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An impedance measuring and lowering device which does not enlarge micropipette tips
Phystology and Behavtor, Vol 11, pp 585-587 Brain Research Pubhcatmns Inc, 1973 Printed in the U S.A
BRIEF COMMUNICATION An Impedance Measuring and Lowering Device Which does not Enlarge Micropipette Tips' DENNIS L GLANZMAN AND SARAH A BEYDLER
Department o f Psychobtolog3,, UniversiO' o f California, Irvlne, Cahfornia 92664
( R e c e i v e d 13 F e b r u a r y 1973) GLANZMAN, D. L AND S A BEYDLER An tmpedance measuring and lowermg devtce whtch does not enlarge mwroplpette taps PHYSIOL BEHAV 11(4) 5 8 5 - 5 8 7 , 1973 -This report describes a sm~ple circuit which allows rapid measuring and lowering of the impedances of glass micropipettes, without enlarging the pipette tips Currents of 10-100 nA are used for testing pipette impedances, larger currents, up to 10 ~A, are used for impedance lowering The device imparts no damage to pipettes, and may be constructed for less than $10 Mlcroplpette
Impedance measuring
Impedance lowering
USE IMPEDANCE MEASURING
THIS clrcmt as designed for rapadly testing and l o w e n n g t h e ampedances of glass m a c r o p l p e t t e s w h i c h are filled w i t h a h q u l d e l e c t r o l y t e and used for antracellular electrophysaological r e c o r d i n g Its operatxon r e q m r e s b o t h a dual b e a m o s c d l o s c o p e ( T e k t r o n i x T y p e 5 0 2 ) a n d a V a n a c variable t r a n s f o r m e r . The carcult ~s h o u s e d m an a l u m i n u m m l n a b o x and is c o n n e c t e d b y shielded cables to the oscilloscope. S t a n d a r d 18 gauge lamp cord m a y be used for c o n n e c t a o n to t h e Varlac. A hole a b o u t 2 m m daa. is p u n c h e d t h r o u g h the t o p of t h e m m l b o x , and a small glass jar as installed t h r o u g h at so t h a t t h e b u l k o f t h e jar rests ms,de t h e b o x Thas allows f o r a good electrical shield s u r r o u n d i n g the testlng e l e c t r o l y t e . A c h l o n d e d salver ware is passed t h r o u g h a small hole drilled t h r o u g h the b o t t o m of the jar, and as c e m e n t e d an place w i t h an e p o x y glue. The jar is filled wath n o r m a l s a h n e (0.9%) NaC1), and t h e c h l o r i d e d silver wire serves as t h e r e t u r n clrcuat for i m p e d a n c e m e a s u r e m e n t s . A c h l o r l d e d salver ware is also i n s e r t e d i n t o t h e barrel of the p i p e t t e , to yaeld a n o n p o l a n z a b l e c o n n e c t i o n to t h e rest of the clrcmtry Fagure 1 as a s c h e m a t i c r e p r e s e n t a t m n of t h e device as c u r r e n t l y used in o u r l a b o r a t o r y . T h e oscilloscope provxdes an o u t p u t c a h b r a t a o n sagnal of 1 KHz s q u a r e waves, stepwlse a d j u s t a b l e f r o m 5 m V to 50 V Thas f r e q u e n c y a n d wavef o r m as ~deal for t e s t i n g e l e c t r o d e s because at possesses a wide range of f r e q u e n c y c o m p o n e n t s samflar t o those e n c o u n t e r e d an e l e c t r o p h y s m l o g l c a l u m t r e c o r d i n g
