Printed in Sweden Copyright @ 1977 by Academic Press. Inc. Al/ rights ofreproduction in any form reserved ISSN WI44827
Experimental
A SIMPLE
DEVICE
FLUIDS
AT
Cell Research 105 (1977) 281-284
FOR THE
HARVESTING MICROSCOPIC
BIOLOGICAL LEVEL
M. BRENh’ER Biological Laboratories,
Harvard University, Cambridge, MA 02138, USA
SUMMARY A simple aspirating device is described which permits repeated samples of biological materials to be taken near or at the microscopic level. It features an inner coaxial tube which delivers a harvesting fluid into the very tip of the collecting needle, permitting metabolic activity in the sample to be quenched immediately. The device is easily constructed from common laboratory mat&al.
For a variety of studies it is useful to be able to sample cells from a particular area of a piece of tissue, or even to sample the cytoplasm from certain parts of large cells. For some of these studies, such as in the analysis of pools of glycolytic intermediates or of nucleotides, it is crucial to stop all metabolic activity at the instant the sample is taken. The primary procedure used to obtain such samples is rapid freezing of the tissue in question. This may be followed by histological staining to reveal the distribution of the compound of interest [ 1,2], or by microdissection of particular sections for quantitative analyses [3-6]. This latter procedure especially has been brought to a fine art by Lowry and his collaborators, who have also developed highly sensitive assays for several classes of compounds [4-71. Many cases remain, however, for which these procedures are not satisfactory, particularly those requiring that multiple samples be taken to accumulate sufficient material for analysis. In this paper a simple aspirating device is described which facili-
tates collection of multiple samples from tissues which are accessible to a micro-needle. A special feature of this micro-sampler is an inner coaxial tube which delivers a harvesting fluid into the very tip of the collecting needle, permitting metabolic activity in the sample to be halted immediately. The device can be constructed in less than 1 h from common laboratory material. Construction of the micro-sampler The micro-sampler is diagrammed in fig. 1. It is made from two pieces of polyethylene tubing, an inner piece 150 cm long (Intramedic PE 10, 0.011” IDx0.024” OD) and an outer piece 100 cm long (Intramedic no. 7425, 0.034” IDX0.060” OD). One end of the outer tubing is enlarged a distance of at least 3 mm by softening in boiling water and inserting a capillary tube (Drummond micrecaps, 50 I.LI size). Once the end is enlarged the capillary tube is removed. The inner tubing is then threaded through the outer tubing such that about 5 cm extends beyond the expanded (collecting) end and 45 cm exExp
Cd
Rcs
IO5 (1977)
282
M. Brenner
open outer
a
Gloss capillary extension tube
b
LJ
tends beyond the other (receiving) end. The receiving end is passed through a short piece of glass tubing inserted through a stopper which will fit on the receiving vessel; in the example shown in the fig. 1 the receiving vessel is a 15 ml sidearm test tube and it is fitted with a no. 3 rubber stopper pierced with a 7 cm length of l/8” OD glass tubing. The end of the outer polyethylene tubing should extend down somewhat past the sidearm of the receiving vessel and if possible rest against a side of the vessel, so that the aspirated samples drain directly to the bottom of the vessel. The remaining inner tubing is looped back out through the glass tubing. Melted paraffin is used to seal the strands of polyethylene tubing into the glass tubing traversing the rubber stopper (the paraffin should not be too hot or it will melt the thinner polyethylene tubing). The sidearm of the receiving vessel is connected to a vacuum source, and the free end of the inner tubing is inserted into the reservoir of harvesting fluid. A small glass capillary extension tube, about 1.5 cm in length, is inserted into the end of the inner tubing at the collecting end. These extensions may be easily made by drawing out one of the 50 ~1 Exp Cell
Res 105 (1977)
Fig. I. (a) Collecting end of the micro-sampler. Not drawn to scale (horizontal distances increased about 8 X , vertical distances about 3 X); (6) receiving end of the microsampler. Not drawn to scale (horizontal distances increased about 1.7X , vertical distances about 1.2X). The thick arrows show the direction of flow of harvesting fluid from the reservoir to the tip of the collecting needle. The thin arrows show the direction of flow of the collected sample from the needle tip to the receiving vessel.
