- Email: [email protected]
Serial sectioning of thick tissue with a novel vibrating blade microtome
Brain Research Protocols 3 Ž1999. 302–307
Protocol
Serial sectioning of thick tissue with a novel vibrating blade microtome Glen T. Prusky
)
, John E. McKenna
Department of Psychology and Neuroscience, The UniÕersity of Lethbridge, 4401 UniÕersity DriÕe, Lethbridge, Alberta, Canada T1K 3M4 Accepted 30 September 1998
Abstract Vibrating blade microtomes are used extensively in biological research to section non-frozen tissue. There are a wide variety of commercial instruments available for this purpose, however, they are designed to cut thin sections primarily from a tissue block less than one centimeter in height. Herein is described a simple modification of a microscope frame that creates a vibrating blade microtome capable of producing a sequential series of sections through three centimeters of tissue. We illustrate the use of this device to identify and reconstruct a column of rat spinal motor neurons retrogradely labeled from a peripheral muscle. q 1999 Elsevier Science B.V. All rights reserved.
Themes: Cellular and molecular biology Topics: Staining, tracing and imaging techniques Keywords: Vibratome; Brain slice; Tissue section; Microtome; Vibrating blade; Microscope; Spinal cord; Motor neurons
1. Type of research
3. Materials
Ø Ø Ø Ø Ø Ø
3.1. Vibrating blade microtome
Neuroanatomy Structurerfunction studies Cell differentiation Development and plasticity Neural patterning Reconstruction
2. Time required The time required to setup the machine, mount the specimen, cut 200 mm sections through ; 3 cm of tissue and clean up, is approximately 1.5 h. This does not include the time required to design and construct the vibrating blade microtome.
) Corresponding [email protected]
author.
Fax:
q 1-403-329-2555;
E-mail:
An early 1970s vintage Carl Zeiss Jena Amplival compound microscope was used as a stand for the vibrating blade microtome. This microscope was mechanically sound, however, it had outlived its use as an optical device. The illuminator, condenser, nosepiece, tube carrier and binocular tube assembly were all removed from the microscope and the X-axial stage control was disabled by removing its hand-operated pinion head. This left the microscope stand with a dovetail mount on the top and a stage with 5 cm of horizontal movement in the Y-axis, controlled by rotating the associated pinion head, and 3 cm of vertical movement in the Z-axis, controlled by rotating either the coarse or fine knob on the coaxial focusing assembly. A rectangular tissue bath Ž9.5 cm = 7.5 cm = 4 cm. is made of 3 mm Plexiglas and a square plate of stainless steel Ž4.5 cm = 4.5 cm = 0.1 cm. is glued to the bottom of the bath to create a durable surface for mounting tissue. The bath is mounted on the microscope stage by sliding it
1385-299Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 5 - 2 9 9 X Ž 9 8 . 0 0 0 5 2 - X
G.T. Prusky, J.E. McKennar Brain Research Protocols 3 (1999) 302–307
into guides formed by two grooved rails bolted to the stage. A thumbscrew on each rail can be tightened to secure the bath in place. Fig. 1 illustrates the assembled vibrating blade microtome. No physical modifications are made to the microscope itself, so it can easily be reassembled if desired. A simple jig to accurately set the blade angle is fabricated from 7 mm Plexiglas; it consists of a regular triangular solid with a 258 acute angle Ž53 mm base =20 mm height.. A vibrating blade assembly is manufactured and mounted on the microscope stand in order to produce a functional instrument ŽFig. 2.. This attachment, made from a rectangular piece of plate steel Ž13.2 cm = 7 cm = 1 cm. welded to a rectangular Ž8.2 cm = 6 cm = 3.1 cm. steel block, provided a platform to anchor other components, and sufficient mass to effectively dampen unwanted vibrations. Two free-standing steel arms were constructed Ž12.9 cm = 3.2 cm = 0.16 cm. to carry the blade cutting head and travel rail. One arm was bolted to each side of the block and extended forward. The front ends of the arms were connected by a steel rail Ž8.75 cm = 0.75 cm dia. on which a stainless steel blade holder Ž6.5 cm = 3 cm = 1 cm. was positioned. The blade holder could rotate around the rail to allow for angle adjustments and was held in place with a thumbscrew. A razor blade was secured in place at a 1058 angle at the end of the holder by a small rectangular Ž1.4 cm = 3 cm = 0.15 cm. stainless steel plate anchored with stainless steel screws. A solenoid Ž11-C120A; Guardian. was placed between the front of the steel block and the rail connecting the arms, and was secured to the bottom of the block with a steel plate. The solenoid’s plug was fixed to one of the two steel arms 3.5 cm behind the rail and 7.2 cm in front of the screws that anchored the arms to the steel block. The completed vibrating blade attachment was installed on the dovetail mount Žon top of
Fig. 1. Assembled vibrating blade microtome, including: ŽA. microscope stand, ŽB. vibrating blade assembly, ŽC. drive control, ŽD. tissue bath, ŽE. Y-axial control, ŽF. Z-axial control.
