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A device for enhancing the visibility of the tip of a glass micropipette: Application in neuroanatomy
0361-9230188 $3.00 + .m
Brain Research Bulletin, Vol. 21, pp. 917-918. * Pergamon Press ptc, 1988. Printed in the U.S.A.
LABORATORY
INSTRUMENTATION
AND COMPUTING
A Device for Enhancing the Visibility of the Tip of a Glass Micropip~tt~: Application in Neuroanatomy G. J. TER HORST,
J. G. MAST
AND
K. VAARTJES
Urofacial Research Group, Faculty Medicine, University of Groningen Bloemsingel 10, 9712 KZ Groningen, The Netherlands Received
18 July
1988
TER HORST, G. J., J. G. MAST AND K. VAARTJES. A device for enhancing the visibilify of the tip of a glass Applicafion in neuroanatomy. BRAIN RES BULL 21(6) 917-918, 1988.-In neuroanatomical tract tracing
micropipette:
glass micropipettes are used to make small injections of tracer in the brain. A problem is that, due to the poor visibility of the tip, the tip of a micropipette often breaks during tilling with tracer solution. Therefore, a device was developed to enhance the visibility of the tip of glass micropipettes. The device is based on the principle that light of a light emitting diode (LED) is transmitted through the glass of the wall to the tip of a micropipette. Here, light converges and gives a brightly illuminated spot. In this set up micropipettes can be positioned accurately into a vial with tracer solution even without microscopic guidance. Tract-tracing
Glass micropipettes
Light emitting diode
-
For filling the micropipette, the tracer solution is sucked up from a volume tracer which commonly is kept in a transparent vial (12x38 mm) with a concentric bottom. With this procedure the poor visibility of the tip of the micropipette is a problem. Even under microscopic guidance it is hard to see whether the tip of the micropipette is above the tracer or in it (and how deep). Therefore, the tip of the micropipette often touches the vial and breaks without being noticed. This leads to unsuccessful experiments or, at best, to a large diameter tracer deposit. We therefore developed a device to enhance the visibility of the tip of the glass micropipettes during filling with tracer solution. The device is based on the principle that light of a light emitting diode (LED) is transmitted through the glass of the wail to the tip of a micropipette. At the tip, due to the shape of a pipette, light converges and gives a brightly illuminated spot. Attached to a micromanipulator, micropipettes in this device can be positioned accurately into a vial with tracer solution even without microscopic guidance. A photograph of the device is shown in Fig. 1. Figure 2 is an assembly drawing of the device.
HORSERADISH peroxidase (HRP), tritiated amino-acids, and Phaseolus vulgaris leuco-agglutinin (PHA-L), are used by neuroanatomists to identify afferent and efferent connections of neurons in the central nervous system (1). After injection into the brain these substances are incorporated in the nerve cell by endocytocis, either at the level of the soma of neurons or at the level of presynaptic endings. Depending on the site of entry into the nerve cell they are transported in an anterograde or a retrograde direction. Horseradish peroxidase is retrogradely transported after incorporation at the presynaptic level and therefore used for the identification of afferent connections of cell groups in the brain. Tritiated amino acids and Phaseolus vulgaris leuco-a~utinin are incorporated at the level of the soma of neurons and then transported in an anterograde direction. Therefore, these tracers are used to visualize efferent connections of nerve cells. Tracer is injected into the brain either by pressure or by iontophoretic injection procedures. The major disadvantage of pressure injections is that tracer deposits have a large diameter. With such large deposits, connections at a cellular level cannot be identified accurately. Therefore, tracers are preferentially brought into the brain iontophoretically by means of small glass micropipettes. This procedure gives much smaller tracer deposits (if desired in the order of a group of 10 to 15 cells). A condition of success of this method, however, is an undamaged beveled tip (10 to 20 pm) of the glass micropipette.