capillary tubes to a long thin capillary whose diameter is intermediate between that of the inner and outer polyethylene tubing, and then pulling it into about 1.5 cm segments over a small flame. The microsampler is completed by slipping a pointed glass needle over the inner tubing and into the expanded end of the outer tubing. It is important that the inner capillary extension tube reach as near to the tip of the glass needle as possible. The glass needles are made by pulling out 50 ~1 capillary tubes. Although this may be done by hand, a glass electrode puller is far more convenient. Should a collecting needle be broken it is a simple matter to remove it and attach another. Allowance for differing lengths is made by inserting the needle to a greater or lesser extent into the enlarged end of the outer tubing (hence, the longer the enlarged portion, the better). In most cases this should be the only adjustment necessary. However, if a needle is still too short, it can be accommodated by cutting back the end of the inner tubing, whereas needles which are too long can be accommodated by cutting away an end segment of the outer tube with a scalpel or razor blade. If fairly large
A simple sampling
device
283
volumes of fluid are aspirated (several ml), the inner tube may be partially sucked back into the outer one, so that the tip of the inner glass capillary no longer extends into the tip of the collecting needle. This is easily remedied by removing the collecting needle and gently pulling the inner tubing back out while the line to the receiving vessel is held straight. Operation
of the micro-sampler
The micro-sampler works as a simple aspirating device with the added feature that a harvesting solution is mixed with the sample as soon as it is collected. When vacuum is applied to the receiving vessel, the space between the inner and outer tubing is evacuated, this being the space opening into the receiving vessel. At the other end of the sampler the intertubing space terminates at the tip of the collecting needle, so the vacuum acts to draw samples into the needle. The inner tubing also opens into the intertubing space at the collecting tip, so in addition the vacuum draws harvesting fluid through the inner tubing towards the collecting tip (thick arrows, fig. I). Thus, samples are drawn in at the tip of the collecting needle, immediately mixed with harvesting fluid, and washed back through the space between the inner and outer tubing into the receiving vessel (thin arrows, fig. 1). The precise details of operation of the micro-sampler will depend on the material being sampled. In our laboratory it is being used for harvesting separately the anterior and posterior portions of pseudoplasmodia disof the cellular slime mold Dictyostefium coideum for the determination of cyclic AMP (CAMP) levels. The pseudoplasmodia are composed of about 50000 amebas formed into a sausage-shaped structure about 0.1 mm in diameter and 1.5 mm long
Fig. 2. Photograph of the micro-sampler being used to halest tips of migrating D. discoideum ps&doplasmodia. The tip of the collecting needle is about 0.3 mm, and thk length of the pseudoplasmodia about 1.5 mm. To increase contrast the pseudoplasmodia have been vitally stained with neutral red, and the collecting needle coated with paint.
which is encased by an outer slime sheath. These dimensions are sufficiently large that the collecting needle may be held by hand and the organisms viewed under a low power dissecting microscope (see fig. 2). A water aspirator is used as the vacuum source, and 1 M formic acid for the harvesting fluid. Both the receiving vessel and the harvesting fluid reservoir are kept in ice. The anterior or posterior portions of the pseudoplasmodia are harvested by simply touching the tip of the collecting needle to the appropriate part of the pseudoplasmodia. Using this procedure 100 pseudoplasmodia can be harvested in about 10 min. The volume of harvesting fluid used can be quite small. For example, in the case above, only about 1 ml is used to harvest 100 pseudoplasmodia. Greatly reducing the volume of fluid required is the fact that the end of the collecting needle acts as a valve; when it is open (no sample being taken) the E.rp Cd
Res 105 (1977)
284
M. Brenner