303
Fig. 2. Schematic drawing of the vibrating blade assembly, in plane Žtop. and side Žbottom. views. Significant components are: ŽA. steel arms, ŽB. blade holder, ŽC. travel rail, ŽD. solenoid, ŽE. steel mounting block, ŽF. locking cam, ŽG. power coupling, and ŽH. angle-setting jig. Scale bar s10 cm.
the stand. with the cam locking screw that originally secured the tube carrier. An AC drive circuit Ž120 volt. for the instrument was located in a separate box and consisted of a variable output transformer, the output of which was current limited and half-wave rectified before being applied to the AC solenoid ŽFig. 3.. When current was applied to the solenoid, it vibrated the blade assembly in an arcuate path, the amplitude of oscillation being proportional to the drive voltage Žneglecting losses.. Since the drive frequency was constant Ž60 Hz., it was necessary to tune the resonant frequency of the vibrating assembly to maintain the efficiency of the solenoid. The resonant frequency is equal to the square root of Ž KrM ., where K equals the spring constant of the arms and M equals the mass of the assembly. We found the resonant frequency of the vibrating mass empirically by attaching a vibration sensor to the mass and exciting it, and then watching the output on an oscilloscope. Adjustments to the length, thickness and material type of the arms, as well as the mass of the assembly were made to alter its resonant frequency and produce a lateral displacement of about 1 mm.
Fig. 3. Schematic drawing of the electrical drive circuitry.
304
G.T. Prusky, J.E. McKennar Brain Research Protocols 3 (1999) 302–307
3.2. Maintenance Lithium grease.
4. Detailed procedure 4.1. Motor neuron labeling A detailed procedure for labeling motor neurons has been presented previously w7x. Briefly, vacuum-load a 10% solution Žin dimethylformamide. of DiI w6x into a siliconcoated w10x glass micropipet pulled to a long taper with the tip broken to a diameter of about 50 mm. Anaesthetize the animals Ž65 mgrkg sodium pentobarbitol. and make a surgical incision over the spinodeltoideus forelimb muscle. Isolate the muscle and inject it with approximately 0.5 ml of the dye solution. In animals used as controls, instead of injecting into the muscle, approximately 2 ml of DiI solution was ejected onto the surface of the spinodeltoideus muscle, and then wiped away with a cotton swab a few minutes later. Recover the animals from surgery and allow one week of survival for axonal terminals contacting this muscle to absorb the dye and transport it retrogradely to the cell bodies of origin in the spinal cord.
