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Thermoelectric freezing stage for serial brain sectioning
Electroencephalography and Chmcal Neurophysiology Elsevier P u b h s h l n g C o m p a n y , A m s t e r d a m Printed in T h e N e t h e r l a n d s
TECHNICAL NOTES THERMOELECTRIC FREEZING STAGE FOR SERIAL BRAIN SECTIONING1 F
C . CHESHIRE AND E. EIDELBERG 2
Dtt, t~lon o f Neurobtology, Barrow Neurologwal Instttute o f St. Joseph's Hospttal, Phoenix, Artz ( U S A ) (Accepted for p u b h c a t l o n J a n u a r y 5, 1966)
Peltier U
n
i
t
~
Board FLg. 1 D m g r a m of the complete umt, minus the power supply. T h e Peltler unit is m o u n t e d directly over the heat sink (copper) and the m o u n t i n g post fits into the m i c r o t o m e chuck. Thick-walled rubber hose connects the heat sink to a source of cold water a n d d r a m P r e p a r a t i o n o f frozen sections of brains for checking electrode placements a n d for special stain studies is a n i m p o r t a n t part of neurophysiologlcal e x p e r i m e n t a t i o n It IS also a time c o n s u m i n g routine, which is n o t m a d e easier by the use of eRher dry Ice In c h u n k s or liquefied c a r b o n dioxide as the freezing m e d m m , since it is h a r d to keep the tissue c o n s t a n t l y at the proper t e m p e r a t u r e for cutting. This p r o b l e m has been solved in our laboratories by the use o f a thermoelectric (Peltier) unit as the freezing plate for the b r a i n specimens. T h e t e m p e r a t u r e of the tissue c a n be set at any level desired a n d it will stay at that level for as long as needed. It is r e m a r k a b l y fast, freezing a 1 cm thick block o f b r a i n tissue m a b o u t 20 m i n T h e thermoelectric u m t e m p l o y e d (Fig. l) is available 1 S u p p o r t e d by U S P H S G r a n t B-3496 a n d G e n e r a l R e s e a r c h S u p p o r t G r a n t FR-5575 ( U S P H S ) 2 U S P H S Career D e v e l o p m e n t A w a r d K3-NB-15,437
commercially f r o m the Ferroxcube C o m p a n y (Model 20/20) It was modified by m d h n g 1/16" deep, 1/8" wide grooves on its smaller (top) side m a checkerboard p a t t e r n to increase the contact surface with the tissue for better grip. T h e b o t t o m side of the unit was m o u n t e d o n a heat sink by screws, with silicone grease in between T h e function o f the heat sink is to reduce the temperature of the " h o t " side o f the t h e r m a l j u n c t i o n It was m a d e by milling a double S-pattern o n two 1/4" copper plates a n d silver-soldering short (1") pieces of copper tubing at the ends, after the two plates h a d been sweated together so that a coil was formed. Cold water, preferably chdled water f r o m a drinking f o u n t a i n suppl~ hne, is circulated via rubber tubing connections t h r o u g h this heat sink T h e thermoelectric unit f u n c t i o n s as a thermal p u m p , carrying heat away f r o m one side (specimen) to the other (heat sink) of the unit D C c u r r e n t o f high intensity, low voltage a n d low A C ripple is used to p r o d u c e this w o r k
Electroenceph. chn Neurophy~tol_, 1966, 21 85-86
86
F. C. CHESHIRE AND E. EIDELBERG
(Peltier effect). W e d r a w a c u r r e n t o f 10-15 A, at 1.5 V, obtained f r o m a D C power supply (Gates Electronic Co., M o d e l G 50.8). T h e leads f r o m the p o w e r supply to t h e Peltier unit m u s t be o f a heavy, well-insulated, multis t r a n d c o n d u c t o r kind since the c u r r e n t they m u s t carry is very high. T h e y are connected to the leads o f t h e thermoelectric unit by heavy insulated binding posts, with the blue lead in the u m t going to the negative side o f the power supply o u t p u t , a n d the red lead to the p o s l h v e side. T h e base o f the c o o h n g unit is securely a n c h o r e d to a 3 " piece o f metal rod m a d e to fit t h e c h u c k o f the m l c r o t o m e T h e o p e r a h o n of the unit is very simple: cold water is m a d e to circulate t h r o u g h the heat sink u n d e r the t h e r m o electric unit first T h e n the current f r o m the power supply is increased f r o m 0 until IO-18 A are reached. T h e specim e n is placed on the top o f the freezing stage a n d a little water ~s p o u r e d a r o u n d it to g~ve good heat transfer T h e proper c u r r e n t levels a n d cooling water flow rates m u s t be determined by experimentation, since they depend o n the size o f the specimen a n d the t e m p e r a t u r e o f the cooling water However, the current m u s t n o t - - for t h e type o f