Glass m l c r o p a p e t t e s are d r a w n f r o m c a p d l a r y t u b i n g w h i c h has b e e n stuffed with f r o m f o u r to six s t r a n d s of twelve m i c r o n d i a m e t e r fiberglass [3] N o r m a l l y , t h e e n d s of t h e fiberglass e x t e n d a p p r o x i m a t e l y 1 cm b e y o n d t h e l e n g t h of the capillary to facdatate h a n d h n g The p i p e t t e s are e x a m i n e d u n d e r light m i c r o s c o p e to d e t e r m i n e acceptabahty of t h e s h a p e a n d tip size T h e y are t h e n filled wath e l e c t r o l y t e s o l u t i o n [ 1 ] , e.g 3 M KC1, b y i n j e c t i n g t h e s o l u t i o n i n t o t h e p~pette barrel w i t h a 30 gauge h y p o d e r m i c needle [ 3 ] , and are r e e x a m i n e d b y m i c r o s c o p e for f r a c t u r e of the tip or o t h e r d a m a g e The c h l o r l d e d salver wire ~s i n s e r t e d i n t o the s h a n k o f t h e p i p e t t e and the tip ~s b a r e l y i m m e r s e d i n t o t h e s a h n e b a t h o f the testing a p p a r a t u s . S w i t c h SW1 xs o p e n e d and s w i t c h SW2 is set to t h e testing p o s i t i o n ( b o t h s w i t c h e s as illustrated m F~g. 1). T h e square wave c a h b r a t l o n o u t p u t of t h e oscilloscope as set to 5 0 0 m V , a n d p o t e n t a o m e t e r R1 as adjusted untal a reading of 10 m V as o b t a i n e d o n C h a n n e l 1 o f the oscilloscope. W h e n these a d j u s t m e n t s have b e e n m a d e , t h e a m p e d a n c e o f the p i p e t t e b e i n g tested is calculated as follows the c u r r e n t flowing t h r o u g h t h e p i p e t t e cxrcmt as k n o w n - a 10 m V sagnal m e a s u r e d across t h e oscilloscope i n p u t a m p e d a n c e of 1 M ~ i n d i c a t e s a c u r r e n t of 10 mV/1 Ms2 or 10 nA. The a m o u n t of c u r r e n t flowing t h r o u g h each e l e m e n t o f t h e r e c o r d i n g c l r c m t , consastlng o f the oscilloscope i n p u t resastor, p i p e t t e , Ag/AgC1 r e f e r e n c e e l e c t r o d e , resistor R2,
We wish to thank Dr Richard k Thompson, Umverslty of Cahforma, Irvme, for encouragement and tire use of laboratory facdltles Supported m part by Research Scientist Award MH06650 (RFT) and Research Grant MH19314 (RFT) from the National Institute of Mental Health, Grant NS07661 from the National Institutes of Health (RFT), and Biological Sciences Research training Grant MH11095 from the National Institute of Mental Health 585
586
G L A N Z M A N AND B E Y D L E R
cables to oscdloscope input a rnphflers
Shielded
To Vorlac variable Transformer Banana plugs (2) via standard 18 gauge ~ w,re to
Ch 2
Ch I
To square wave cal~brahon pulse
output
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0
SbleldedVarla~.,0~
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0
T
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"-', SW 1
L sK
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Normal Saline % Solutmn
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,~..