cutting head ventrally until the lower surface of the blade rests against the 258 angle of the jig. Steady the blade holder against the jig with one hand and tighten the thumb screw of the cutter head with the other hand. The blade is now at a 258 angle to the plane of the stage. Remove the jig by sliding it laterally. Move the tissue to the most extreme position in front of the blade, by rotating the Y-axial pinion head, and raise it to the level of the blade by rotating the coarse focusing knob. Fill the bath with enough PBS to cover the tissue specimen. Switch the circuit drive on and manually advance the tissue toward the vibrating blade and cut a section. Conclude the process by moving the tissue to the most extreme position in front of the blade. Remove the section from the bath with a paint brush and mount it on a subbed Ž2%
4.2. Perfusion and tissue preparation Anaesthetize animals with an overdose of sodium pentobarbitol, canulate the heart and transcardially perfuse the vasculature with 48C phosphate buffered saline ŽpH 7.4; PBS. until the blood is cleared. Replace the PBS with a 48C buffered fixative solution containing 4% paraformaldehyde and continue perfusing the vasculature at a slow rate Žapproximately 35 mlrmin.. until the musculature is firm Žapproximately 5 min.. Remove a 3 cm length of spinal cord Žfirst cervical ŽC1. to second thoracic ŽT2. vertebrae. and postfix it for at least 12 h in the perfusion fixative. Embed the spinal cord in a large cylinder of agar Ž7% in PBS. by filling a 30 ml traditional shot glass Žwith tapered walls. with warm liquid agar, and suspending the spinal cord vertically in the center of the glass, then place the glass in crushed ice to rapidly cure the agar. Once the agar is set, remove it from the mold and trim it on each end to the approximate length of the spinal cord. 4.3. Sectioning and reconstruction Glue Žcyanoacrylate. the larger end of the agar cylinder to the plate in the center of the tissue bath. Move the stage of the microtome to its lowest vertical position by rotating the coarse focusing knob, and secure the bath in position. Rotate the cutting head vertically and insert a razor blade into the blade holder; fasten the blade in place by tightening the 2 stainless steel screws. Place the jig in the tissue bath, with the right angle against the back wall. Rotate the
Fig. 4. Ža. Low magnification photograph of a series of spinal cord sections cut with the vibrating blade microtome. 138 sections Ž200 mm. were cut and mounted in sequence Župper left to lower right. on three slides Žtop to bottom.. Žb. Schematic drawings of a rat spinal cord in horizontal and coronal planes. A column of motor neuron cell bodies Žsmall dots. in the spinal cord that innervate the left spinodeltoideus muscle is illustrated. Labeled cells were found in the ventrolateral horn of C2–C6. Coronal sections are shown on the right to illustrate the relative position of the labeled cells in the ventral horn of the spinal cord at different levels. Scale bar s1 mm.
G.T. Prusky, J.E. McKennar Brain Research Protocols 3 (1999) 302–307
gelatin. slide. Then raise the stage 200 mm vertically, by rotating the fine focusing knob Ž100 mmrrotation., and cut another section using the same procedures as described above. After all sectioning is completed, switch the circuit drive off, move the stage of the microtome to its lowest position, and drain and clean the tissue bath. Coverslip the slides Ž1:1 8% PFA, glycerol., then view and digitize the sections under rhodamine optics w9x.
5. Results The vibrating blade microtome we made was capable of producing a sequential series of 200 mm transverse sections through almost 3 cm of spinal cord ŽFig. 4a.. The sections were consistent in thickness and quality, and no tissue was lost as a result of blocking. Drawings of DiI labeled motor neurons made from digitized images allowed us to reconstruct a complete, discrete longitudinal column in the lateral ventral horn ŽFig. 4b. that extended through 5 adjacent spinal segments and was spatially restricted to a 0.25–0.5 mm diameter locus in the transverse plane. Control animals showed no cell-specific labelling Ždata not shown..
