberAmp. Press <Enter> when you have entered allthe desired settings. Note that this menu is with reference tothe CyberAmp controlling (via the RS-232 connection) theprocessing applied to each CyberAmp INPUT channel, anddoes not contain any information about where the output ofthe CyberAmp is to go.

Secondly, select “MapChannels”:

to relate the CyberAmp channels to the digitizer data inputchannels (BNC connections between CyberAmp outputchannels and Digidata (or TL-1) input channels).

The ANALOG IN channel in MapChannels is the channel ofthe digitizer that pCLAMP 6 is using; MapChannels is necessary to relate the CyberAmp and the digitizer. Press<Enter> when you have entered your desired mappingbetween CyberAmp output channels to digitizer input channels.If MapChannels is not filled in, there is no way for pCLAMPto meaningfully use the input to its digitizer channels.

Filling in both of these menus will allow you to successfullycontrol and use the CyberAmp with pCLAMP 6.

Axon RepliesTo Commonly Received Questions

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Axon Replies . . .

Q: In my experiments, I like to leave the patch clampat maximum bandwidth and filter after the fact with an 8-pole Bessel filter for maximum noise reduction. However,the 100 kHz bandwidth of the Axopatch 200B makes itimpossible to simultaneously use the maximum bandwidthand the maximum gain of 500 mV/pA for single-channelrecording. This is because the wideband noise at maximumgain causes saturation of the Scaled Output current and, ofcourse, artifacts in the externally filtered current. So, evenwith good low-noise techniques, I cannot record with theAxopatch 200B gain set larger than 200 mV/pA. This is notenough to fully utilize the A/D range of my Digidata 1200Afor small single channels. My filter does not have gain afterthe filter, so I cannot use it to boost my signal. Do you havesuggestions regarding how to solve this problem using Axonhardware and software?

A: Although it is the simplest solution, it is clear thatyou do not want to simply reduce the patch clamp band-width to 10 kHz so you can use all of the patch clamp'sgain, given your understandable desire to use as much band-width as you can. Filtering the amplifier output at 10 kHzwould make the transfer function of the Axopatch 200B filter contribute to the overall transfer function of the signalemerging from the external 8-pole Bessel filter; however,you want the transfer function affecting your signal to bedominated by your 8-pole Bessel filter. One solution is torecord at 100 or 200 mV/pA and 100 kHz bandwidth fromthe Axopatch 200B, filter as desired with your 8-pole Besselfilter and simply increase the programmable gain of theDigidata 1200A when you are sampling. If you are usingthe 8-pole Bessel filter of a CyberAmp 320 to set the frequency response, you can specify a CyberAmp gain of 2or 5 in pCLAMP to increase your signal prior to digitization(this will be post-filter gain). This would allow you to useeither 100 or 200 mV/pA on the patch clamp and the fullbandwidth of 100 kHz. Do not specify a gain of 10 or higher since this will increase pre-filter gain in theCyberAmp and recreate your original problem.

Axon University: A Note from the Provost

Axon Instruments will be sponsoring two workshops on the daypreceding the Society for Neuroscience meeting in Washington,D.C. These workshops provide practical advice, technicalinformation and leading-edge encounters with important techniques in the neurosciences. The talks will be given by recognized experts in their fields. These workshops will be:

Experimental Techniques in Electrophysiology, to be heldSaturday morning, November 16, 1996; and

Imaging Techniques: From Cells to CNS, to be heldSaturday afternoon, November 16, 1996.

The morning session will cover: Optimizing an ElectrophysiologySet-Up, Designing Electrophysiological Experiments for Results,

Data Analysis Techniques, Oocyte Recording, Brain-Slice Recording,and Electrical Recording in the Operating Room.

The afternoon session, chaired by Dr. Brian MacVicar ofUniversity of Calgary, will cover Fluorescence Microscopy,Basic Imaging Techniques, Ion Concentration Imaging,Imaging of Single Cells, Imaging of Brain Slices, Imagingusing Voltage-Sensitive Dyes, Volume Regulation Studied withImaging, and Imaging with Confocal Microscopes.