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Circuit ground To Ag/AgCI
reference
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Aluminum
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# biG. 1 Schematic diagram ol an Impedance measuring and loitering device Connections to (hannel 1, Channel 2, and the square wave calibration pulse of the oscilloscope are made with shmlded cable, connechons to the Varlac transformer are made with standard 18 gauge lamp cord Capamtor C1 prevents rather electrode from becoming polarized during testing and etching, but may be om]tted ff good Ag/AgC1 connections exist on both electrodes Resistors are all one-hall W, capacitor is t0 V nonelectroly tic and part o f p o t e n t l o m e t e r R l , l s t h e s a m e Slncethecarcmt current ts k n o w n , it as possible t o calculate the mrcult ~mpedance by measuring the voltage required to pass the (10 n A ) c u r r e n t That voltage is m e a s u r e d indirectly on Channel 2 o f the oscilloscope by m e a s u n n g the voltage b e t w e e n the arm of p o t e n t m m e t e r R1 and g r o u n d , this as t a k e n to be equal to the voltage d r o p p e d across the reference e l e c t r o d e , p i p e t t e and oscilloscope i n p u t resistor The voltage d r o p p e d across resistor R2 is substantmlly less t h a n 1% o f that d r o p p e d across the rest o f the circuit, and is neglected. (Resistor R2 prevents accxdental direct short clrcmtlng o f the oscilloscope square wave o u t p u t ) Thus the voltage measured on Channel 2 o f the oscilloscope as directly related to the clrcmt I m p e d a n c e , where i m p e d a n c e equals the voltage measured on Channel 2 diwded by 10 -~ A For e x a m p l e , ff the mrcmt as adjusted as described, and a voltage o f 400 m V is measured at Channel 2 o f the oscilloscope, the total i m p e d a n c e o f the mrcult is calculated as 400 m V / 1 0 -" A, or 40 Mxz Of this total m l p e d a n c e , 1 M~2 is due to the oscilloscope i n p u t resistor and should be subtracted to ymld the correct p i p e t t e i m p e d a n c e m this case, 39 Mfz For p i p e t t e s with i m p e d a n c e s greater than 20 Mgz, the error t e r m is maximally 5:?, and decreases with increasing p i p e t t e I m p e d a n c e (error equals the oscilloscope input i m p e d a n c e divided by total circmt i m p e d a n c e ) If the error term as small, or if a high degree o f accuracy is n o t r e q m r e d , the error may be neglected and the total mrcmt ~mpedance a c c e p t e d as being equal to the i m p e d a n c e of the pipette.
For p i p e t t e s o f less t h a n a b o u t 20 ]M~z i m p e d a n c e , the accuracy o f the m e a s u r e m e n t can be increased by closing switch SW1 This shunts the oscilloscope i n p u t o f Channel 1 with a 110 k ~ resistor, whach lowers the eflectave i n p u t ampedance to 100 k~2 The result is to decrease the Impedance contrabuted by the oscalloscope i n p u t resistor With swatch SWl closed the m e a s u r e m e n t is altered as follows p o t e n t l o m e t e r R1 is adlusted as b e f o r e , but the total carcmt is now 10 m V / 1 0 0 kgz or 100 n A The I m p e d a n c e o f the entire carcuat is t h e n calculated as follows ampedance equals the voltage measured at Channel 2 divaded by 10 -7 A For e x a m p l e , If a voltage reading of 300 mV is made at Channel 2, a carcuit i m p e d a n c e o f 300 m V / 1 0 -7 A or 3 M~-z would be indicated The effective oscilloscope i m p e d a n c e is now 100 k~2, and should be s u b t r a c t e d from the circuit ampedance to yield a correct ampedance o f the p i p e t t e o f 2 0 M~2 F o r p l p e t t e s w i t h ~ m p e d a n c e s o v e r 5 \I~2, the