6. Discussion Tissue sectioning is central to many anatomical and physiological techniques used in biological research. Freezing or embedding tissue before sectioning is useful, especially when slices as thin as 1 mm are desired; however, these treatments can create artifacts, alter morphology, destroy enzyme activity and produce other deleterious effects. In addition, these methods are incompatible with the preparation of tissue for in vitro experiments. An alternative tissue slicing method Žpatented under the name Vibratome w ŽTechnical Products International, USA.., enabled the sectioning of fresh, living and unfrozen tissue by moving a vibrating blade through a specimen below the surface of a liquid bath. Although this method does not normally allow for sections as thin as 1 mm to be cut, thicker sections of soft tissue from 30–500 mm can be produced reliably. Most importantly, this method preserves ultrastructure, which is essential for experiments utilizing electron microscopy w3x, immunohistochemical w4x, cell tracing w5x, and living brain slice techniques w1x. Several companies other than TPI, including Energy Beam Sciences Žthe ‘MicroCut’., Dosaka Žthe ‘MicroSlicer’., Camden Instruments Žthe ‘Vibroslice’., and Leica Ž‘VT 1000’., manufacture excellent vibrating blade microtomes. In general, these instruments are designed to cut sections from relatively small blocks of tissue and are not compatible with procedures that require generating serial sections from a piece of tissue more than 1 cm in height. We had such a requirement when we set out to serially
305
section motor neuron columns in the rat spinal cord, retrogradely labeled from individual muscles in the forelimb w7x. These columns of cells are located between C1 and T2; however, their precise location can vary in relation to vertebral landmarks in individual animals w7,8x. Because the vertical travel of the standard Vibratome is limited to 0.9 cm, a 2–3 cm length of spinal cord had to be blocked into at least 3 segments and individually mounted and cut, to produce a set of consecutive spinal cord sections. No matter how precise these blocking procedures were, 200– 300 mm of tissue, which often contained labeled cells, was invariably lost at the cut surfaces. In order to overcome this problem, we required a vibrating blade microtome with more vertical travel. Since modifications of our existing Vibratome was not mechanically or economically feasible, we decided to design and build our own instrument. The basic mechanical components of a vibrating blade microtome are: Ž1. A stationary vibrating blade; Ž2. A mechanism to move tissue horizontally toward and away from the vibrating blade in a controlled fashion; and Ž3. Micrometric control of tissue movement vertically. While searching for the equipment to fulfill these requirements, we reasoned that an upright compound microscope stand had two of these three components, Ži.e. an X–Y specimen stage and a coaxial focusing device.. This paper describes an inexpensive modification of an upright microscope to produce a vibrating blade microtome. This microtome provides precise control of horizontal and vertical specimen movements and is capable of producing a sequential series of uniformly thick sections through up to 3 cm of nervous system tissue. 6.1. Troubleshooting There are a number of parameters under the control of the operator that contribute to the quality of sections generated by the vibrating blade microtome described in this paper. First, steps must be taken to ensure stability of the tissue before sectioning. The spinal cord that we sectioned and illustrated in this paper was about 3 cm in length; however, it was only 0.25–0.4 cm in diameter. Therefore, it was necessary to encase this long, slender piece of tissue in a much larger cylinder of agar to provide sufficient support while sectioning. This meant that a sizable slice of agar surrounded each section of spinal cord and had to be removed from the tissue before the section was mounted on a slide. As the height of the block was reduced with sectioning, less support for the tissue was necessary and agar was trimmed from the edge of the column to reduce the amount of agar that surrounded each section. In general, whenever tissue of atypical shape or consistency is to be cut with this instrument, it must be adequately supported to produce consistent sections. Second, the characteristics of the blade must be matched to the physical properties of the tissue to be sectioned. Cosio w2x recommends that an angle of 158–208 is appro-
306
G.T. Prusky, J.E. McKennar Brain Research Protocols 3 (1999) 302–307
priate for cutting fixed tissue. In our laboratory, however, we have found that an angle of 258 or more is best to produce high quality sections of neural tissue. We built a series of jigs to reliably reproduce angles of 158, 208 and 258 on our microtome, but jigs corresponding to other angles could also be easily produced. Unfixed or living tissue may section better if a 108–158 blade angle is used, but in any case, the specific cutting angle should be determined empirically. We prefer to use double edged razor blades ŽTatra. broken in half lengthwise, rather than single edged blades ŽShick., since their added thickness creates more drag through the tissue. Third, the amplitude of blade vibration must be matched to the physical properties of the tissue to be sectioned. Although the amplitude of the blade is continually adjustable on our instrument, in practice we almost always slice fixed brain tissue at high amplitude. Softer tissue may break up from over-agitation, so the amplitude of the blade should be reduced in this circumstance. Overall, we have found that blade sharpness contributes more to section quality than vibration amplitude. Fourth, the advance speed of the tissue should be altered during a cutting stroke. In most cases, the tissue can be moved quickly toward the blade and then slowed while the blade is cutting a section. We have found that well fixed, firm tissue, such as the spinal cord used in our study, can be cut at relatively high speeds; however, a reduced cutting speed will likely improve the quality of sections in softer tissue w2x. There is also an interaction between vibration amplitude and cutting speed. Rigid specimens may be sectioned with a low amplitude-to-advance speed ratio, while soft specimens are often best sectioned with a high amplitude-to-advance speed ratio. In addition, when specimen distortion occurs in the direction of advance, cutting speed should be reduced andror the amplitude setting increased. Finally, commercial vibrating blade microtomes often provide semi-automatic control over the speed of the cutting stroke; however, the manual control of the advance speed in our instrument can provide more precise manipulation over the amplitude-to-advance speed ratio throughout the entire cutting stroke, to produce superior results.