vacuum primarily draws in air through that opening rather than drawing liquid from the harvesting fluid reservoir; when the tip is closed (sample being taken) harvesting fluid flows readily. If necessary, the rate of flow of harvesting fluid may be controlled by sealing the reservoir and regulating its pressure with a pump. When particularly small portions of tissue are being sampled it will be necessary to hold the collecting needle in a micromanipulator. Use of a finer-tipped capillary will increase the likelihood of clogging the tip. Even in the case of the fairly large tip used for collection of pseudoplasmodia segments (about 0.1 mm) the sticky slime sheath occasionally obstructs the opening. Siliconizing the glass parts of the micro-sampler does not alleviate this problem. However, the problem is virtually eliminated by proper choice of harvesting fluid, in this case 1 M formic acid. This solvent prevents the breakdown of CAMP in the collected sample (whereas water or physiological buffers do not), while not causing any material to precipitate in the needle tip (as trichloroacetic acid solutions do). When material does occasionally begin to collect in the tip it can be readily dissolved by aspirating small (25 ~1) volumes of concentrated (88 %) formic acid. For some applications, such as collecting cytoplasm or organelles from an internal position in a cell, it would be necessary to have the needle properly positioned before
Exp Cell
Res 105 (1977)
applying suction. This may be easily accomplished by having a valve between the vacuum source and the collection vessel; particularly useful would be one which could be controlled by the operator’s foot. Although the emphasis in this note has been on using the micro-sampler in situations in which one would like to halt metabolic activity in the sample being collected, it clearly is also useful for obtaining materials in their native state: stabilizing buffers may be mixed with the sample as soon as it is collected, while the flushing action of the harvesting solution rapidly brings the sample into a temperature-controlled receiving vessel. Hence the device should be useful for a wide range of applications in which samples must be obtained near or at the microscopic level. This work was supported by the NSF grant BMS7201830-A02.
REFERENCES 1. Shida, H, Exp cell res 63 (1970) 385. 2. Wedner, H J; Hoffer, B J, Battenberg, E, Steiner, A L, Parker, C W &Bloom. F E. J histochem cytothem 20 (1972) 293. 3. Kato, T & Lowry, 0 H, J biol them 248 (1973) 2044. 4. Lowry, 0 H, Bull NY acad med 38 (1%2) 788. 5. Matchinsky, F M, Passonneau, J V &Lowry, 0 H, J histochem cytochem 16 (1968) 29. Lowry, 0 H, Harvey lect 58 (l&2-1%3) 1. f : Kato, T, Berger, S J, Carter, J A & Lowry, 0 H, Anal biochem 53 (1973) 86. Received July 5, 1976 Accepted November 3, 1976
Experimental
A SIMPLE
DEVICE
FLUIDS
AT
Cell Research 105 (1977) 281-284
FOR THE
HARVESTING MICROSCOPIC
BIOLOGICAL LEVEL
M. BRENh’ER Biological Laboratories,
Harvard University, Cambridge, MA 02138, USA
SUMMARY A simple aspirating device is described which permits repeated samples of biological materials to be taken near or at the microscopic level. It features an inner coaxial tube which delivers a harvesting fluid into the very tip of the collecting needle, permitting metabolic activity in the sample to be quenched immediately. The device is easily constructed from common laboratory mat&al.
For a variety of studies it is useful to be able to sample cells from a particular area of a piece of tissue, or even to sample the cytoplasm from certain parts of large cells. For some of these studies, such as in the analysis of pools of glycolytic intermediates or of nucleotides, it is crucial to stop all metabolic activity at the instant the sample is taken. The primary procedure used to obtain such samples is rapid freezing of the tissue in question. This may be followed by histological staining to reveal the distribution of the compound of interest [ 1,2], or by microdissection of particular sections for quantitative analyses [3-6]. This latter procedure especially has been brought to a fine art by Lowry and his collaborators, who have also developed highly sensitive assays for several classes of compounds [4-71. Many cases remain, however, for which these procedures are not satisfactory, particularly those requiring that multiple samples be taken to accumulate sufficient material for analysis. In this paper a simple aspirating device is described which facili-
tates collection of multiple samples from tissues which are accessible to a micro-needle. A special feature of this micro-sampler is an inner coaxial tube which delivers a harvesting fluid into the very tip of the collecting needle, permitting metabolic activity in the sample to be halted immediately. The device can be constructed in less than 1 h from common laboratory material. Construction of the micro-sampler The micro-sampler is diagrammed in fig. 1. It is made from two pieces of polyethylene tubing, an inner piece 150 cm long (Intramedic PE 10, 0.011” IDx0.024” OD) and an outer piece 100 cm long (Intramedic no. 7425, 0.034” IDX0.060” OD). One end of the outer tubing is enlarged a distance of at least 3 mm by softening in boiling water and inserting a capillary tube (Drummond micrecaps, 50 I.LI size). Once the end is enlarged the capillary tube is removed. The inner tubing is then threaded through the outer tubing such that about 5 cm extends beyond the expanded (collecting) end and 45 cm exExp
Cd
Rcs
IO5 (1977)
282
M. Brenner
open outer
a
Gloss capillary extension tube
b
LJ
