12
Continued on Page 2. 1 June 1999 Axon Instruments, Inc. Integrated Solutions for Electrophysiology, Imaging and Genomics New Product News In This Issue New Product News .......................................................................... 1 The Genepix 4000 Microarray Scanner .......................................... 1 pCLAMP 8, Ready to Go! .............................................................. 1 Digidate 1320 Series Digitizers ...................................................... 1 Axon Replies ................................................................................... 4 Focus on Methods ........................................................................... 5 Techniques for Neuronal Recording and Microstimulation in Humans ................................................................................ 5 Measurement of Organelle pH in Single Living Cells ....................... 8 Visit the Axon Instruments Booth .................................................... 7 pCLAMP 8, Ready to Go! Clampfit 8 for Windows is ready for your research labs! Whole-cell analysis is now easier and more comprehensive than ever before. New fitting functions, power spectra analysis, and much more have been added. Plus, cooperativity with Clampex 8 brings you automatic on-line I-V graphing! The GenePix 4000 Microarray Scanner Axon is proud to announce the GenePix 4000 microarray scanner, its first offering in the field of genomics. Axon has applied its substantial expertise in biological imaging technology to provide a fully-integrated scanner and software package that sets new standards in the acquisition and analysis of gene expression data from microarrays. Developed by Axon engineers to the requirements of leading experts in microarray research, the GenePix 4000 offers unparalleled perfor- mance and affordability in a compact system. What are Microarrays? Microarrays have opened up tremendous new research opportunities Continued on Page 3. Digidata 1320 Series Digitizers The New Standard in Data Acquisition Axon Instruments is proud to announce the new Digidata 1320 Series data acquisition systems. These digitizers offer a better combination of high-speed, high-resolution, and low-noise than other data acquisition systems. Continued on Page 2.

mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

  • Upload
    others

  • View
    3

  • Download
    0

Embed Size (px)

Citation preview

Page 1: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

�������������� �

�����������������������������

����������������������������������������������������������

����� �������

�������������

����� ������� �������������������������������������������������������������������������� ������������ ������������������ ������������������������������������������ ���������������������� �������������������������������������������������������������� �� ������!" �����#�� �$��# ������������������������������������������������������ �

������������ ����������������������������������������������������������������������������������� �

�������������� � ��������������������������������������������������������������������������� %�����&'�#�(���)�'����*�������� ���������#�+'*������,'+��# �������������������������������������������������������������������������������� %

���#'��+�����(�-� ���**��,����� *���.� ���**# ����������������������� �

�������������������������������� ���������������������������������������������������� /

�� ���!"���# $����%�&

Clampfit 8 for Windows is ready for your research labs!Whole-cell analysis is now easier and more comprehensivethan ever before. New fitting functions, power spectraanalysis, and much more have been added. Plus,cooperativity with Clampex 8 brings you automatic on-lineI-V graphing!

����%������'(((������#��#$�)�#����

Axon is proud to announce the GenePix 4000 microarrayscanner, its first offering in the field of genomics. Axon hasapplied its substantial expertise in biological imagingtechnology to provide a fully-integrated scanner andsoftware package that sets new standards in the acquisitionand analysis of gene expression data from microarrays.

Developed by Axon engineers to the requirements ofleading experts in microarray research, the GenePix 4000offers unparalleled perfor-mance and affordability ina compact system.

*�#��#�������#��#$�+

Microarrays haveopened uptremendous newresearch opportunities

����������������

,�-� #�#��./(�)������,�-���0���

The New Standard in Data Acquisition

Axon Instruments is proud to announce the new Digidata1320 Series data acquisition systems. These digitizers offera better combination of high-speed, high-resolution, andlow-noise than other data acquisition systems.

�������������� �

Page 2: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

Clampex 8 forWindows hasalso had its maincapabilitiesextended. Dualwaveforms and100,000 samplesper sweep persignal meanmore flexibilityin your stimulat-ing and recording of cells.

pCLAMP 8 is the result of extensive development and testing over thepast year. The main goal was to port Clampfit from a DOS programto a full Windows program. Along the way, several major new corefeatures were added, as Clampfit was converted from a pure analysisprogram to an analysis/graphing package.

Existing features were greatlyenhanced in both Clampfit 8and Clampex 8. However, afeature trade-off was made,and macros are no longerincluded in Clampfit 8 -instead, third-party macroutilities are recommended.

The Fetchan 6 and pSTAT 6single-channel analysisprograms continue to run underDOS, and are unchangedexcept for minor bug fixes.

pCLAMP 8 includes full on-line Help and printed manualswith extensive tutorials. Alsounder the Help menu, theClampex and Clampfit 6Assistants map all version 6commands to their pCLAMP 8equivalents.

pCLAMP 8 runs under Win-dows 95 / 98 / NT 4.0 on arecommended minimum of a133 MHz Pentium computerwith 32 MB of RAM, and a CDdrive to install the programs.All previous ABF and DATAxon data files are supported.

�!������ �!����

/

��"�#$����������������%�

There are two models; both offer high resolution16-bit analog inputs and analog outputs. TheDigidata 1320A offers a maximum 250 kHzsampling rate (aggregate) with ±1 mV noise in a±10 V input range. The Digidata 1321A offers amaximum 500 kHz sampling rate (aggregate) with±0.5 mV noise in a ±10 V input range, a ten-foldreduction in noise compared to 12-bit data acquisi-tion systems.

The Digidata 1320 Series digitizers contain theBNC interface and the digitizer circuitry in oneunit. Communication and data transfer to thecomputer is accomplished via a SCSI interface. ASCSI cable and SCSI host adapter are included withthe digitizer.

These systems come complete with AxoScope forWindows 95, 98, and NT 4.0. AxoScope ispowerful, easy-to-use data acquisition softwarewith four distinct acquisition modes. BothAxoScope and the Digidata 1320 Series digitizerssupport up to sixteen analog inputs.

Clampex 8(purchasedseparately as partof the pCLAMP8 suite)extendsAxoScope’sabilities withanalog anddigitaloutput command waveforms. Clampex supports theDigidata 1320’s sixteen analog inputs, two analogoutputs (with two independent stimulus wave-forms), the Tag and Start trigger inputs, and thefour instrument telegraph inputs, which are inde-pendent of the analog inputs. Clampex alsosupports eight of the sixteen digital outputs.