Look to this space for more details in the next AxoBits, whenspeakers, times and locations will be published.

Sincerely,

James Fox, Ph.D.Head, Educational Programs

Visit the Axon Instruments Booth

Experimental Biology 96Washington, D.C.April 14-17, 1996Booths 1062 and 1064

The Society for NeuroscienceWashington, D.C.November 16-21, 1996

8

Axon Seeks to Hire...Confocal Microscope Technical Sales Support

Axon instruments is the world leader in the design andmanufacture of instrumentation and software for electro-physiology and biophysics research. Our products includevoltage-clamp and patch-clamp amplifiers, imaging systems, and data acquisition and analysis systems.

Axon Instruments has a mature neurophysiology productline that is welcoming a new Confocal Microscope lineinto its product basket. We are looking to add technical aswell as sales staff to support this exciting new product.

Requirements: Energetic individual to take a challengingrole in the confocal microscopy world. Candidate musthave a biological/biotechnology background. Have atleast five years’ experience in the laboratory environment.Optics and microscopy knowledge is a plus.

Axon Instruments offers a technologically innovativeatmosphere, opportunities for professional growth, a competitive salary and an attractive benefits package.

Please fax, mail, or e-mail your resume to:Axon Instruments, Inc.1101 Chess Drive, Foster City CA 94404fax: (415) 571-9500e-mail: enriquec@axonet.comPrincipals only please

9

(continued from page 6)

as their primary and alternate addresses respectively. AllSoundBlaster cards and most SoundBlaster compatible cardsuse 220h as the default address. Many Network Interface Cards(NIC) use 300h or 310h as their default address. While the pos-sibility for conflict certainly exists, the Digidata offers more thanenough alternatives to resolve an I/O address conflict.

How Can You Free Up Resources that are Currently Being Used?The best time to consider what configuration will work thebest is before you purchase a new system. By carefully considering what you need your computer to do now andover the next two years, you can save yourself money andavoid headaches.

Start by choosing the component interface you want yourboot hard drive to use. If a system contains an IDE drive,the system must boot from an IDE drive. IDE/ATAPI is atype of component interface that allows you to connect multiple devices like hard drives and CD-ROMs.

If there are no other types of hard drives present, your system can boot from a SCSI drive. SCSI is another type ofcomponent interface that allows you to connect multipledevices. While there is a wider variety of SCSI devices thanIDE/ATAPI devices, SCSI is more complicated, more diffi-cult to setup and it is slightly more expensive.

If you know you are going to buy a SCSI tape drive or scanner, choosing an all-SCSI system could save resources. Inmost systems, you can save an IRQ or two by having the SCSIcomponent interface replace the IDE bus functions (i.e.,boothard drive). Be careful, this does not always work; some sys-tems will release IRQ 14 (IDE controller) and others will not.You need to ask the manufacturer. If it does release IRQ 14,set your SCSI adapter to IRQ 14 so you have more IRQsavailable for the Digidata 1200A board. Be aware, however,SCSI devices come with their own set of trials and tribulations.

Next, when purchasing accessories, try to get the computermanufacturer to pre-install them. Avoid proprietary inter-faces; try to buy components that use your existing interfaces(i.e., IDE or SCSI). Resources are frequently wasted on CD-ROMs and multimedia kits where the CD-ROM uses asound card or proprietary interface. On many sound cards,you must disable the CD-ROM interface (even if you do nothave a CD-ROM) to free the resources. Moreover, manysound cards offer emulation modes and MIDI synthesiswhich use even more resources. To preserve resources andminimize conflicts, opt for an 8-bit sound card.

Planning ahead and asking suppliers questions can reallymake your life easier. Better still, get the manufacturer toinstall all of the additional components and specify that youneed one IRQ (10, 11, 12 or 15) and two DMAs (5, 6 or 7)free. If that opportunity already passed, reconsider what useyou plan for your computer. If getting the most accurateand reliable data is most important, you may need to removesome other devices.