75 mm. If one wanted to optimize the microtome for thinner sections, a shock absorbing interface between the vibrating blade attachment and the stand could be installed, or the blade attachment could be mounted on a separate, isolated stand. We have never cut living tissue on this instrument but we can see no reason why the cutting apparatus would not work for this purpose. When cutting living tissue, some investigators mount the tissue in a box that is surround by another box filled with ice. This allows the tissue to be cooled, without being in direct contact with the ice itself. Since our instrument has a large amount of open space on the stage, constructing and using such a cooling box should not be problematic. In addition, boxes of different shapes could be manufactured to accommodate tissue in different orientations or of dramatically different sizes. We have compared the consistency of section thickness between our instrument and a vibratome and found them to be very similar Žapproximately "1 mm.. This is not surprising given the accuracy of the coaxial focusing units on most microscopes. We have found, however, that our instrument is easier to clean than a vibratome, because the cutting box can be quickly removed from the stage and drained, and the glue holding the specimen can be quickly scraped-off of the metal plate with a razor blade. The instrument described in this paper has been remarkably free of mechanical or electrical breakdown for 2 years. We periodically lubricate the coaxial gears with lithium grease, but we have found no need to provide any other servicing. We expect that the solenoid will have to be replaced at some point, as it eventually does on all vibrating blade microtomes. However, if the vertical travel of the blade assembly is dampened as described above, the solenoid should work reliably for a long period of time. The reconstruction of motor neuron columns in spinal cord is not the only application for the instrument described in this paper. The size of many biological structures exceeds the vertical travel of commercially available vibrating blade microtomes. The accurate reconstruction of these structures would benefit from using this simple instrument.
6.2. AlternatiÕe and support protocols
7. Quick procedure
The vibrating blade microtome described in this paper was designed primarily to provide more vertical cutting capability than existing instruments; it was not optimized to cut exceptionally thin sections. On our instrument, the vibrating blade attachment is not physically isolated from the stand, so some horizontal vibrations are translated through the stand and tissue bath to the tissue. Consequently, there are undulations Ž- 1 mm. on the cut surface of the sections. This has not noticeably reduced the quality of data in our labeled spinal cord sections, but it may reduce the quality of information in sections thinner than
ŽA. Inject a muscle with DiI and allow sufficient time for it to retrogradely transport to motor neuron somata in the spinal cord. ŽB. Perfuse animals with a fixative and remove spinal cord. ŽC. Section, mount and reconstruct labeled tissue.