error term is le~s than 5~; and decreases w i t h l n c r e a s m g p i p e t t e i m p e d a n c e (see above) The accuracy o f this t e s t m g d e v t c e was c h e c k e d by s u b s t i t u t i n g several 1% precasaon resastors for the papette, and the results o f thas m e a s u r e m e n t s h o w e d that at the lower ampedance range {SW1 closed), accuracms of + 3(;f were generally o b t a i n e d for resistors o f f r o m 1 to 20 Mgz At the highm i m p e d a n c e range (SWI o p e n e d ) , accuracies of , 5:'; were o b t a i n e d for resistors from 10 100 Mrz. The major source ot error e n c o u n t e r e d in using this device appears to be the a d j u s t m e n t of p o t e n t a o m e t e r RI to
IMPEDANCE MEASURING AND LOWERING DEVICE yield exactly 10 m V at C h a n n e l 1 o f t h e oscilloscope T h e circuit s h o u l d be calibrated initially w i t h p r e c i s i o n resistors, to allow for c o m p o n e n t t o l e r a n c e s and oscilloscope i n p u t ~mpedances USE-IMPEDANCE LOWERING By passing small c u r r e n t s (1 to 10 u A ) t h r o u g h a p i p e t t e , it is possible to r e d u c e greatly its i m p e d a n c e (or increase its c u r r e n t carrying c a p a b i l i t y ) ([ 2 ], p 6 3 ) w i t h o u t n o t i c e a b l y increasing the tip d m m e t e r F o r this p u r p o s e , t h e o u t p u t of a Varlac variable t r a n s f o r m e r is c o n n e c t e d via s w i t c h SW2 b e t w e e n the p i p e t t e e l e c t r o d e and t h e saline b a t h . A f t e r m a k i n g an m m a l i m p e d a n c e test, t h e p i p e t t e can be e t c h e d electrically b y passing an a.c c u r r e n t t h r o u g h it as follows, amtially, t h e Variac s h o u l d b e a d j u s t e d t o a b o u t 40 V S w i t c h SW2 is t h e n t h r o w n to c o n n e c t the Varlac o u t p u t to t h e p i p e t t e and testing s o l u t i o n for a few seconds, and t h e n r e t u r n e d to the n o r m a l or t e s t i n g p o s i t i o n . P o t e n t l o m e t e r R1 is t h e n r e a d j u s t e d to yield a reading of 10 m V at C h a n n e l 1 of t h e oscilloscope, and t h e new, lowered p i p e t t e i m p e d a n c e IS m e a s u r e d as b e f o r e This process
587 m a y be r e p e a t e d several t i m e s In cases o f e x t r e m e l y high p i p e t t e i m p e d a n c e s , full line voltage has b e e n applied directly across the p i p e t t e s u n d e r g o i n g e t c h i n g w i t h o u t a p p a r e n t l y h a r m i n g the tips. M a x i m u m i m p e d a n c e reduct i o n s are usually of t h e o r d e r of o n e - h a l f to t h r e e - f o u r t h s t h e original i m p e d a n c e , 1.e, an initially 60 M a p i p e t t e can be e t c h e d to b e t w e e n 15 and 30 M a P i p e t t e s s h o u l d always be r e e x a m i n e d u n d e r the m i c r o s c o p e to c h e c k for m e c h a n i c a l damage, b u t t h e r e is n o r m a l l y n o n o t i c e a b l e change in t h e tip d i a m e t e r s or shapes We have had successful results using this t e c h m q u e w i t h p o t a s s m m chloride, p o t a s s i u m citrate and p o t a s s i u m a c e t a t e e l e c t r o l y t e s C A U T I O N W h e n SW2 is in the e t c h p o s i t i o n , o n e o f the t w o e l e c t r o d e s will be c o n n e c t e d directly to t h e p o w e r line, since t h e Varlac o u t p u t is n o t isolated. T h e r e f o r e , e x t r e m e care m u s t be t a k e n in always r e t u r n i n g s w i t c h SW2 to t h e test p o s i t i o n w h e n installing or r e m o v i n g p i p e t t e s f r o m t h e c h l o r l d e d silver wire Failure to d o so m a y result in dangerous electrical s h o c k to t h e o p e r a t o r .