In many cases it is best if you can dedicate a computer todata acquisition and use your network or personal computerfor analysis, e-mail, write-ups, etc. Where this is not possi-ble, you must prioritize your needs because we all must livewithin the limits of these resources.

BMT Research ServicesIntegrated Recording Chambers and Temperature ControllersBMT Research Services offers several models of integratedrecording chamber and temperature control platforms whichcan be used for combined electrophysiological and opticalexperiments with isolated cell preparations.

BioTherm-1 mounts on standard stages of a range of invertedmicroscopes; custom adapting to other types of microscopes isalso available. This system features an easily removable, small-volume (0.2 ml)open chamber with a thin (0.17 mm) glass bottom for good optical imaging with high numerical aperture,short working distance microscope objectives. The chamber ismade from a biologically inert metal with excellent chemical,mechanicaland thermal characteristics. Plastic recording cham-bers are also available. The stage-mounted temperature controlplatform is ≈100 mm in diameter, and has a low profile allowingfor shallow angles of electrode placement. The platform isavailable for heating only (BioTherm-1A) or both heating andcooling (BioTherm-1B). BioTherm-1A uses a foil resistanceelement to heat both the recording chamber and the superfusionsolutions with a pre-heater operating between 20 - 40 °C.BioTherm-1B produces heating and cooling with Peltier "heatpumps" operating within 2 - 40 °C. An internal water-cooledheat exchanger in the temperature control platform increasescooling efficiency below about 10 °C.

Power supplies for BioTherm-1 feature microprocessor-basedproportional control which automatically optimizes control loopcharacteristics. The circuits are low-noise, even for single-channel recordings. Output current levels are limited to preventthermal damage to the temperature control platform elements,and alarms and automatic power shut-down are provided forinappropriate set-points, temperature-sensor open-circuit conditions, or inadvertent placement of temperature-sensorsoutside the control loop.

BMT Research Services also produces BioFlow-1, a solenoid-controlled solution flow system. This system uses tube-pinchsolenoid valves to avoid contamination of either solutions orvalves, and for easy cleaning of the system. Solutions in up to12 reservoirs can be controlled. The solenoid valves can beoperated independently either manually via front panel switches,or remotely by computer via parallel digital lines,e.g.,fromAxon Instruments pCLAMP.

For more complete information on these and other productsfrom BMT Research Services, contact the company at 172 Silverview Way NW, Calgary, Alberta, Canada, T3B 3K3;(phone) 403-247-9829, (fax) 403-286-5519.

CV-4-1/100 Headstage Adapted for Scanning Tunneling Microscopy

David Dunlap, Ricardo Garcìa#, Steve Smith*, and CarlosBustamante*

DIBIT 3°A3, San Raffaele Scientific Institute, via Olgettina 58,20132 Milan, Italy

#Centro Nacional de Microelettronica, CSIC, Serrano 144, 28006Madrid, Spain

*Institute of Molecular Biology, University of Oregon, Eugene,Oregon 97403

In as little as a decade of development, the scanning tunneling microscope has emerged as a powerful tool fornanometer-scale microscopy of surfaces. Atomic-scaleimages of hard, clean, and conductive or semiconductivesurfaces are routine in many laboratories worldwide. Theapplication of this technique to the usually soft, complexsurfaces of biological specimens is more difficult. Forexample, it has been shown that the electronic conductivityof DNA is too low for visualization with standard scanningtunneling microscopes (1). However, further investigationhas shown that DNA can be imaged with tunneling currentsin the picoampere range.

A scanning tunneling micrograph of a surface can be simplydescribed as a color-encoded topograph. It is a digitalrecording of the separation necessary to maintain a constantcurrent between a finely pointed, conductive probe and asample surface throughout a two-dimensional scan of thesurface. The relative separations are then arranged as a digi-tal image on a computer in which height is usually encodedin gray-scale values. The topographic nature of the imageresults from the decaying exponential dependence of thecurrent,I, upon the separation between the probe and thesurface,s. For two conductors separated by a slight gap in avacuum that dependence is:

Obviously changes in the "conductivity" at different pointson the surface (the work function,φ) also influence currentand therefore a scanning tunneling micrograph must be carefully interpreted to separate topographic from electronicfeatures.