unit we use - - exceed 20 A, at 2 V, or It will be d a m a g e d . T h e h o t side o f the unit m u s t be cooled by circulating water so long as current is flowing t h r o u g h it_ To defrost the specimen after cutting, the p o w e r is turned off first a n d t h e n the water flow. To keep the Ussue at cutting temperature, e n o u g h c u r r e n t m u s t be s u p p h e d to offset the h e a t gained by t h e tissue f r o m the a t m o s p h e r e . This device h a s functioned m o u r laboratory for over 2 years without fadure. T h e Pelher u m t is virtually mdestructible, unless allowed to overheat (due to fadure o f heat sink water supply or tf c u r r e n t h m l t s are exceeded). It Is noiseless a n d clean, a n d c o n s t a n t attention to the s p e c . m e n is n o t reqmred. O n occasion, a tissue block h a s been left frozen m o s t o f the day w~thout c h a n g e m its cutting properties. SUMMARY A freezing stage is described, using thermoelectric (Peltler effect) cooling, t h a t c a n be fitted into a n y s t a n d a r d m l c r o t o m e . It is m a d e f r o m a c o m m e r c m l l y available Peltier unit to which a heat sink is attached.
Reference CHESHIRE, F. C. a n d EIDELBERG, E. Thermoelectric freezing stage for serial brain sectioning. Electroenceph. chn Neurophysiol , 1966, 21" 85-86
A SYSTEM FOR SELECTION OF RESPONSES FOR
AVERAGING
1
J. H . SATTERFIELD
Washington Unwerstty, School o f Medicme, Department o f Psychiatry, St. Louts, M o ( U S A.) (Accepted for publication: J a n u a r y 6, 1966)
INTRODUCTION T h e a m o u n t o f artifact potential c o n c u r r e n t with single responses selected for averaging c a n influence the average evoked response This m particularly true of u n w a n t e d potentials t h a t are time locked to the stimulus, as averaging does n o t reduce n o n - r a n d o m noise. O n e e x a m p l e o f n o n - r a n d o m noise is the eye blink potential which occurs following a s t i m u l u s to which a subject IS a t t e n d i n g (Satterfield 1965). W h e n presented with s h o c k s t l m u h to left a n d right wrxsts a n d asked to attend to only o n e o f t h e two stimuli, s o m e subjects b h n k their eyes m o r e frequently following the s t i m u l u s to w h i c h they are attending. T h e s e eye blink potentials are time locked to the s t i m u l u s and, therefore, do n o t average out, as does r a n d o m noise. T h e a m o u n t o f r a n d o m no~se ( m o v e m e n t artifact, m u s c l e potentials, etc.) m a n u n c o o p e r a t i v e 1 S u p p o r t e d in part by the following U S P H S G r a n t s MH05806, MH04808, MH5804, MH7081, FR-00161.
subJeCt m a y also affect the a m p l i t u d e a n d wave f o r m o f the average evoked response. O n e way to reduce b o t h n o n - r a n d o m a n d r a n d o m noise is the selection o f artifact-free responses for averaging. This can be d o n e by utilizing a tape recorder 2 as a delay m e c h a n i s m . Such a system operates as follows the E E G response a n d s t i m u l u s m a r k e r pulse are recorded o n tape a n d a p p r o x i m a t e l y 0.5 sec later (the time it takes for the tape to travel f r o m record head to playback head) the response is played back into the c o m p u t e r 3 a n d s a m p l e d for averaging. T h e 0.5 sec tape delay provides time e n o u g h to m a k e a decision as to whether or n o t large a m p h t u d e z T h e tape recorder used was a n A m p e x SP-300 r u n m n g at 7 5 l.p.s A n y F M recorder with a delay time between writing a n d reading h e a d s equal to the response interval plus t h e few m i c r o s e c o n d s necessary to operate the sense system, would work satisfactorily. 3 A special p u r p o s e digital c o m p u t e r similar to M n e motron's CAT.