Note to European Customers: the Digidata 1320Series digitizers have not yet been CE certified, sothey are not immediately available to our Europeancustomers. Please be assured that Axon is seekingCE certification and expects it to be granted in thenear future.

The Digidata 1320A price is $3,500, and theDigidata 1321A price is $4,900.

������� 0!��

��������1�� ���2��" 03��

��������1�� ������!" � 03�3

��������1�� ������!"�� 0��

4 ������������/��������� 0%

4 ������������3��������� 0���

4 ������������%��������� 0��/

4 ���������������������� 0"�"

�� ���!������-

&�������%� '����������������%�

��������#��1���!

�'��+����567�����#

89'�2:�����#

"�������(����;��� �;'�����#

4#��6��(�����'#��+�;���

��<��������'+����*�##

�����#���������+���

���*�##��(�7������

�����*����#

�� ������'��=���<

, ��##�1�>����##�1�)�����;*���#

?*������*�5����(�������;*���#

��������#�����!

�'�*�=�.�(��+����*� �-'�'�#

� � ���+*�#�1��<���1�� ��*

-�6*�������#��#����##�����

���*6�+����*� ��������*

�� ���!�2�-���-���

Page 3: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

.

��� �!������ �!�

in virtually all fields of biology. In fact, they are consideredby some as the next revolution in molecular biology afterthe polymerase chain reaction (PCR). The breakthroughthat microarrays, or DNA chips as they are sometimescalled, provide is that researchers can now study geneexpression of thousands of genes, up to the entire genome,at once. There are already microarray chips containing theentire genome of yeast and C. Elegans, and within a fewshort years, complete mouse and human chips will beavailable.

Microarrays are fabricated by arraying, or spotting, cDNA,mRNA or protein onto standard microscope slides, coatedwith a substance to allow the spotted substance to stick.The ‘spots’ are on the order of 100 – 200 mm diameter andliterally thousands of spots, each representing a single genesequence, can fit onto a subregion of the slide.

Fluorescently-tagged complementary DNA from control andtest samples is then hybridized onto the microarray.Differential gene expression can then be measured asdifferential labeling of each spot.

Besides expression studies, there are a number of otherapplications for microarrays including virus/bacteriadetection, single nucleotide polymorphism (SNP) determi-nation, sequencing, drug discovery, and gene discovery. Inthe future, microarray technology will likely be appliedclinically to hone in on the genetic bases of disease, drugresponsiveness, and diagnosis.

����%������'(((�)$����

The GenePix 4000 system is a fully-featured microarrayscanner and a tightly-integrated software package that:

· Provides dual-laser simultaneous scanning· Displays images from two wavelengths and calculates a

ratio in real-time· Precisely controls the scan area, reducing scan time and

image storage requirements· Collects emitted light at close to the theoretical limit· Offers outstanding sensitivity and image quality· Includes a complete set of intuitive and powerful data

acquisition, display and analysis tools· Automatically archives events in a Lab Book· Facilitates assistance with built-in HTML help· Is backed by unlimited free telephone technical support

Some technical specifications of the system are:

· Accepts standard microscope slides· Dual laser excitation at 532 nm and 635 nm· 10 mm and 40 mm scan resolutions· Acquires 16-bit TIFF gray scale images· Extremely high sensitivity (better than 0.5 fluors / mm2)

and wide dynamic range (four orders)· SCSI connection to computer· Windows 95, 98 and NT compatible· Flexible ‘block’ or ‘template’ making, saving, retrieving· Multiple methods of ratio calculation· Hardware, acquisition, analysis and display settings

saved as separate file

The GenePix system has been tested and installed in theleading academic, biotech and pharmaceutical laboratoriesusing microarray technology. The response to and accep-tance of GenePix has been overwhelming. If you wouldlike a full description of the system and its capabilities,please refer to our web site at www.axon.com and lookunder ‘Genomics’. Call our GenePix support group at(650) 571-9400 to arrange for us to scan a slide for you.

�����������#�(�<���� ����(����#��<� � ������**� ('������*��� #��+@������ ���@'����#����*���2��*����.����@��A�#������#��4��;�

���� ����BBB�������5�#��'+���#��5���

����������������������%�

Editor ......................................... Heather EvansAxon Contributors ...................... Burt Maertz

Dave GallegosDavid Wellis, Ph.D.

Additional Contributors ............. A.M. Lozano, Ph.D.G. Chandy, Ph.D.J.O. Dostrovsky, Ph.D.K.D. Davis, Ph.D.M. Manduch, B.Sc.M.M. WuW.D. Hutchison, Ph.D.

Page 4: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

'

����������������������$������3� �4��������

Q: Can I install pCLAMP /AxoScope for Windowsonto Windows NT 4.0 systems running on DEC Alpha,MIPS or PowerPC processors?

A: No, only computers with Intel Pentium-classprocessors are supported.

Q: Will pCLAMP 6 run under Windows NT?

A: The pCLAMP 6 analysis programs Clampfit,Fetchan and pSTAT will run fine. However, the pCLAMP 6data acquisition programs Clampex and Fetchex will onlyrun in demo mode. You may consider partitioning yourhard disk for NT and DOS.

Q: Can I install pCLAMP /AxoScope for Windowsonto computers with multiple processors running?

A: No. pCLAMP and AxoScope will not run withmultiple processors, and are only certified for singleprocessor systems.

Q: Can I install pCLAMP /AxoScope for Windowsonto dual-boot systems with both Windows NT 4.0 andWindows 95?

A: Yes. Guidelines on setting this up are available onour web site. See the Technical Note for pCLAMP 7 or 8on ‘Running Axon Hardware and Software Under WindowsNT 4.0’.

Q: Which file systems are pCLAMP /AxoScope forWindows compatible with?

A: pCLAMP/AxoScope for Windows runs fine on allWindows file systems (FAT16, FAT32 and NTFS).

Q: Will installing pCLAMP/AxoScope for Windowsonto a system with Windows NT 4.0 improve its perfor-mance?

A: Some customers running Windows 95b/98 oncomputers 200 MHz or faster may experience problemswith Clampex/AxoScope for Windows. During dataacquisition, they get the error message “Cannot keep up dueto Windows activity”. Switching to Windows NT 4.0 oftensolves this problem.

Q: Does it matter into which slot on the motherboardthe Digidata 1200A/B board is placed?