8. Essential literature references References w1,2,6,8,9x.
G.T. Prusky, J.E. McKennar Brain Research Protocols 3 (1999) 302–307
Acknowledgements w4x
The authors would like to thank Greg Tompkins, Frank Klassen, and Tatiana Arjannikova for their technical assistance on this project. This work was supported by a Natural Sciences and Engineering Research Council of Canada research grant to G.P.
w5x
w6x
w7x
References w8x w1x P.G. Aitken, G.R. Breese, F. Edwards, M.T. Espanol, P.M. Larkman, P. Lipton, G.C. Newman, T.S. Nowak Jr., K.L. Panizzon et al., Preparative methods for brain slices: a discussion, J. Neurosci. Meth. 59 Ž1995. 139–149. w2x L. Cosio, Modifications to the Vibratome w , J. Histotechnology 4 Ž1981. 166–168. w3x H.D. Dellmann, R.L. Denadel, C.D. Jacobson, Preservation of fine
w9x
w10x
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structure in vibratome-cut sections of the central nervous system stained for light microscopy, Stain Technol. 58 Ž1983. 319–323. A. Herzog, C. Brosamle, ‘Semifree-floating’ treatment: a simple and fast method to process consecutive sections for immunohistochemistry and neuronal tracing, J. Neurosci. Meth. 72 Ž1997. 57–63. S.J. Hill, D.L. Oliver, Visualization of neurons filled with biotinylated-lucifer yellow following identification of efferent connectivity with retrograde transport, J. Neurosci. Meth. 46 Ž1993. 59–68. M.C. Honig, R.I. Hume, Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures, J. Cell Biol. 103 Ž1986. 171–187. J.E. McKenna, G.T. Prusky, I.Q. Whishaw, Spinal motoneuron columns related to forelimb function in the rat, Soc. Neurosci. Abstr. 23 Ž1997. 763. S. Nicolopoulos-Stornos, J.F. Iles, Motor neuron columns in the lumbar spinal cord of the rat, J. Comp. Neurol. 217 Ž1983. 75–85. D.K. Simon, D.D.M. O’Leary, Development of topographic order in the mammalian retinocollicular projection, J. Neurosci. 12 Ž1992. 1212–1232. D. Yu, F. Gordon, A simple method to improve the reliability of iontophoretic administration of tracer substances, J. Neurosci. Meth. 52 Ž1994. 161–164.
Protocol
Serial sectioning of thick tissue with a novel vibrating blade microtome Glen T. Prusky
)
, John E. McKenna
Department of Psychology and Neuroscience, The UniÕersity of Lethbridge, 4401 UniÕersity DriÕe, Lethbridge, Alberta, Canada T1K 3M4 Accepted 30 September 1998
Abstract Vibrating blade microtomes are used extensively in biological research to section non-frozen tissue. There are a wide variety of commercial instruments available for this purpose, however, they are designed to cut thin sections primarily from a tissue block less than one centimeter in height. Herein is described a simple modification of a microscope frame that creates a vibrating blade microtome capable of producing a sequential series of sections through three centimeters of tissue. We illustrate the use of this device to identify and reconstruct a column of rat spinal motor neurons retrogradely labeled from a peripheral muscle. q 1999 Elsevier Science B.V. All rights reserved.
Themes: Cellular and molecular biology Topics: Staining, tracing and imaging techniques Keywords: Vibratome; Brain slice; Tissue section; Microtome; Vibrating blade; Microscope; Spinal cord; Motor neurons
1. Type of research
3. Materials
Ø Ø Ø Ø Ø Ø
3.1. Vibrating blade microtome
Neuroanatomy Structurerfunction studies Cell differentiation Development and plasticity Neural patterning Reconstruction
2. Time required The time required to setup the machine, mount the specimen, cut 200 mm sections through ; 3 cm of tissue and clean up, is approximately 1.5 h. This does not include the time required to design and construct the vibrating blade microtome.
) Corresponding [email protected]
author.