REFERENCES 1
2
Coombs, J S, J C Eccles and P Fatt The specific lomc conductances and the ionic movements across the motoneuronal membrane that produce the Inhibitory post-synaptlc potential J Physiol. 130: 326-373, 1955 Frank, K and M C Becker Mlcroelectrodes for recording and stimulation In Phystcal Techntques m Btologtcal Research, Volume 5, Electrophystologlcal Methods, Part A, edited by Wllham L Nastuk New York Academic Press, 1964, pp. 23 84
3. Tasakl, K , Y. Tsukahara, S lto, M. J. Wayner and W. Y. Yu A simple, direct and rapid method for filling mlcroelectrodes. Physlol Behal' 3 : 1 0 0 9 1010, 1968
BRIEF COMMUNICATION An Impedance Measuring and Lowering Device Which does not Enlarge Micropipette Tips' DENNIS L GLANZMAN AND SARAH A BEYDLER
Department o f Psychobtolog3,, UniversiO' o f California, Irvlne, Cahfornia 92664
( R e c e i v e d 13 F e b r u a r y 1973) GLANZMAN, D. L AND S A BEYDLER An tmpedance measuring and lowermg devtce whtch does not enlarge mwroplpette taps PHYSIOL BEHAV 11(4) 5 8 5 - 5 8 7 , 1973 -This report describes a sm~ple circuit which allows rapid measuring and lowering of the impedances of glass micropipettes, without enlarging the pipette tips Currents of 10-100 nA are used for testing pipette impedances, larger currents, up to 10 ~A, are used for impedance lowering The device imparts no damage to pipettes, and may be constructed for less than $10 Mlcroplpette
Impedance measuring
Impedance lowering
USE IMPEDANCE MEASURING
THIS clrcmt as designed for rapadly testing and l o w e n n g t h e ampedances of glass m a c r o p l p e t t e s w h i c h are filled w i t h a h q u l d e l e c t r o l y t e and used for antracellular electrophysaological r e c o r d i n g Its operatxon r e q m r e s b o t h a dual b e a m o s c d l o s c o p e ( T e k t r o n i x T y p e 5 0 2 ) a n d a V a n a c variable t r a n s f o r m e r . The carcult ~s h o u s e d m an a l u m i n u m m l n a b o x and is c o n n e c t e d b y shielded cables to the oscilloscope. S t a n d a r d 18 gauge lamp cord m a y be used for c o n n e c t a o n to t h e Varlac. A hole a b o u t 2 m m daa. is p u n c h e d t h r o u g h the t o p of t h e m m l b o x , and a small glass jar as installed t h r o u g h at so t h a t t h e b u l k o f t h e jar rests ms,de t h e b o x Thas allows f o r a good electrical shield s u r r o u n d i n g the testlng e l e c t r o l y t e . A c h l o n d e d salver ware is passed t h r o u g h a small hole drilled t h r o u g h the b o t t o m of the jar, and as c e m e n t e d an place w i t h an e p o x y glue. The jar is filled wath n o r m a l s a h n e (0.9%) NaC1), and t h e c h l o r i d e d silver wire serves as t h e r e t u r n clrcuat for i m p e d a n c e m e a s u r e m e n t s . A c h l o r l d e d salver ware is also i n s e r t e d i n t o t h e barrel of the p i p e t t e , to yaeld a n o n p o l a n z a b l e c o n n e c t i o n to t h e rest of the clrcmtry Fagure 1 as a s c h e m a t i c r e p r e s e n t a t m n of t h e device as c u r r e n t l y used in o u r l a b o r a t o r y . T h e oscilloscope provxdes an o u t p u t c a h b r a t a o n sagnal of 1 KHz s q u a r e waves, stepwlse a d j u s t a b l e f r o m 5 m V to 50 V Thas f r e q u e n c y a n d wavef o r m as ~deal for t e s t i n g e l e c t r o d e s because at possesses a wide range of f r e q u e n c y c o m p o n e n t s samflar t o those e n c o u n t e r e d an e l e c t r o p h y s m l o g l c a l u m t r e c o r d i n g