tends beyond the other (receiving) end. The receiving end is passed through a short piece of glass tubing inserted through a stopper which will fit on the receiving vessel; in the example shown in the fig. 1 the receiving vessel is a 15 ml sidearm test tube and it is fitted with a no. 3 rubber stopper pierced with a 7 cm length of l/8” OD glass tubing. The end of the outer polyethylene tubing should extend down somewhat past the sidearm of the receiving vessel and if possible rest against a side of the vessel, so that the aspirated samples drain directly to the bottom of the vessel. The remaining inner tubing is looped back out through the glass tubing. Melted paraffin is used to seal the strands of polyethylene tubing into the glass tubing traversing the rubber stopper (the paraffin should not be too hot or it will melt the thinner polyethylene tubing). The sidearm of the receiving vessel is connected to a vacuum source, and the free end of the inner tubing is inserted into the reservoir of harvesting fluid. A small glass capillary extension tube, about 1.5 cm in length, is inserted into the end of the inner tubing at the collecting end. These extensions may be easily made by drawing out one of the 50 ~1 Exp Cell
Res 105 (1977)
Fig. I. (a) Collecting end of the micro-sampler. Not drawn to scale (horizontal distances increased about 8 X , vertical distances about 3 X); (6) receiving end of the microsampler. Not drawn to scale (horizontal distances increased about 1.7X , vertical distances about 1.2X). The thick arrows show the direction of flow of harvesting fluid from the reservoir to the tip of the collecting needle. The thin arrows show the direction of flow of the collected sample from the needle tip to the receiving vessel.
capillary tubes to a long thin capillary whose diameter is intermediate between that of the inner and outer polyethylene tubing, and then pulling it into about 1.5 cm segments over a small flame. The microsampler is completed by slipping a pointed glass needle over the inner tubing and into the expanded end of the outer tubing. It is important that the inner capillary extension tube reach as near to the tip of the glass needle as possible. The glass needles are made by pulling out 50 ~1 capillary tubes. Although this may be done by hand, a glass electrode puller is far more convenient. Should a collecting needle be broken it is a simple matter to remove it and attach another. Allowance for differing lengths is made by inserting the needle to a greater or lesser extent into the enlarged end of the outer tubing (hence, the longer the enlarged portion, the better). In most cases this should be the only adjustment necessary. However, if a needle is still too short, it can be accommodated by cutting back the end of the inner tubing, whereas needles which are too long can be accommodated by cutting away an end segment of the outer tube with a scalpel or razor blade. If fairly large
A simple sampling
device
283
volumes of fluid are aspirated (several ml), the inner tube may be partially sucked back into the outer one, so that the tip of the inner glass capillary no longer extends into the tip of the collecting needle. This is easily remedied by removing the collecting needle and gently pulling the inner tubing back out while the line to the receiving vessel is held straight. Operation
of the micro-sampler
The micro-sampler works as a simple aspirating device with the added feature that a harvesting solution is mixed with the sample as soon as it is collected. When vacuum is applied to the receiving vessel, the space between the inner and outer tubing is evacuated, this being the space opening into the receiving vessel. At the other end of the sampler the intertubing space terminates at the tip of the collecting needle, so the vacuum acts to draw samples into the needle. The inner tubing also opens into the intertubing space at the collecting tip, so in addition the vacuum draws harvesting fluid through the inner tubing towards the collecting tip (thick arrows, fig. I). Thus, samples are drawn in at the tip of the collecting needle, immediately mixed with harvesting fluid, and washed back through the space between the inner and outer tubing into the receiving vessel (thin arrows, fig. 1). The precise details of operation of the micro-sampler will depend on the material being sampled. In our laboratory it is being used for harvesting separately the anterior and posterior portions of pseudoplasmodia disof the cellular slime mold Dictyostefium coideum for the determination of cyclic AMP (CAMP) levels. The pseudoplasmodia are composed of about 50000 amebas formed into a sausage-shaped structure about 0.1 mm in diameter and 1.5 mm long
Fig. 2. Photograph of the micro-sampler being used to halest tips of migrating D. discoideum ps&doplasmodia. The tip of the collecting needle is about 0.3 mm, and thk length of the pseudoplasmodia about 1.5 mm. To increase contrast the pseudoplasmodia have been vitally stained with neutral red, and the collecting needle coated with paint.