Fax:
q 1-403-329-2555;
E-mail:
An early 1970s vintage Carl Zeiss Jena Amplival compound microscope was used as a stand for the vibrating blade microtome. This microscope was mechanically sound, however, it had outlived its use as an optical device. The illuminator, condenser, nosepiece, tube carrier and binocular tube assembly were all removed from the microscope and the X-axial stage control was disabled by removing its hand-operated pinion head. This left the microscope stand with a dovetail mount on the top and a stage with 5 cm of horizontal movement in the Y-axis, controlled by rotating the associated pinion head, and 3 cm of vertical movement in the Z-axis, controlled by rotating either the coarse or fine knob on the coaxial focusing assembly. A rectangular tissue bath Ž9.5 cm = 7.5 cm = 4 cm. is made of 3 mm Plexiglas and a square plate of stainless steel Ž4.5 cm = 4.5 cm = 0.1 cm. is glued to the bottom of the bath to create a durable surface for mounting tissue. The bath is mounted on the microscope stage by sliding it
1385-299Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 5 - 2 9 9 X Ž 9 8 . 0 0 0 5 2 - X
G.T. Prusky, J.E. McKennar Brain Research Protocols 3 (1999) 302–307
into guides formed by two grooved rails bolted to the stage. A thumbscrew on each rail can be tightened to secure the bath in place. Fig. 1 illustrates the assembled vibrating blade microtome. No physical modifications are made to the microscope itself, so it can easily be reassembled if desired. A simple jig to accurately set the blade angle is fabricated from 7 mm Plexiglas; it consists of a regular triangular solid with a 258 acute angle Ž53 mm base =20 mm height.. A vibrating blade assembly is manufactured and mounted on the microscope stand in order to produce a functional instrument ŽFig. 2.. This attachment, made from a rectangular piece of plate steel Ž13.2 cm = 7 cm = 1 cm. welded to a rectangular Ž8.2 cm = 6 cm = 3.1 cm. steel block, provided a platform to anchor other components, and sufficient mass to effectively dampen unwanted vibrations. Two free-standing steel arms were constructed Ž12.9 cm = 3.2 cm = 0.16 cm. to carry the blade cutting head and travel rail. One arm was bolted to each side of the block and extended forward. The front ends of the arms were connected by a steel rail Ž8.75 cm = 0.75 cm dia. on which a stainless steel blade holder Ž6.5 cm = 3 cm = 1 cm. was positioned. The blade holder could rotate around the rail to allow for angle adjustments and was held in place with a thumbscrew. A razor blade was secured in place at a 1058 angle at the end of the holder by a small rectangular Ž1.4 cm = 3 cm = 0.15 cm. stainless steel plate anchored with stainless steel screws. A solenoid Ž11-C120A; Guardian. was placed between the front of the steel block and the rail connecting the arms, and was secured to the bottom of the block with a steel plate. The solenoid’s plug was fixed to one of the two steel arms 3.5 cm behind the rail and 7.2 cm in front of the screws that anchored the arms to the steel block. The completed vibrating blade attachment was installed on the dovetail mount Žon top of
Fig. 1. Assembled vibrating blade microtome, including: ŽA. microscope stand, ŽB. vibrating blade assembly, ŽC. drive control, ŽD. tissue bath, ŽE. Y-axial control, ŽF. Z-axial control.
303
Fig. 2. Schematic drawing of the vibrating blade assembly, in plane Žtop. and side Žbottom. views. Significant components are: ŽA. steel arms, ŽB. blade holder, ŽC. travel rail, ŽD. solenoid, ŽE. steel mounting block, ŽF. locking cam, ŽG. power coupling, and ŽH. angle-setting jig. Scale bar s10 cm.
the stand. with the cam locking screw that originally secured the tube carrier. An AC drive circuit Ž120 volt. for the instrument was located in a separate box and consisted of a variable output transformer, the output of which was current limited and half-wave rectified before being applied to the AC solenoid ŽFig. 3.. When current was applied to the solenoid, it vibrated the blade assembly in an arcuate path, the amplitude of oscillation being proportional to the drive voltage Žneglecting losses.. Since the drive frequency was constant Ž60 Hz., it was necessary to tune the resonant frequency of the vibrating assembly to maintain the efficiency of the solenoid. The resonant frequency is equal to the square root of Ž KrM ., where K equals the spring constant of the arms and M equals the mass of the assembly. We found the resonant frequency of the vibrating mass empirically by attaching a vibration sensor to the mass and exciting it, and then watching the output on an oscilloscope. Adjustments to the length, thickness and material type of the arms, as well as the mass of the assembly were made to alter its resonant frequency and produce a lateral displacement of about 1 mm.