Glass m l c r o p a p e t t e s are d r a w n f r o m c a p d l a r y t u b i n g w h i c h has b e e n stuffed with f r o m f o u r to six s t r a n d s of twelve m i c r o n d i a m e t e r fiberglass [3] N o r m a l l y , t h e e n d s of t h e fiberglass e x t e n d a p p r o x i m a t e l y 1 cm b e y o n d t h e l e n g t h of the capillary to facdatate h a n d h n g The p i p e t t e s are e x a m i n e d u n d e r light m i c r o s c o p e to d e t e r m i n e acceptabahty of t h e s h a p e a n d tip size T h e y are t h e n filled wath e l e c t r o l y t e s o l u t i o n [ 1 ] , e.g 3 M KC1, b y i n j e c t i n g t h e s o l u t i o n i n t o t h e p~pette barrel w i t h a 30 gauge h y p o d e r m i c needle [ 3 ] , and are r e e x a m i n e d b y m i c r o s c o p e for f r a c t u r e of the tip or o t h e r d a m a g e The c h l o r l d e d salver wire ~s i n s e r t e d i n t o the s h a n k o f t h e p i p e t t e and the tip ~s b a r e l y i m m e r s e d i n t o t h e s a h n e b a t h o f the testing a p p a r a t u s . S w i t c h SW1 xs o p e n e d and s w i t c h SW2 is set to t h e testing p o s i t i o n ( b o t h s w i t c h e s as illustrated m F~g. 1). T h e square wave c a h b r a t l o n o u t p u t of t h e oscilloscope as set to 5 0 0 m V , a n d p o t e n t a o m e t e r R1 as adjusted untal a reading of 10 m V as o b t a i n e d o n C h a n n e l 1 o f the oscilloscope. W h e n these a d j u s t m e n t s have b e e n m a d e , t h e a m p e d a n c e o f the p i p e t t e b e i n g tested is calculated as follows the c u r r e n t flowing t h r o u g h t h e p i p e t t e cxrcmt as k n o w n - a 10 m V sagnal m e a s u r e d across t h e oscilloscope i n p u t a m p e d a n c e of 1 M ~ i n d i c a t e s a c u r r e n t of 10 mV/1 Ms2 or 10 nA. The a m o u n t of c u r r e n t flowing t h r o u g h each e l e m e n t o f t h e r e c o r d i n g c l r c m t , consastlng o f the oscilloscope i n p u t resastor, p i p e t t e , Ag/AgC1 r e f e r e n c e e l e c t r o d e , resistor R2,
We wish to thank Dr Richard k Thompson, Umverslty of Cahforma, Irvme, for encouragement and tire use of laboratory facdltles Supported m part by Research Scientist Award MH06650 (RFT) and Research Grant MH19314 (RFT) from the National Institute of Mental Health, Grant NS07661 from the National Institutes of Health (RFT), and Biological Sciences Research training Grant MH11095 from the National Institute of Mental Health 585
586
G L A N Z M A N AND B E Y D L E R
cables to oscdloscope input a rnphflers
Shielded
To Vorlac variable Transformer Banana plugs (2) via standard 18 gauge ~ w,re to
Ch 2
Ch I
To square wave cal~brahon pulse
output
of
osmlloscope BNC connectors(:3)
0
SbleldedVarla~.,0~
i-
0
T
(~
v~a shielded cable to osc=lloscope
"-', SW 1
L sK
co ,e,o
pipette conductar
~
e
IIOK NOTE Chassis ground IS
Normal Saline % Solutmn
~ ~ ,",, , , .
,~..
-" .