The current measured by a standard microscope ranges from0.1 to 1.0 nA and is determined by the operator along withthe bias voltage applied between the probe and the surface.The microscope then executes a scan modulating s in orderto keep I at the determined value. In response to insufficientcurrent, the instrument will iteratively reduce s and mayplunge the probe into the surface. In the case of poorly

conducting biomolecules adsorbed on conducting surfaces,the probe may stab through the adsorbate to reach the underlying, conductive support. In many experiments,biomolecules are swept from the scan area and are not detected.

One attempt to overcome this limitation was to employ alow-current scanning tunneling microscope. A very low-current microscope was assembled which operates at currents as low as 0.07 pA (1000-fold lower than standardvalues) yet has sufficiently broad bandwidth to record a 400 × 400 point image in several minutes (2). A CV-4-1/100headstage from Axon Instruments was incorporated into aNanoScope II-III scanning probe microscope (DigitalInstruments Inc., Santa Barbara, California). In addition, thelow-current scanning tunneling microscope required twocustom components: the scanner support base, which housesthe stepper motor for automated approach of the tip to thesample surface, and a probe housing which contains the tipholder and carries the low-current amplifier electronics. Thescanner support base of the low-current scanning tunnelingmicroscope is a replica of the scanner support base of theNanoScope small-sample scanning force microscope inwhich the force microscope circuitry was replaced by simpleelectrical connections between the DB-25 pin connector andthe stepper motor or the boost box.

The amplifier was a key factor for simultaneously achievingvery low currents and wide bandwidth feedback response.Maintaining an adequately broad bandwidth was necessaryto maximize the scan speed and thereby minimize the distortions produced by low-frequency mechanical instabili-ties and thermal drift. The scan speed multiplied by thehighest spatial frequency in the image constitutes a high frequency input signal that must fall within the bandwidth ofthe feedback that modulates separation of the tip and thesample. The largest gain stage in the feedback loop is thecurrent-to-voltage converter, and therefore the current amplifier constrains the bandwidth of the feedback. If thebandwidth of the amplifier is too low, the feedback is slowand the probe may not faithfully follow the surface profile.The simple relationship, v=∆f/ωx, between the scan speed, v,the feedback bandwidth,∆f, and the highest spatial frequencycomponent in the image,ωx, defines an upper limit for thescan speed. A spatial frequency of 10 cycles/nm at 20 kHzbandwidth establishes a maximum scan speed of 200 nm/s.

The CV-4-1/100 headstage consists of a high-gain, current-to-voltage converter attached to the probe input, and a"boost box" that houses circuitry to tune the frequencyresponse of the amplifier and select the gain. For use as apatch-clamp amplifier, the boost box is connected via a DB-25 pin plug to the controlling electronics. For scanningtunneling microscopy, those connections and some circuitrywere modified to provide the appropriate power and controlsignals from the NanoScope electronics (3). The CV-4-1/100

Focus on Methods

10

was mounted on the probe housing that was machined froma single block of aluminum (see figure below). The frontside is a hinged aluminum frame electrically connected withan inset indium tin oxide-coated gals window. The bottomis a steel plate that articulates with the three magneticadjustment screws of the kinematic mount of the piezoelectric scanner of the NanoScope.

The amplifier was calibrated for a DC input and separatelytuned to optimize the frequency response. A variable voltage from a potentiometer connected to a 9 V battery wasapplied to a 2 GΩ resistor that was connected to the amplifier input. The applied voltage and the amplifier output were displayed simultaneously as the DC gain wasadjusted with a gain potentiometer in the boost box. Tooptimize the frequency response (bandwidth) of the amplifier, a triangular voltage waveform was applied acrossa capacitor present in the headstage circuitry connected tothe input of the amplifier. The amplifier response was tunedusing a series of seven potentiometers in the boost box toachieve the squarest response possible. At room temperature,and for a measured bandwidth of 7 kHz, the thermal noiseof the amplifier was calculated to be 0.05 pA.