A: Yes. If there are multiple ISA slots to choose from,install the Digidata 1200A/B board into the first ISA slot inthe computer. This provides the best performance.

Q: Is pCLAMP 8 Y2K compliant?

A: Yes, as are all Axon products. For an officialstatement on the Y2K conformity of Axon products pleaserefer to our web site:

www.axon.com/MR_Y2Kconformity.html

����5�� #�����)�1�#�����������

Imaging Workbench 2.2 AxoGraph 4.0AxoScope 8.0 Fetchan 6.0.5.02Clampex 8.0 pSTAT 6.0.5.07Clampfit 8.0 GenePix 1.0

If you would like to update to the current version,please see our Web site at www.axon.com for moreinformation.

Page 5: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

6

�������������� �

������7����1���������#������� ��-�#� �����������#��������2��#��

J.O. Dostrovsky, Ph.D., M. Manduch, B.Sc.,K.D. Davis, Ph.D., W.D. Hutchison, Ph.D., andA.M. Lozano, Ph.D.Departments of Physiology and SurgeryUniversity of Toronto, Toronto, Ontario, Canada.

����� ������The use of microelectrode recording of neuronal actionpotentials in functional stereotactic neurosurgery is becom-ing increasingly more common as part of the procedure forthe treatment of a variety of motor and pain syndromes. Thetechnique can provide the surgeon with valuable informa-tion regarding the localization of targets for placement ofdeep brain stimulation electrodes or radio frequencylesions3,9. In addition to being a useful clinical tool,microelectrode recording can be of benefit in contributingto our understanding of normal brain function, as well as thepathological states that underlie the patient’s disorder.

Microelectrode recordings are usually used for localizingtargets in the thalamus and the basal ganglia although theyhave also been occasionally used in cortex and rostralbrainstem. The patient is usually awake during the surgicalprocedure, which allows a unique opportunity for clinicaland scientific study. For instance, it is possible to have thepatient perform a variety of mental tasks and voluntarymovements while recording the cellular activity within aparticular brain region. Furthermore, one can stimulatethrough the microelectrode and induce stimulation-evokedmotor effects and sensations that the patient can report.

The techniques and equipment involved are generallysimilar to those used in electrophysiological studies inanimals and are summarized below. In addition, there aresome technical issues that relate primarily to humanrecording in the operating room and these will be addressedin more detail. The article is intended solely as a generaloverview and is not intended to explain the detailedmethods of extracellular recordings. Also, the surgicalprocedure and how the findings in various regions canprovide information on the location of the electrode isbeyond the scope of this article. For additional informationregarding microelectrode recordings in humans see refer-ences2,3,7.

8������ ��The microelectrodes used for human recordings aregenerally constructed from tungsten or platinum-iridium andcoated with glass, parylene-C or some other insulatingsubstance. These can be purchased from several sources(e.g., A-M Systems or Frederick Haer, Inc.). The length ofthe exposed microelectrode tip typically ranges between 15and 40 mm and has an initial impedance of 1 - 2 MW . Sincethese electrodes are too short to reach most targets in thehuman brain, it is necessary to extend them. To do this, theupper shank of the microelectrode is stripped of theinsulation and the electrode is back-loaded into a longstainless steel tube (e.g., 25 gauge, Small Parts, Inc.); toimprove electrical contact the upper part of the shank isbent somewhat. The stainless steel tube is insulated with acovering of polyimide Kapton tubing (e.g., 23 gauge, MicroML). Finally, epoxy resin glue is used to permanently sealand insulate the microelectrode, electrode shaft andpolyimide tubing at their junction. To check for possiblebreaks in the insulation, it is a good idea to pass electriccurrent through the microelectrode whose tip is immersed insaline, using a 3-5V DC battery. This results in the forma-tion of bubbles at the electrode tip. Accumulation ofbubbles at any place other than the tip of the electrodeindicates faulty insulation. To improve the quality of therecording, the electrode tip impedance can be reduced byplating it with platinum. Since platinum does not plate welldirectly onto tungsten it is best to first plate the tip with goldand then platinum4. This plating usually reduces theelectrode impedance below 0.2 MW . The electrode tip isthen immersed into a saline solution just below the junctionof the electrode and polyimide tubing and an impedancecheck is performed to ensure that there are no breaks in theinsulation. The tested microelectrodes are stored inappropriately labeled protective carrier tubes.

Long electrode assemblies suitable for human recordingsare now available from several companies (e.g., FrederickHaer, Inc., Radionics, ARS). Electrodes with a larger tipare sometimes also used for macrostimulation and/ormicroinjection of lidocaine1. These macroelectrodes areconstructed by insulating the 25-gauge stainless steel tubingto within 15 mm of the tip, which is cut at an angle andpolished.

Page 6: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

9

(������#���������

87��������������#�$�1�������� ��-�#� )�����#����

This description assumes the use of the Axon GuidelineSystem 3000 which provides an electrically isolatedintegrated system comprised of an amplifier, signal condi-tioner, microelectrode stimulator, computer with monitorfor display and a CD writer for data storage. Alternatively,one can assemble separate components: a high impedanceamplifier, headstage, and, if it is not part of the mainamplifier, a signal conditioner to filter and further amplifythe signals. Also required are an oscilloscope, audiomonitor, rate meter, simple spike discriminator, stimulator(capable of producing 1 – 10 s trains of 100-300 Hzmonophasic or biphasic pulses of 0.1 – 0.5 ms duration) andstimulus isolation unit capable of delivering 1 – 100 mAconstant current pulses. It should be noted, however, thatmost of the individual components that are available fromvarious manufacturers have been designed for animal useand approval for human use has not been obtained. Inaddition to this equipment, a stereotactic frame, amicrodrive adapted to fit on the stereotactic frame andassociated guide tubes are required. In our center, we use amanually controlled hydraulic microdrive.