Electroenceph. chn. Neurophystol , 1966, 21" 86-88
TECHNICAL NOTES THERMOELECTRIC FREEZING STAGE FOR SERIAL BRAIN SECTIONING1 F
C . CHESHIRE AND E. EIDELBERG 2
Dtt, t~lon o f Neurobtology, Barrow Neurologwal Instttute o f St. Joseph's Hospttal, Phoenix, Artz ( U S A ) (Accepted for p u b h c a t l o n J a n u a r y 5, 1966)
Peltier U
n
i
t
~
Board FLg. 1 D m g r a m of the complete umt, minus the power supply. T h e Peltler unit is m o u n t e d directly over the heat sink (copper) and the m o u n t i n g post fits into the m i c r o t o m e chuck. Thick-walled rubber hose connects the heat sink to a source of cold water a n d d r a m P r e p a r a t i o n o f frozen sections of brains for checking electrode placements a n d for special stain studies is a n i m p o r t a n t part of neurophysiologlcal e x p e r i m e n t a t i o n It IS also a time c o n s u m i n g routine, which is n o t m a d e easier by the use of eRher dry Ice In c h u n k s or liquefied c a r b o n dioxide as the freezing m e d m m , since it is h a r d to keep the tissue c o n s t a n t l y at the proper t e m p e r a t u r e for cutting. This p r o b l e m has been solved in our laboratories by the use o f a thermoelectric (Peltier) unit as the freezing plate for the b r a i n specimens. T h e t e m p e r a t u r e of the tissue c a n be set at any level desired a n d it will stay at that level for as long as needed. It is r e m a r k a b l y fast, freezing a 1 cm thick block o f b r a i n tissue m a b o u t 20 m i n T h e thermoelectric u m t e m p l o y e d (Fig. l) is available 1 S u p p o r t e d by U S P H S G r a n t B-3496 a n d G e n e r a l R e s e a r c h S u p p o r t G r a n t FR-5575 ( U S P H S ) 2 U S P H S Career D e v e l o p m e n t A w a r d K3-NB-15,437
commercially f r o m the Ferroxcube C o m p a n y (Model 20/20) It was modified by m d h n g 1/16" deep, 1/8" wide grooves on its smaller (top) side m a checkerboard p a t t e r n to increase the contact surface with the tissue for better grip. T h e b o t t o m side of the unit was m o u n t e d o n a heat sink by screws, with silicone grease in between T h e function o f the heat sink is to reduce the temperature of the " h o t " side o f the t h e r m a l j u n c t i o n It was m a d e by milling a double S-pattern o n two 1/4" copper plates a n d silver-soldering short (1") pieces of copper tubing at the ends, after the two plates h a d been sweated together so that a coil was formed. Cold water, preferably chdled water f r o m a drinking f o u n t a i n suppl~ hne, is circulated via rubber tubing connections t h r o u g h this heat sink T h e thermoelectric unit f u n c t i o n s as a thermal p u m p , carrying heat away f r o m one side (specimen) to the other (heat sink) of the unit D C c u r r e n t o f high intensity, low voltage a n d low A C ripple is used to p r o d u c e this w o r k
Electroenceph. chn Neurophy~tol_, 1966, 21 85-86
86
F. C. CHESHIRE AND E. EIDELBERG
(Peltier effect). W e d r a w a c u r r e n t o f 10-15 A, at 1.5 V, obtained f r o m a D C power supply (Gates Electronic Co., M o d e l G 50.8). T h e leads f r o m the p o w e r supply to t h e Peltier unit m u s t be o f a heavy, well-insulated, multis t r a n d c o n d u c t o r kind since the c u r r e n t they m u s t carry is very high. T h e y are connected to the leads o f t h e thermoelectric unit by heavy insulated binding posts, with the blue lead in the u m t going to the negative side o f the power supply o u t p u t , a n d the red lead to the p o s l h v e side. T h e base o f the c o o h n g unit is securely a n c h o r e d to a 3 " piece o f metal rod m a d e to fit t h e c h u c k o f the m l c r o t o m e T h e o p e r a h o n of the unit is very simple: cold water is m a d e to circulate t h r o u g h the heat sink u n d e r the t h e r m o electric unit first T h e n the current f r o m the power supply is increased f r o m 0 until IO-18 A are reached. T h e specim e n is placed on the top o f the freezing stage a n d a little water ~s p o u r e d a r o u n d it to g~ve good heat transfer T h e proper c u r r e n t levels a n d cooling water flow rates m u s t be determined by experimentation, since they depend o n the size o f the specimen a n d the t e m p e r a t u r e o f the cooling water However, the current m u s t n o t - - for t h e type o f