which is encased by an outer slime sheath. These dimensions are sufficiently large that the collecting needle may be held by hand and the organisms viewed under a low power dissecting microscope (see fig. 2). A water aspirator is used as the vacuum source, and 1 M formic acid for the harvesting fluid. Both the receiving vessel and the harvesting fluid reservoir are kept in ice. The anterior or posterior portions of the pseudoplasmodia are harvested by simply touching the tip of the collecting needle to the appropriate part of the pseudoplasmodia. Using this procedure 100 pseudoplasmodia can be harvested in about 10 min. The volume of harvesting fluid used can be quite small. For example, in the case above, only about 1 ml is used to harvest 100 pseudoplasmodia. Greatly reducing the volume of fluid required is the fact that the end of the collecting needle acts as a valve; when it is open (no sample being taken) the E.rp Cd
Res 105 (1977)
284
M. Brenner
vacuum primarily draws in air through that opening rather than drawing liquid from the harvesting fluid reservoir; when the tip is closed (sample being taken) harvesting fluid flows readily. If necessary, the rate of flow of harvesting fluid may be controlled by sealing the reservoir and regulating its pressure with a pump. When particularly small portions of tissue are being sampled it will be necessary to hold the collecting needle in a micromanipulator. Use of a finer-tipped capillary will increase the likelihood of clogging the tip. Even in the case of the fairly large tip used for collection of pseudoplasmodia segments (about 0.1 mm) the sticky slime sheath occasionally obstructs the opening. Siliconizing the glass parts of the micro-sampler does not alleviate this problem. However, the problem is virtually eliminated by proper choice of harvesting fluid, in this case 1 M formic acid. This solvent prevents the breakdown of CAMP in the collected sample (whereas water or physiological buffers do not), while not causing any material to precipitate in the needle tip (as trichloroacetic acid solutions do). When material does occasionally begin to collect in the tip it can be readily dissolved by aspirating small (25 ~1) volumes of concentrated (88 %) formic acid. For some applications, such as collecting cytoplasm or organelles from an internal position in a cell, it would be necessary to have the needle properly positioned before
Exp Cell
Res 105 (1977)
applying suction. This may be easily accomplished by having a valve between the vacuum source and the collection vessel; particularly useful would be one which could be controlled by the operator’s foot. Although the emphasis in this note has been on using the micro-sampler in situations in which one would like to halt metabolic activity in the sample being collected, it clearly is also useful for obtaining materials in their native state: stabilizing buffers may be mixed with the sample as soon as it is collected, while the flushing action of the harvesting solution rapidly brings the sample into a temperature-controlled receiving vessel. Hence the device should be useful for a wide range of applications in which samples must be obtained near or at the microscopic level. This work was supported by the NSF grant BMS7201830-A02.
REFERENCES 1. Shida, H, Exp cell res 63 (1970) 385. 2. Wedner, H J; Hoffer, B J, Battenberg, E, Steiner, A L, Parker, C W &Bloom. F E. J histochem cytothem 20 (1972) 293. 3. Kato, T & Lowry, 0 H, J biol them 248 (1973) 2044. 4. Lowry, 0 H, Bull NY acad med 38 (1%2) 788. 5. Matchinsky, F M, Passonneau, J V &Lowry, 0 H, J histochem cytochem 16 (1968) 29. Lowry, 0 H, Harvey lect 58 (l&2-1%3) 1. f : Kato, T, Berger, S J, Carter, J A & Lowry, 0 H, Anal biochem 53 (1973) 86. Received July 5, 1976 Accepted November 3, 1976