)������#��������� ���

A stereotactic frame is fitted to the patient’s head followingan application of a local anaesthetic. The 3-dimensionalcoordinates of the patient’s anterior and posterior commis-sures (AC and PC) are determined relative to the frameusing CT or MRI scans. A standard stereotactic map of thebrain based on the Schaltenbrand and Wahren atlas 6 isconstructed using a computer program (e.g., SNS, Solve-It,Toronto) and is then stretched or shrunk to conform to theintercommissural distance of the patient. The microelec-trode trajectories can be drawn on these sagittal maps to aidin the determination of the initial target. Alternatively,estimated coordinates of the target can be determineddirectly from the MRI scan. A small access hole is drilledin the skull of the patient under local anesthesia and theunderlying dura matter is resected to allow the insertion of aguide tube. The electrode is then inserted into the guidetube and is stereotactically directed toward the chosen targetsite by means of a hydraulic microdrive so that its tip restsabout 10 mm above the target. Single and multi-unitrecordings are then obtained as the microelectrode is slowlydriven through the brain over a distance of 15 to 20 mm.Generally it useful to perform microstimulation (1 s, 300 Hztrain of 0.2 ms pulses of 1-100 mA) at selected intervals

along the length of the trajectory. Any stimulation-inducedmotor effects and/or sensory sensations reported by thepatient are carefully noted.

�������:������

The injection of a local anesthetic or muscimol at the levelof the target site can be useful in assessing the potentialeffects of a lesion in that area. The microinjection setupconsists of a steel tube of the same diameter as the micro-electrode that is connected to a 25 mL Hamilton syringe viafine polyethylene tubing (PE50). The tube is filled with a2% lidocaine solution without preservatives or muscimol[Sigma, St. Louis, MO; 1 mg/ml (8.8 mM) in sterile saline]and the injection apparatus is attached to the frame adapterin place of the microelectrode. The injection cannula isthen stereotactically guided into the brain and 1 to 5 mL istypically injected initially. Additional injections may bedelivered if no effect is seen within a few minutes1,5.

)����#������� ��#������#� �����;���������-

Sterility of Electrodes: Gas sterilization is the method ofchoice, but on occasion we have also sterilized electrodesby autoclaving or immersing in sterilizing fluid (e.g., 2%glutaraldehyde - Cidex, Metricide) followed by sterile waterrinse. For gas sterilization the electrodes are placed in aperforated box and sent for sterilization prior to the opera-tion and must be prepared beforehand. If more than oneprocedure is to be carried out within a day, then it isessential to have at least one additional box of microelec-trodes, as once a box is opened it is generally not permis-sible to use other electrodes from that box on anotherpatient.

Everything that comes in contact with the stereotactic frameand electrode carrier needs to be sterile as well. We gassterilize the leads that connect the electrode and ground theframe, and the guide tube. The electrode/microdrive carrierwhich is all metal is autoclaved immediately before theprocedure. Sterilizing the microdrive may be more difficult.In our case we use a hydraulic microdrive and the slavecylinder which attaches to the frame is sterilized in glutaral-dehyde and rinsed in sterile water immediately before theprocedure.

Electrical noise, connections and electrode quality:Because of its design, the Guideline System 3000 circum-vents many of the common problems encountered whenrecording extracellularly from single units. Even so, it isimportant to consider some common sources of problems.

Page 7: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

<

The various types of electrical equipment in the operatingroom act as sources of electromagnetic fields. Probably theworst source of electrical noise is the monopolar cauteryequipment frequently used in adjacent operating rooms.One way of eliminating the electrical noise is by means of ashielded room; however, this might not always be a plau-sible option due to the high cost involved, and we success-fully obtain recordings without a specially shielded room.Heart rate and other monitors sometimes also contribute toelectrical noise and may have to be turned off. Further-more, unlike regular incandescent lamps and surgical lights,fluorescent lights are frequent sources of interference andmay have to be shielded with a wire mesh or turned off.The Humbug filter (Quest, Inc.) can be used to effectivelyeliminate the 50 or 60 Hz noise arising from the powermains.

Problems may also result from faulty connections anddiminished electrode quality. High noise level is usually aresult of a loose electrode connection or increased electrodeimpedance. Line frequency noise can be caused by avariety of factors including poor ground connection, groundloop, and interference from the operating room equipmentor the fluorescent lights. Noise can also easily occur ifthere is intermittent contact of metal parts of the frame witheach other or with retractors. If the electrode insulation isbroken or its tip becomes bent, then it may be impossible toobtain neuronal recordings. Finally, a lack of stimulation-induced effects where expected (e.g., stimulation-evokedparesthesia in the core of Ventralis caudalis), usuallyindicates a potential short somewhere between the stimula-tor output and the electrode. Alternatively, if the electrodeimpedance is high there may not be enough voltage to drivesufficient current for stimulation.

Microphonics and speech: Occasionally, coupling betweenthe audio monitor loudspeaker and the microelectrode orstereotactic frame may produce feedback oscillations knownas microphonics. This phenomenon is most commonlycaused by loose junctions in the frame or oscillations of theelectrode within the guide tube. The problem is easily dealtwith by turning the volume down or, better still, by usingwireless head phones. Interference in the recordings mayalso be caused by patient’s speech during recording.Changing the electrode may also help resolve these prob-lems.

Stability: Even though the electrode is firmly fixed in placeby the stereotactic frame and, thus, there is no movement ofthe electrode relative to the skull, minute displacements ofthe brain can create stability problems. For example,cardiac and/or respiratory induced movements of the brain

can result in poor stability and fluctuating spike amplitude.Furthermore, large active or passive movements of thepatient’s limbs or coughing will usually induce brainmovement resulting in an unstable recording or even in theloss of the unit.

Brain shift: One final issue deserving mention and consider-ation is that of brain shift. It is possible that the brain mayshift between the time the CT or MRI scan was taken andthe time at which the brain is explored during surgery. Thiscan result from differences in patient position betweenacquisition of images and the operating room and/or the lossof CSF through the burr hole that is made in the skull.