Circuit ground To Ag/AgCI
reference
electrode
Aluminum
mmlbox
# biG. 1 Schematic diagram ol an Impedance measuring and loitering device Connections to (hannel 1, Channel 2, and the square wave calibration pulse of the oscilloscope are made with shmlded cable, connechons to the Varlac transformer are made with standard 18 gauge lamp cord Capamtor C1 prevents rather electrode from becoming polarized during testing and etching, but may be om]tted ff good Ag/AgC1 connections exist on both electrodes Resistors are all one-hall W, capacitor is t0 V nonelectroly tic and part o f p o t e n t l o m e t e r R l , l s t h e s a m e Slncethecarcmt current ts k n o w n , it as possible t o calculate the mrcult ~mpedance by measuring the voltage required to pass the (10 n A ) c u r r e n t That voltage is m e a s u r e d indirectly on Channel 2 o f the oscilloscope by m e a s u n n g the voltage b e t w e e n the arm of p o t e n t m m e t e r R1 and g r o u n d , this as t a k e n to be equal to the voltage d r o p p e d across the reference e l e c t r o d e , p i p e t t e and oscilloscope i n p u t resistor The voltage d r o p p e d across resistor R2 is substantmlly less t h a n 1% o f that d r o p p e d across the rest o f the circuit, and is neglected. (Resistor R2 prevents accxdental direct short clrcmtlng o f the oscilloscope square wave o u t p u t ) Thus the voltage measured on Channel 2 o f the oscilloscope as directly related to the clrcmt I m p e d a n c e , where i m p e d a n c e equals the voltage measured on Channel 2 diwded by 10 -~ A For e x a m p l e , ff the mrcmt as adjusted as described, and a voltage o f 400 m V is measured at Channel 2 o f the oscilloscope, the total i m p e d a n c e o f the mrcult is calculated as 400 m V / 1 0 -" A, or 40 Mxz Of this total m l p e d a n c e , 1 M~2 is due to the oscilloscope i n p u t resistor and should be subtracted to ymld the correct p i p e t t e i m p e d a n c e m this case, 39 Mfz For p i p e t t e s with i m p e d a n c e s greater than 20 Mgz, the error t e r m is maximally 5:?, and decreases with increasing p i p e t t e I m p e d a n c e (error equals the oscilloscope input i m p e d a n c e divided by total circmt i m p e d a n c e ) If the error term as small, or if a high degree o f accuracy is n o t r e q m r e d , the error may be neglected and the total mrcmt ~mpedance a c c e p t e d as being equal to the i m p e d a n c e of the pipette.
For p i p e t t e s o f less t h a n a b o u t 20 ]M~z i m p e d a n c e , the accuracy o f the m e a s u r e m e n t can be increased by closing switch SW1 This shunts the oscilloscope i n p u t o f Channel 1 with a 110 k ~ resistor, whach lowers the eflectave i n p u t ampedance to 100 k~2 The result is to decrease the Impedance contrabuted by the oscalloscope i n p u t resistor With swatch SWl closed the m e a s u r e m e n t is altered as follows p o t e n t l o m e t e r R1 is adlusted as b e f o r e , but the total carcmt is now 10 m V / 1 0 0 kgz or 100 n A The I m p e d a n c e o f the entire carcuat is t h e n calculated as follows ampedance equals the voltage measured at Channel 2 divaded by 10 -7 A For e x a m p l e , If a voltage reading of 300 mV is made at Channel 2, a carcuit i m p e d a n c e o f 300 m V / 1 0 -7 A or 3 M~-z would be indicated The effective oscilloscope i m p e d a n c e is now 100 k~2, and should be s u b t r a c t e d from the circuit ampedance to yield a correct ampedance o f the p i p e t t e o f 2 0 M~2 F o r p l p e t t e s w i t h ~ m p e d a n c e s o v e r 5 \I~2, the error term is le~s than 5~; and decreases w i t h l n c r e a s m g p i p e t t e i m p e d a n c e (see above) The accuracy o f this t e s t m g d e v t c e was c h e c k e d by s u b s t i t u t i n g several 1% precasaon resastors for the papette, and the results o f thas m e a s u r e m e n t s h o w e d that at the lower ampedance range {SW1 closed), accuracms of + 3(;f were generally o b t a i n e d for resistors o f f r o m 1 to 20 Mgz At the highm i m p e d a n c e range (SWI o p e n e d ) , accuracies of , 5:'; were o b t a i n e d for resistors from 10 100 Mrz. The major source ot error e n c o u n t e r e d in using this device appears to be the a d j u s t m e n t of p o t e n t a o m e t e r RI to