DESCRIPTION
OF THE DEVICE
A Toshiba, TLRA 150 (angle e), light emitting diode is built into a metal case (8x0.8 cm) that fits to a micromanipulator. The metal case is formed from 4 parts that
917
TER HORST, MAST AND VAARTJES
____
I
____
I
holder
LED: Toshiba TLRA
150
!$?&I &-&-.---
O-ring(s)
L ---------
.I
FIG. 1. Photograph of a device that during filling with tracer solution enhances the visibility of the tip of glass micropipettes. The apparatus is connected to a current source and held by a micromanipulator. The inset shows the brightly illuminated tip of a micropipette.
are screwed onto each other. An air-tight seal is secured by O-rings (l-3) that are placed between the parts of the metal case. The power of the LED is supplied by a 1.5Volts source that is connected to the light emitting diode via a 480 $1 resistance (also mounted in the tube). A glass micropipette can be inserted into a small opening, with an inside diameter of 1.5 mm, at the bottom end of the tube. The smaller diameter (1.4 mm) of the chamber (B) underneath the LED blocks the insertion of the pipette and ensures a proper positioning of the LED on top of the glass micropipette. The pipette is held by two O-rings (numbers 2 and 3) of the metal case. The chamber (B) between the second O-ring and the LED has an outlet to which a syringe (A) can be connected. Care should be taken that the diameter of
I “._ -~_-
glass micropipette
10-20um
FIG. 2. Assembly drawing of the device shown in Fig. I. The device consists of a tube containing a light emitting diode (LED) mounted above a glass micropipette. The light of the LED is transmitted to the tip of the pipette through the glass of the wall. At the tip light converges and gives a brightly illuminated spot. The micropipette can be tilled by sucking up the tracer fluid with the syringe (A) that is connected to the air-tight chamber (B).
the pipette, the O-rings and the nozzle of the syringe is such that the chamber is air-tight. After placing the tip of the micropipette into the tracer solution the desired amount of tracer is sucked up into the micropipette by the syringe. After filling, a silver wire can be inserted into the tracer solution via the syringe outlet, for iontophoresis. It is beneficial to place the described device in front of a dark background to obtain more contrast with the su~oundings,
REFERENCE 1. Luiten, P. G. M.; Ter Horst, G. J.; Steffens, A. B. The hypothaiamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and metabolism. Prog. Neurobiol. 2&l-54; 1987.
Brain Research Bulletin, Vol. 21, pp. 917-918. * Pergamon Press ptc, 1988. Printed in the U.S.A.
LABORATORY
INSTRUMENTATION
AND COMPUTING
A Device for Enhancing the Visibility of the Tip of a Glass Micropip~tt~: Application in Neuroanatomy G. J. TER HORST,
J. G. MAST
AND
K. VAARTJES
Urofacial Research Group, Faculty Medicine, University of Groningen Bloemsingel 10, 9712 KZ Groningen, The Netherlands Received
18 July
1988
TER HORST, G. J., J. G. MAST AND K. VAARTJES. A device for enhancing the visibilify of the tip of a glass Applicafion in neuroanatomy. BRAIN RES BULL 21(6) 917-918, 1988.-In neuroanatomical tract tracing
micropipette:
glass micropipettes are used to make small injections of tracer in the brain. A problem is that, due to the poor visibility of the tip, the tip of a micropipette often breaks during tilling with tracer solution. Therefore, a device was developed to enhance the visibility of the tip of glass micropipettes. The device is based on the principle that light of a light emitting diode (LED) is transmitted through the glass of the wall to the tip of a micropipette. Here, light converges and gives a brightly illuminated spot. In this set up micropipettes can be positioned accurately into a vial with tracer solution even without microscopic guidance. Tract-tracing
Glass micropipettes
Light emitting diode
-
For filling the micropipette, the tracer solution is sucked up from a volume tracer which commonly is kept in a transparent vial (12x38 mm) with a concentric bottom. With this procedure the poor visibility of the tip of the micropipette is a problem. Even under microscopic guidance it is hard to see whether the tip of the micropipette is above the tracer or in it (and how deep). Therefore, the tip of the micropipette often touches the vial and breaks without being noticed. This leads to unsuccessful experiments or, at best, to a large diameter tracer deposit. We therefore developed a device to enhance the visibility of the tip of the glass micropipettes during filling with tracer solution. The device is based on the principle that light of a light emitting diode (LED) is transmitted through the glass of the wail to the tip of a micropipette. At the tip, due to the shape of a pipette, light converges and gives a brightly illuminated spot. Attached to a micromanipulator, micropipettes in this device can be positioned accurately into a vial with tracer solution even without microscopic guidance. A photograph of the device is shown in Fig. 1. Figure 2 is an assembly drawing of the device.