��1�������

������#���.#2���A�-�����������������.#��C��������������������,'���#����=����������#2��������������D�������(�*������������'+������*�+'#E���'#�('*����*���#�����������#'� ����������������;'������*�)�'��#'� �����3 E�3�6�/����BB!�"������$��;�������#���.#2���A�-���C<����,�������#2����������F�+�#�����C�������'�����A����������#�(���+���#�+'*���������������� ��(�#� *����'���#������.�2���������*#�������'+���������*����.�'#�#�#��+��A�'���*��(�)�'��#'� �����3�E3! 63!����B���!�����$�����������,'���#����=�����C##��G�,�������#2������������.#��C����������#���.#2��A�-���������#�(���+����*�������6 '�����#����.�����*��**����+���A�'���*��()�'��#'� �������E�B�6" "���BB3���������**��?��������#<�����������*�##6�������*���'+6*������'� #����+����*�������#������*�>�*� ��*�?� ����� ��� E33"63/"���B/"�%�������**����������.���������#���.#2���A�-�����.#��C�������$�����������#2��������������$��������������+�������#��<������*�+��+����D�����#��(�+'#�+�*��������#�<���##����*����+��������*#��(�)�'��*� �������##�3�������*���@�������������=�������=�����*�#�(���������������(�����,'+���>��������+����'�� ������B//�/�����#2������������#���.#2���A�-������,'���#����=����������*��������������� ������*� ��������&'�#���)�'��#'� �����%H�IE�363����BBB�������#2�������������$��;�����F�+�#�����C��������2��A���,����+������������#���.#2���A�-�������*�������������&'�#���*���*$������(�#�������������� ��#��)�'��*� ��*���#������BE� %6��"���B�/�B�����#2������������.#��C�����,'���#����=�����������#���.#2���A�-�����'@������*�������*�+��+�� ���('������*���'��#'� �����5�E�����@��2��(�����������������;'������*)�'��#'� �������#��������*���@�� ������������#2���������<6,**����!6B B��BB��

���������������������������������#��=

)������#����"���������#��#� �������#��3����������-��$New York City, NY � June 10-12, 1999

�����1���#�����#� �%����������#���San Francisco, CA � June 14-15, 1999

�� ��#������1�8�����#�����������#��)��������Nice, France � June 19-24, 1999

%�������"���������#��%��������#� ����������Paris, France � June 24-25, 1999

������#��)�����$��1�)������#�����#� ���������#����������-��$Snowbird, UT � July 7-10, 1999

>����������#����#�����-������1�#�?�����5��,���#��Vancouver, Canada � July 26-28, 1999

���-������1��������-��#��)��-����Boston, MA � October 30 - November 4, 1999

����)�����$�1����������������5��Miami, FL � October 24-27, 1999

������#��)�����$�1�������������-$

Washington, DC � December 12-15, 1999

���(������#������

Page 8: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

!

��#����������1�@�-#�������2����)��-�� �3��-������

G. Chandy, Ph.D. and M.M. WuDepartments Molecular BiologyUniversity of California BerkeleyBerkeley, CA 94720

����� ������

This article briefly summarizes recently developed methodsthat measure pH in specific cellular organelles by targetingpH-sensitive fluorophores. Many important biochemicalreactions in organelles are pH-dependent. Bulk fluidmarkers in the extracellular medium are endocytosed andpass through sorting endosomes and late endosomes beforeentering lysosomes for degradation by pH-dependenthydrolases (pH < 5.0) (see Fig. 1A). The transferrin-transferrin receptor complex (Tf-TfR) initially enters thesame endosomal pathway as bulk fluid molecules, and theTf-TfR complex is segregated from the sorting endosomesinto tubular extensions that exclude the soluble markers.These recycling endosomes then carry the Tf-TfR complexback to the cell surface. Secretory and membrane proteinsare synthesized in the endoplasmic reticulum (ER; see Fig.1A) and post-translational modifications(including deglycosylation,glycosylation, sulfation and sialylation)are made as the proteins transit throughthe Golgi, trans-Golgi network (TGN)and secretory vesicles on the way to theplasma membrane. Each biochemicalstep exhibits some pH-dependence. Forinstance, the TGN is proposed to be thelocation where constitutively secretedproteins are segregated from regulatedproteins in a process that requiresproper acidification of the TGN lumen.Proteins in secretory granules are notfully processed until the secretorygranule attains a pH < 5.5. Our interestin organelle pH arises from a proposalthat some of the pathologies of thedisease cystic fibrosis result from analtered pHTGN, which in turn leads toan alteration in the normal biochemicalprocessing reactions in this organelle.We have begun investigating themechanisms by which the organelles areable to maintain pH. Some data

presented are from our recent paper 20 and other data arefrom manuscripts that are in preparation. Several reviewson the endocytic pathway are available 5, 11, 13.

����#�������1�����������-�@�-#�������2

There is a consensus that organelles in the secretory andendosome-lysosome pathways generate their various levelsof acidity in part by the activity of an electrogenic vacuolartype proton ATPase (V-ATPase). The membrane potentialgenerated by the V-ATPase will be affected by the magni-tudes of Cl-, K+ or Na+ conductances. The presence of theelectrogenic Na+/K+-ATPase in organelles could alsocontribute to a lumen-positive potential that would inhibitthe V-ATPase. Of course, any organelle membranepotentials will also affect H+ leak rates. Therefore,organelle pH will be affected by a complicated interactionamong the V-ATPase, other H+ pumps/transporters, H+

leaks, counterion conductances and membrane potential.The specific mechanisms that give rise to the 100-folddifferent acidities (pH 7 to pH 5) have not been determinedfor any organelle. The recent methods developed formeasuring pH in identified organelles will help in answeringsuch questions.

FITC- Anti-hIg F(ab)Ig

TMCellubrevin

Sortingendosomes

Recyclingendosomes

Ig

N CTM

Cellubrevin

Cb-Ig

TGN

Golgi

pH ~6.5

pHER 7.0-7.2

EE

pH ~6.0

SE

pH

RE

CbTf

pH ~6.0

CbTf

pH ~6.5

LE

Lysosomes

pH <5.0

~5.5pH 6.2 to 6.5

pH ~7.4A B

��-���������������+��������#���������(������������������@�#���������� ���**�#�<��������+��#'���,���??�����*������#�+�#J��?��#���� �����#�+�#J��?��*��������#�+�#J��?�������*� �����#�+�#J��)�������6��* ����<��2J�?�������*�#+������'*'+J��@����**'@��.�J��(������#(���������������+�������#���������(��@65 ����#��'������������ ��� ���;'**6*�� ��������@�<�#�('#�����(��+����������,"�����,!���+��#��(��'+���5 ���������#'*�� ��@65 ����#��'�����'#������������*'+��*�5 ��������� (�**�<� ���������#+�+@�����H��I���+����(��@����@65 �����@��.#'�*$�����*.� ���**#�@�������������(�;5��6���D' ��������6�5 ��;H�@I����@���#����������'@�����+��'+�����������'�*����*� ��(��@65 @��<������������#�+�#�����*�#+��+�+@�������#'*�#�����������#�������#'����(�����5 ���� ��������������**'*���+��'+��=������#����'�#������;5��6;H�@I����@���#�@����������������������.�*�<���@65 �����' ����������#�+�*�#�#��+�������������#������#������@65 ���#��#������������*� ����#�+�#������(*'���#�����# ��*���#�#�����+����*��(��+���#�*�������

(������#���������

Page 9: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

���(������#������

�#�-����-�����������������@�-#������

Previous methods of labeling organelles with ion-sensitivedyes relied on poorly selective accumulation of the dyes inthe targeted organelles. These methods provided muchuseful information, but the lack of selective targeting alwayspresented problems of interpretation of the generated data.The approach that we and others have used to target ion-sensitive fluorophores to selected organelles relies onexpressing chimera proteins that consist of an organelletargeting sequence fused to a lumenal portion that is eitherintrinsically fluorescent (i.e. green fluorescent protein, GFP)or is a “receptor” that will bind a fluorescently labeledmolecule. The enzymes and other organelle-specificproteins that reside at particular locations along theendosomal or secretory pathway have been used to targetfluorophores to different locations. A summary of somerecent applications is shown in the Table below. Thesemolecular targeting methods allow pH measurements to anyorganelle, not just those that are accessible by endocytosis.In addition, it should be possible to target different “recep-tors” to distinct compartments in the same cell and simulta-neously label each compartment.

��� 8

@�-#����� �#�-����-��������� �#�-��� ���������A��2�������

?� *���*� C�?�1���;6@6#'@'����(#� �����K��

C�?� �.��1+�+@�������+����(*'���#����@���L"�

��* ��*�*����#(���#� ,�#��#�.����������������@*�(*'�'��#����������L��

�.��1+�+@�������+����(*'���#����@���L�"�

b6���6 �*����#�*����#(���#� ���6�-M����@���1�-M6(*'���#���L�

+'������;�L�B��

*���*�# ���;6@6#'@'����(�#� �����L�

;5��6.�������K�/

�����D�������(�*�#�+�# ;*'���#����#'*(���������#'*(������+����3��/

��) ��)!� ?�������������@*��+'������;�L�"

��)!�����;'�� ��"%1�;5��6���6��"%����@���K"

�������.�#�*�#� 7���6"6,*'��� ?�������������@*��+'������;�L��"

��������������'*�#

?���#�+�# ��**'@��.� ?�������������@*��+'������;�L��"

5 1;5��6���65 �6;�@K�!��B

����#(������������ ����#(����6;5��K�!�3��B�" �""

L� ������**����� ���@*�J�K���������##6������������ ���

�#�-����-����$����-�@�-#������

As their name implies, recycling endosomes cycle between acytosolic location and the plasma membrane. Previousexperiments measuring endosomal pH have employedfluorescein-labeled Tf 3,6,19,20,22 which has the disadvantagethat fluorophore is lost as it is released from cells when itcycles to the plasma membrane. We used the moleculartargeting approach to allow long-term localization of a dyeto the recycling endosomes. The CH3 domain of human Igwas appended to the lumenal surface of the transmembrane-cytosolic domain of the resident protein Cb (Fig. 1B). Incells stably transfected with this construct, extracellularlyapplied FITC-labeled anti-Ig F(ab) fragment accumulated inperinuclear recycling endosomes (Fig. 2). The high affinityinteraction between the F(ab) and the hIg led to stablelabeling of recycling endosomes.

��-����/B�� #;����-������;C�-�����#�������;$�#���;� $����#?�B����@���'��2��#���������������������##���5 6���������**#�#��@*������#(������<���@65 �<������'@�������+��'+�������� �� �+ 1+*�;5��6 �������6�5 �;H�@I�(�� +��������������+��6�(�����**#�<����(�����(������"���!/�N���'@����������������'�*���#������(�*�@�*#��-�*����������#(��������**#�D�E��2��'�;5��6;H�@I�<������6*���*$�#�<������������+��6�(�D�EB

We measured lumenal pH by ratioing the emission(520-560 nm) images of cells obtained during excitation at490 nm (the pH-sensitive wavelength) followed by 440 nm(the relatively pH-insensitive wavelength) using AxonInstruments’ Imaging Workbench. Data were collected onlyfrom the fluorescence that was located in the perinuclearregion. After each experiment nigericin (K+/H+ exchanger)and monensin (Na+/H+ exchanger) were applied in asolution containing equal concentrations of [Na+] and [K+]to equilibrate pH of the organelle lumen with differentextracellular pH levels to obtain an “in vivo” calibrationcurve (Fig. 3). Average pH in Cb-containing endosomeswas ~6.0, and bafilomycin caused rapid alkalinization (Fig.4A). Membrane-permeant ouabain analogs had no effect onthe pH of Cb-labeled endosomes but caused Tf-containingendosomes to acidify from pH 6.5 to 6. Based on thesedifferences we proposed that recycling endosomes are a

Page 10: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

(������#���������

�(

heterogeneous population of vesicles with differingamounts of Cb, Tf and Na/K ATPase (Fig. 1) and also thatmembrane potential or the lumenal [Na+] or [K+] may beimportant in pH regulation of some, but not all, recycling

endosomes.

��-����.�������;��3����#��;�#�����B� ��'++�����(���*@�����#� (��+�!%���**#���**#�<����'*#���<���#�*'���#����.���'#�,�.�*'�#�������� �� �+�� ���������+����#���������B 61�� 6�+�(*'���#�����������(����������**�<�#*������.��#'#�,��(�������*@������#�*'�������������+��+'+�.�*'�#�(��+��.�'�*�(�#�<����#���������������<���*@�����������<�������������##��

��*��.�� ��� ���� (�����+��+'+�����**�<���+��#����+�� �����+���#��������C��<�#�O3�3�

Two similar methods that depend on endocytosis of proteinlabeled with pH-sensitive fluorophores have been used tomeasure pH in the TGN, Golgi and ER. The b-subunits ofverotoxin and shigatoxin bind surface glycolipids and areendocytosed and trafficked retrograde to the Golgi 7, 8.Modification of shigatoxin with an appended C-terminalKDEL sequence facilitated its accumulation in the ER 8.The TGN-resident proteins TGN38 and furin, which cyclewith the plasma membrane, were fused to the lumenallypresented CD25, which allowed endocytosis of labeled anti-CD25 antibody 2.