Initial efforts to image DNA using this low-current amplifieralso failed because the DNA was much less conductive thanindicated by rare, previous images from several laboratories(1). However a new operating mode employing unusuallyhigh bias voltages, high relative humidity, and low tunnelingcurrents has been successful (3,4). Under such conditions,DNA adsorbed on mica, an insulator, has been imaged byvirtue of a weakly conductive hydration layer over the surface and the adsorbed DNA. The effective resistance ofthe 0.4 nm thick hydration layer has been estimated to be 10 GΩ. Under these conditions on a conductive support, theprobe would usually remain distant from the surface and resolution would be reduced. Instead, with the DNA spreadon mica, the probe scans closely enough to image singleDNA molecules. The critical parameters appear to be high

bias voltages (6-10 V) which may also induce near fieldemission of electrons for imaging (5), relative humidity inthe range 30-70%, and tunneling currents of 2 pA or less.Incorporating the CV-4-1/100 headstage into the NanoScopesystem was relatively straightforward and extended therange of investigations with scanning tunneling microscopy.

1. Dunlap D., Garcìa R., Schabtach E., and Bustamante C. Maskinggenerates contiguous segments of metal-coated and bare DNA forscanning tunneling microscopy. Proc. Natl. Acad. Sci. USA.90:7652-7655, 1993.

2. Dunlap D., Smith S., Bustamante, C., Tamayo J., and Garcìa R.A very low current scanning tunneling microscope.Review ofScientific Instruments. 66(10):4876-4879, 1995.

3. Guckenberger R., Heim M., Cevc G., Knapp H., Wiegräbe W.,and Hillebrand A. Scanning tunneling microscopy of insulatorsand biological specimens based on lateral conductivity of ultrathinwater films. Science. 266:1538-1540, 1994.

4. Zuccheri G., Garcìa R., and Bustamante C. unpublished results.

5. Garcìa R., Tamayo J., Soler J. M., and Bustamante C. Physicalparameters that control the imaging of purple membranes with thescanning tunneling microscope.Langmuir. 11:2109-2114, 1995.

Focus on Methods . . .

11

Sales Corner

Customers in Every Country Throughout the World Can Buy Direct from AxonWe would like to remind our international customersthat we can process and ship orders directly to you.We can accept international orders directly from yourinstitution and our experience with export procedureswill facilitate the receipt of your order in a safe,efficient manner. Please contact the Sales Departmentif you have questions regarding international orderingand shipping procedures.

Non-U-Type HeadstagesEventually, we will discontinue the old non-U-typeheadstages that were included with purchases of ouramplifiers. If you order a non-U-type headstage thatwe no longer stock, you will be informed that you willreceive the U-type headstage with a complimentarycompatible electrode holder and electrical interface toconnect the holder to your model cell. Please keep inmind that we will continue to fill orders for non-U-typeheadstages while supplies last.

INTERNATIONAL TOLL-FREE NUMBERSBelgium 11-8201 Italy 1678-74-022Denmark 8001-0306 Netherlands 06-022-6850Finland 9800-10039 Norway 050-12-042France 05-90-1137 Sweden 020-795-661Germany 0130-81-0458 Switzerland 046-05-7323Israel 177-100-1504 United Kingdom 0800-89-1504

Worldwide sales are direct from the factory.

If you would like your name added to the AxonInstruments mailing list or would like to join thepCLAMP Users Group, please photocopy this form, fillit out in print letters and fax or mail it to AxonInstruments.

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JAPANOur customers in Japan may also order fromour local distributor, Inter Medical

Phone: 052-937-7060Fax 052-937-5423

Toll-free fax number from Japan for directsales and technical support from AxonInstruments is 00-66-33-800-102.

Worldwide sales are direct from Axon Instruments, Inc.

Axon Instruments, Inc.1101 Chess Drive, Foster City, CA 94404 USAPhone (415) 571-9400 Fax (415) 571-9500

e-mail: sales@axonet.comhttp://www.axonet.com