HORSERADISH peroxidase (HRP), tritiated amino-acids, and Phaseolus vulgaris leuco-agglutinin (PHA-L), are used by neuroanatomists to identify afferent and efferent connections of neurons in the central nervous system (1). After injection into the brain these substances are incorporated in the nerve cell by endocytocis, either at the level of the soma of neurons or at the level of presynaptic endings. Depending on the site of entry into the nerve cell they are transported in an anterograde or a retrograde direction. Horseradish peroxidase is retrogradely transported after incorporation at the presynaptic level and therefore used for the identification of afferent connections of cell groups in the brain. Tritiated amino acids and Phaseolus vulgaris leuco-a~utinin are incorporated at the level of the soma of neurons and then transported in an anterograde direction. Therefore, these tracers are used to visualize efferent connections of nerve cells. Tracer is injected into the brain either by pressure or by iontophoretic injection procedures. The major disadvantage of pressure injections is that tracer deposits have a large diameter. With such large deposits, connections at a cellular level cannot be identified accurately. Therefore, tracers are preferentially brought into the brain iontophoretically by means of small glass micropipettes. This procedure gives much smaller tracer deposits (if desired in the order of a group of 10 to 15 cells). A condition of success of this method, however, is an undamaged beveled tip (10 to 20 pm) of the glass micropipette.
DESCRIPTION
OF THE DEVICE
A Toshiba, TLRA 150 (angle e), light emitting diode is built into a metal case (8x0.8 cm) that fits to a micromanipulator. The metal case is formed from 4 parts that
917
TER HORST, MAST AND VAARTJES
____
I
____
I
holder
LED: Toshiba TLRA
150
!$?&I &-&-.---
O-ring(s)
L ---------
.I
FIG. 1. Photograph of a device that during filling with tracer solution enhances the visibility of the tip of glass micropipettes. The apparatus is connected to a current source and held by a micromanipulator. The inset shows the brightly illuminated tip of a micropipette.
are screwed onto each other. An air-tight seal is secured by O-rings (l-3) that are placed between the parts of the metal case. The power of the LED is supplied by a 1.5Volts source that is connected to the light emitting diode via a 480 $1 resistance (also mounted in the tube). A glass micropipette can be inserted into a small opening, with an inside diameter of 1.5 mm, at the bottom end of the tube. The smaller diameter (1.4 mm) of the chamber (B) underneath the LED blocks the insertion of the pipette and ensures a proper positioning of the LED on top of the glass micropipette. The pipette is held by two O-rings (numbers 2 and 3) of the metal case. The chamber (B) between the second O-ring and the LED has an outlet to which a syringe (A) can be connected. Care should be taken that the diameter of
I “._ -~_-
glass micropipette
10-20um
FIG. 2. Assembly drawing of the device shown in Fig. I. The device consists of a tube containing a light emitting diode (LED) mounted above a glass micropipette. The light of the LED is transmitted to the tip of the pipette through the glass of the wall. At the tip light converges and gives a brightly illuminated spot. The micropipette can be tilled by sucking up the tracer fluid with the syringe (A) that is connected to the air-tight chamber (B).
the pipette, the O-rings and the nozzle of the syringe is such that the chamber is air-tight. After placing the tip of the micropipette into the tracer solution the desired amount of tracer is sucked up into the micropipette by the syringe. After filling, a silver wire can be inserted into the tracer solution via the syringe outlet, for iontophoresis. It is beneficial to place the described device in front of a dark background to obtain more contrast with the su~oundings,
REFERENCE 1. Luiten, P. G. M.; Ter Horst, G. J.; Steffens, A. B. The hypothaiamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and metabolism. Prog. Neurobiol. 2&l-54; 1987.