�#�-����-��3� ������@�-#������

Compared with the recycling endosomes and the TGN, theER and Golgi compartments are not as accessible from theplasma membrane. Therefore, approaches which dependupon endocytic uptake of extracellular pH indicators are notideal for labeling organelles of the biosynthetic pathway(e.g., ER, Golgi, secretory granules). Combining thegenetic targeting of a receptor with membrane-permeantpH-sensitive ligands has advantages. All organelles are

accessible and labeled ligands that do not dependupon endocytosis. This ensures that localization willnot vary with time, and labeling is not limited to aparticular fluorophore or to measuring pH, sincetheoretically any fluorophore can be modified tointeract with the receptor. In collaboration with RogerTsien, Stephen Adams and Juan Llopis we havegenetically targeted avidin to both the Golgi and ERand subsequently labeled these organelles with amembrane-permeant, pH-sensitive fluorescein-biotin.The biotin-based pH dye bound strongly to avidin(Kd = 10-15), giving strong, stable labeling even afterlong chases with dye-free media. We found that themildly acidic Golgi pH (~6.4) was maintained by abafilomycin-sensitive H+ V-ATPase; in contrast,pHER was neutral and not regulated by a H+ V-ATPase 21. Comparable results have been obtainedusing a Golgi-targeted, single chain anti-haptenantibody that was labeled with a membrane permeant,pH-sensitive hapten 4.

�#�-����-�%�����@�-#������

Since its first characterization a number of mutantGFPs have been developed 18. There are now cyan,blue, green and yellow fluorescent proteins all of

5 98764pH

0.00

1.00

0.75

0.50

0.25

Rel

ativ

e R

atio

��-����'B��811������1;#1����$���� #� ��2.A�2'�����-#�������2�� ���� ���**'@��.�������� � �����*� �����#�+�#���*�� �*2�*�$�#�<���� �������<���@�(*�+���������������(��+�����@65 �����##� ��-����***������<���;5��6;H�@I����B��?�,�#��������'���*������*2�*�$�#<������**#��������#������),!1),��#�*'���#���=����),��#��+�.��������?�����(�#��������.��#�(��+����(������++�2� �����,����� �#��������'������������#�*���������#(��+�����,������**�����##� �.��6C�?������*������<��(*'���#���6@�������B����* �,��,������**#�#�����.��� ��O3�������*2�*�$�#�<������������<��@�(*�+������������* ��*#��*2�*�$�#���������#������(),������<������#���+�.������#���*�����(�#���������#����.��� ���(���* �(��+�����**#<������.��@����*�@�*���<����#�*�*����#(���#���� ����;���+������?����#��*��@������#���#� %�+�'��#�

Bafilomycin

6.0

6.4

6.8

7.2

pHC

b

6.5

7.5

pHE

R

8.5NH4B

A

Baf NH4

6.0

7.0

8.0

pHG

olgi

C

Page 11: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

���(������#������

��

which have fluorescence intensities that vary with pH (pKa5.5-7). These have been used to measure pH in mitochon-dria, Golgi and the ER 9,10,14,15. Targeted fluorescentproteins have many advantages as organelle pH sensors:any organelle can be targeted; small dyes that need to behydrolyzed do not need to be added to cells; there is no“dye” leakage; and GFPs are resistant to photobleaching.Although the single wavelength intensity changes can becalibrated with respect to pH, artifacts can arise whenfluorophore concentration changes and pH does not.Miesenbock et. al 12 overcame this problem bymutagenizing and selecting for a GFP, which had a spec-trum that shifted with pH (pHlorin), thereby allowing themto ratio the intensities at two excitation wavelengths (theratio being proportional to pH).

We have undertaken an alternative method to develop aratioable GFP-based pH sensor for organelles. By fusingtwo mutant GFPs with different spectra and different pHdependencies, we created a chimeric protein that has a pH-dependent wavelength that can be ratioed to a relatively pH-independent excitation wavelength, yielding a ratio that isproportional to pH (pKa of ratio ~6.5). Our results with thisconstruct are similar to those obtained with the targetedavidin constructs1. Golgi pH rapidly alkalinizes when theV-ATPase is inhibited with bafilomycin. Subsequentrecovery from acidification due to an NH4 prepulseconfirms the rapid proton leak out of the Golgi (Fig. 4C).

�������� #�����

1) Choose a fluorophore that has pH sensitivity in theexpected range of the organelle to be measured.

2) It is desirable to have a ratioable indicator so that thefluorescence ratio is proportional only to pH, and artifactsthat give rise to pH-independent changes in intensity can beeliminated. This is best achieved with a fluorophore thathas a shift in its excitation or emission spectrum but it isalso possible to use fluorophores like FITC and the GFP-based protein we have developed that have a pseudo-isoesbestic points.

3) The most reproducible pH measurements are obtainedwhen each experiment is calibrated.

4) Protein chimeras are occasionally mistargeted, andtraditional cell biological approaches should be used todetermine their locations. A related but less obviousproblem is that pH measurements are usually made from thecell’s entire pool of fluorophore, and when a protein is

determined to be in a particular compartment either by lightor electron microscopy, it is usually because this is wherethe protein is most densely localized. If, however, theprotein is present at a lower density in an adjacent compart-ment that has relatively more surface area, the aggregatefluorescence may contribute significantly to the overallsignal. We minimize this problem by thresholding toeliminate low intensities and by collecting data from theregion of the cell that is brightest.