unit we use - - exceed 20 A, at 2 V, or It will be d a m a g e d . T h e h o t side o f the unit m u s t be cooled by circulating water so long as current is flowing t h r o u g h it_ To defrost the specimen after cutting, the p o w e r is turned off first a n d t h e n the water flow. To keep the Ussue at cutting temperature, e n o u g h c u r r e n t m u s t be s u p p h e d to offset the h e a t gained by t h e tissue f r o m the a t m o s p h e r e . This device h a s functioned m o u r laboratory for over 2 years without fadure. T h e Pelher u m t is virtually mdestructible, unless allowed to overheat (due to fadure o f heat sink water supply or tf c u r r e n t h m l t s are exceeded). It Is noiseless a n d clean, a n d c o n s t a n t attention to the s p e c . m e n is n o t reqmred. O n occasion, a tissue block h a s been left frozen m o s t o f the day w~thout c h a n g e m its cutting properties. SUMMARY A freezing stage is described, using thermoelectric (Peltler effect) cooling, t h a t c a n be fitted into a n y s t a n d a r d m l c r o t o m e . It is m a d e f r o m a c o m m e r c m l l y available Peltier unit to which a heat sink is attached.
Reference CHESHIRE, F. C. a n d EIDELBERG, E. Thermoelectric freezing stage for serial brain sectioning. Electroenceph. chn Neurophysiol , 1966, 21" 85-86
A SYSTEM FOR SELECTION OF RESPONSES FOR
AVERAGING
1
J. H . SATTERFIELD
Washington Unwerstty, School o f Medicme, Department o f Psychiatry, St. Louts, M o ( U S A.) (Accepted for publication: J a n u a r y 6, 1966)
INTRODUCTION T h e a m o u n t o f artifact potential c o n c u r r e n t with single responses selected for averaging c a n influence the average evoked response This m particularly true of u n w a n t e d potentials t h a t are time locked to the stimulus, as averaging does n o t reduce n o n - r a n d o m noise. O n e e x a m p l e o f n o n - r a n d o m noise is the eye blink potential which occurs following a s t i m u l u s to which a subject IS a t t e n d i n g (Satterfield 1965). W h e n presented with s h o c k s t l m u h to left a n d right wrxsts a n d asked to attend to only o n e o f t h e two stimuli, s o m e subjects b h n k their eyes m o r e frequently following the s t i m u l u s to w h i c h they are attending. T h e s e eye blink potentials are time locked to the s t i m u l u s and, therefore, do n o t average out, as does r a n d o m noise. T h e a m o u n t o f r a n d o m no~se ( m o v e m e n t artifact, m u s c l e potentials, etc.) m a n u n c o o p e r a t i v e 1 S u p p o r t e d in part by the following U S P H S G r a n t s MH05806, MH04808, MH5804, MH7081, FR-00161.
subJeCt m a y also affect the a m p l i t u d e a n d wave f o r m o f the average evoked response. O n e way to reduce b o t h n o n - r a n d o m a n d r a n d o m noise is the selection o f artifact-free responses for averaging. This can be d o n e by utilizing a tape recorder 2 as a delay m e c h a n i s m . Such a system operates as follows the E E G response a n d s t i m u l u s m a r k e r pulse are recorded o n tape a n d a p p r o x i m a t e l y 0.5 sec later (the time it takes for the tape to travel f r o m record head to playback head) the response is played back into the c o m p u t e r 3 a n d s a m p l e d for averaging. T h e 0.5 sec tape delay provides time e n o u g h to m a k e a decision as to whether or n o t large a m p h t u d e z T h e tape recorder used was a n A m p e x SP-300 r u n m n g at 7 5 l.p.s A n y F M recorder with a delay time between writing a n d reading h e a d s equal to the response interval plus t h e few m i c r o s e c o n d s necessary to operate the sense system, would work satisfactorily. 3 A special p u r p o s e digital c o m p u t e r similar to M n e motron's CAT.
Electroenceph. chn. Neurophystol , 1966, 21" 86-88