IMPEDANCE MEASURING AND LOWERING DEVICE yield exactly 10 m V at C h a n n e l 1 o f t h e oscilloscope T h e circuit s h o u l d be calibrated initially w i t h p r e c i s i o n resistors, to allow for c o m p o n e n t t o l e r a n c e s and oscilloscope i n p u t ~mpedances USE-IMPEDANCE LOWERING By passing small c u r r e n t s (1 to 10 u A ) t h r o u g h a p i p e t t e , it is possible to r e d u c e greatly its i m p e d a n c e (or increase its c u r r e n t carrying c a p a b i l i t y ) ([ 2 ], p 6 3 ) w i t h o u t n o t i c e a b l y increasing the tip d m m e t e r F o r this p u r p o s e , t h e o u t p u t of a Varlac variable t r a n s f o r m e r is c o n n e c t e d via s w i t c h SW2 b e t w e e n the p i p e t t e e l e c t r o d e and t h e saline b a t h . A f t e r m a k i n g an m m a l i m p e d a n c e test, t h e p i p e t t e can be e t c h e d electrically b y passing an a.c c u r r e n t t h r o u g h it as follows, amtially, t h e Variac s h o u l d b e a d j u s t e d t o a b o u t 40 V S w i t c h SW2 is t h e n t h r o w n to c o n n e c t the Varlac o u t p u t to t h e p i p e t t e and testing s o l u t i o n for a few seconds, and t h e n r e t u r n e d to the n o r m a l or t e s t i n g p o s i t i o n . P o t e n t l o m e t e r R1 is t h e n r e a d j u s t e d to yield a reading of 10 m V at C h a n n e l 1 of t h e oscilloscope, and t h e new, lowered p i p e t t e i m p e d a n c e IS m e a s u r e d as b e f o r e This process
587 m a y be r e p e a t e d several t i m e s In cases o f e x t r e m e l y high p i p e t t e i m p e d a n c e s , full line voltage has b e e n applied directly across the p i p e t t e s u n d e r g o i n g e t c h i n g w i t h o u t a p p a r e n t l y h a r m i n g the tips. M a x i m u m i m p e d a n c e reduct i o n s are usually of t h e o r d e r of o n e - h a l f to t h r e e - f o u r t h s t h e original i m p e d a n c e , 1.e, an initially 60 M a p i p e t t e can be e t c h e d to b e t w e e n 15 and 30 M a P i p e t t e s s h o u l d always be r e e x a m i n e d u n d e r the m i c r o s c o p e to c h e c k for m e c h a n i c a l damage, b u t t h e r e is n o r m a l l y n o n o t i c e a b l e change in t h e tip d i a m e t e r s or shapes We have had successful results using this t e c h m q u e w i t h p o t a s s m m chloride, p o t a s s i u m citrate and p o t a s s i u m a c e t a t e e l e c t r o l y t e s C A U T I O N W h e n SW2 is in the e t c h p o s i t i o n , o n e o f the t w o e l e c t r o d e s will be c o n n e c t e d directly to t h e p o w e r line, since t h e Varlac o u t p u t is n o t isolated. T h e r e f o r e , e x t r e m e care m u s t be t a k e n in always r e t u r n i n g s w i t c h SW2 to t h e test p o s i t i o n w h e n installing or r e m o v i n g p i p e t t e s f r o m t h e c h l o r l d e d silver wire Failure to d o so m a y result in dangerous electrical s h o c k to t h e o p e r a t o r .
REFERENCES 1
2
Coombs, J S, J C Eccles and P Fatt The specific lomc conductances and the ionic movements across the motoneuronal membrane that produce the Inhibitory post-synaptlc potential J Physiol. 130: 326-373, 1955 Frank, K and M C Becker Mlcroelectrodes for recording and stimulation In Phystcal Techntques m Btologtcal Research, Volume 5, Electrophystologlcal Methods, Part A, edited by Wllham L Nastuk New York Academic Press, 1964, pp. 23 84
3. Tasakl, K , Y. Tsukahara, S lto, M. J. Wayner and W. Y. Yu A simple, direct and rapid method for filling mlcroelectrodes. Physlol Behal' 3 : 1 0 0 9 1010, 1968