��1�������

����������������������,���������������?�����* �,����'+�����#�����������*�����'#���������������"�����+�'�����)���;'�'����=����P��'$�������>��(������A��������#���������������#+��(���(�������(����������6��* ����<��2�H��)I���5��#�'�+��#'��+���#��(�,�'#� ������.�*�(���)!������('���(��+�������**�#'�(���������������� ��� ������� �������/<.H�IE" ��6%����BB��!����P��'$������������6��@���������F'��;����������C����'2��#�������2����2��C����������,����-�*�<#2��A������#�����������������*�*�#��'+6����� ������������)�Q1,Q������� ��!����'+'*���#�����#�('������*��������*� �����#�+�#������������� ��� ������� �����/<.H�IE" !%6�!���BB������;����#��A���7��2+�����������������6+���������� ��� ��(�(*'���#�������@�#���*.� ��**#������������� ��� ������� ������/<'H�"IE/3 !63���BBB�%�����'��@�� ��A������(�*���;�������+@���������#����������������������<������������� � ��� ������ ������<H�IE%%"63!���BB%�3���A���#�����������'����C�=�������<#2��>��������<����?���?���#�+�����(��������������������((�2� E�@�(*�+�������#*�<����������������*$�����@����+�����#+��.�*.� �����������P#�������*$�����+��(����������� �����������������'H�"IE�"%�633���BB!�/���C+��A�,����� <�����������=**�+#����>���;'�'����=�������*#������;������#�����������+��+��#'��+�����(�����,��(�������* ���+*�����*.� ���**#�'#� ������ ��������#�����(�����.������������������������������� �������.'H3IE�!�/6BB���BB3�����C+��A�,���A������#��������'���>����������������� <���������������+�����������#���������)���.�#.��+��#'��+�����(�����,��(���������*�#+������'*'+������#�������'�� ��*�'+���*��#����������6H3IE"BB/6! "���BB��B���C����������;����#��A������F���7��2+����������������(*'���#������������#�������.�#.�������**'*���,������������ ����� ����������<'H!IE�%B�6B���BB��� ����*�#��A�������((�����A��������<�2������;��&'������������#������F������#'��+�����(����#�*���+���������*��������* �,���#� *��*.� ���**#�<��� �����(*'���#����������#��������6H�"IE3� !6����BB���������**+����5���?�������##�����+�*��'*���#���� ����������� ������������ ������������ �������/E%/%63"%���BB3��"�����#��@��2����������� �*#������������+����A�?���7#'�*$� �#������������#���������#+##���<���,6#��#�.�� �����(*'���#����������#����������.�'H33�BIE�B"6%��BB���!����'2���D�����������#�����)������(�*���;�����?�������##������� ��� ������ ����<<H!IE/%B6� !���BB/������������#������,���C��@�*������������(��=�����C�����������#�������=���4#���(����� ����(*'���#����������������#�+'����#���&'������.��(*'���#������+���#������� ����� ���������<.H%IE"/�"6B ���BB/��%�����@������>����'$��-��������#����7�������A���C�����;���=�� ����A��������A���>�����������������'����A�����,6���������(*'���#�������(���������*� �'#*������##�����&'���� �����(*'���#�����������+'����E���#�'�#�����*����������#��#������*��@*�����������**'*���,��#�+�������� ����� ������.<H"�IEB�B�6B ����BB���3�����2#�2��-���><��#��A���7��2+����������������+��#'��+�����(�����#6��* �,���*.� ��**#������� '*�����@��#������+�##�� ��#������������� ��� ������� �����/<(H� IE�B3/6/ ���BB%��/�����2#�2�-J�><��#�AJ�7��2+��������?.������� ��#����(���.������#6��* ����(���������#���(@��##������������� ��� ������� ������/<�H"3IE�%%�"6����BB3�������#������F������� �����(*'���#��������������������� ������� ����� �������9<E% B6�����BB���B����������C���������������9'����#��>������������C����������������������,�������**'@��.�6��� �����(*'���#������'���.��#������� �����������������*� �����#�+�#������������ ��� ������� ������/<.H!�IE�B3"%6!!���BB��" ���.���=��������=����'����C�=����'�$���,�A������(�*���;����������.� �*��=�������#���(��+�*��������#�+�#����*�#�#�+�#��@'������#���� ��(���� ��*�+�+@����������#������#�+�#�������#��������.��'�*���������'+���������������� �������.(H�IE�"�6!����BB%�"����='���������*�#��A������+#����������((�����A���������������?����������,���,����#�����F�����'��� �,�<����� ���**�6��� ������.��������'#���������������""���G����C���><��#��A�������#�+���)���7��2+�����������������+�##�� ��#��� '*�������#�+�*����(���������<##�!�!�(@��@*�#�#���������������� ���������H�IEBB6�� ��BB"�

Page 12: mdc.custhelp.commdc.custhelp.com/euf/assets/content/AxoBits26.pdf · image storage requirements · Collects emitted light at close to the theoretical limit · Offers outstanding sensitivity

���8�����@�� ��@ C��88��F��8�)Belgium 11-8201Denmark 8001-0306Finland 9800-10039France 05-90-1137Germany 0130-81-0458Israel 177-100-1504Italy 1678-74-022Netherlands 06-022-6850Norway 050-12-042Switzerland 046-05-7323Sweden 020-795-661United Kingdom 0800-89-1504

Worldwide sales are direct from the factory.

����Our customers in Japan may also order from our local distributor,Inter Medical.

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

Toll-free fax number from Japan for direct sales and technical support fromAxon Instruments is 00-66-33-800-102.

�@��� ����� ��@,F��)����8F�@8��@�����G

8�����#��@11����D���������#���EManaging DirectorAxon Instruments Europe GmbHHolzdamm 40D-20099 HamburgGermanyPhone: 40 2805 4979Fax: 40 2805 4999e-mail: [email protected]

� ���8��2��F)�@�8�)� 8�)8��@�����G

�������,�����;����DIPSI IndustrieVecteur-Sud70-86, Avenue de la RepubliqueF-92325 CHATILLON CEDEXFranceTelephone: 01 49 65 67 20Fax: 01 49 65 67 29e-mail: [email protected]

8F�@8

�8%�)�8��H@F���@,F��)�@���,,���@ 8%F8��@�@F����� ��%� �)�

If you would like your name added to the AxonInstruments mailing list or would like to register yourAxon products, please photocopy this form, fill it outin print letters and fax or mail it to Axon Instruments.Or you can register via e-mail to: [email protected]

Check as appropriate:

� Add my name to your mailing list

� Register the products listed below

%����#����1���#����

Name ______________________________________

Department _________________________________

Institute ____________________________________

Street ______________________________________

City, State _________________ Zip _____________

Country ____________________________________

Phone ( ___ ) ______________ Fax ____________

e-mail: _____________________________________

�� ������-����#����

Product name: _______________________________

Serial number: ______________________

Product name: _______________________________

Serial number: ______________________

Product name: _______________________________

Serial number: ______________________

����������������� ��������������������������

������������������������� � ����������������

��!�"#����"��$� ��%&�!

'''%� ��%&�!

��������������������

�/

������������������������)�����������)������������)�*������������*���+������,���-��������