Fig. 3. Schematic drawing of the electrical drive circuitry.
304
G.T. Prusky, J.E. McKennar Brain Research Protocols 3 (1999) 302–307
3.2. Maintenance Lithium grease.
4. Detailed procedure 4.1. Motor neuron labeling A detailed procedure for labeling motor neurons has been presented previously w7x. Briefly, vacuum-load a 10% solution Žin dimethylformamide. of DiI w6x into a siliconcoated w10x glass micropipet pulled to a long taper with the tip broken to a diameter of about 50 mm. Anaesthetize the animals Ž65 mgrkg sodium pentobarbitol. and make a surgical incision over the spinodeltoideus forelimb muscle. Isolate the muscle and inject it with approximately 0.5 ml of the dye solution. In animals used as controls, instead of injecting into the muscle, approximately 2 ml of DiI solution was ejected onto the surface of the spinodeltoideus muscle, and then wiped away with a cotton swab a few minutes later. Recover the animals from surgery and allow one week of survival for axonal terminals contacting this muscle to absorb the dye and transport it retrogradely to the cell bodies of origin in the spinal cord.
cutting head ventrally until the lower surface of the blade rests against the 258 angle of the jig. Steady the blade holder against the jig with one hand and tighten the thumb screw of the cutter head with the other hand. The blade is now at a 258 angle to the plane of the stage. Remove the jig by sliding it laterally. Move the tissue to the most extreme position in front of the blade, by rotating the Y-axial pinion head, and raise it to the level of the blade by rotating the coarse focusing knob. Fill the bath with enough PBS to cover the tissue specimen. Switch the circuit drive on and manually advance the tissue toward the vibrating blade and cut a section. Conclude the process by moving the tissue to the most extreme position in front of the blade. Remove the section from the bath with a paint brush and mount it on a subbed Ž2%
4.2. Perfusion and tissue preparation Anaesthetize animals with an overdose of sodium pentobarbitol, canulate the heart and transcardially perfuse the vasculature with 48C phosphate buffered saline ŽpH 7.4; PBS. until the blood is cleared. Replace the PBS with a 48C buffered fixative solution containing 4% paraformaldehyde and continue perfusing the vasculature at a slow rate Žapproximately 35 mlrmin.. until the musculature is firm Žapproximately 5 min.. Remove a 3 cm length of spinal cord Žfirst cervical ŽC1. to second thoracic ŽT2. vertebrae. and postfix it for at least 12 h in the perfusion fixative. Embed the spinal cord in a large cylinder of agar Ž7% in PBS. by filling a 30 ml traditional shot glass Žwith tapered walls. with warm liquid agar, and suspending the spinal cord vertically in the center of the glass, then place the glass in crushed ice to rapidly cure the agar. Once the agar is set, remove it from the mold and trim it on each end to the approximate length of the spinal cord. 4.3. Sectioning and reconstruction Glue Žcyanoacrylate. the larger end of the agar cylinder to the plate in the center of the tissue bath. Move the stage of the microtome to its lowest vertical position by rotating the coarse focusing knob, and secure the bath in position. Rotate the cutting head vertically and insert a razor blade into the blade holder; fasten the blade in place by tightening the 2 stainless steel screws. Place the jig in the tissue bath, with the right angle against the back wall. Rotate the
Fig. 4. Ža. Low magnification photograph of a series of spinal cord sections cut with the vibrating blade microtome. 138 sections Ž200 mm. were cut and mounted in sequence Župper left to lower right. on three slides Žtop to bottom.. Žb. Schematic drawings of a rat spinal cord in horizontal and coronal planes. A column of motor neuron cell bodies Žsmall dots. in the spinal cord that innervate the left spinodeltoideus muscle is illustrated. Labeled cells were found in the ventrolateral horn of C2–C6. Coronal sections are shown on the right to illustrate the relative position of the labeled cells in the ventral horn of the spinal cord at different levels. Scale bar s1 mm.
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gelatin. slide. Then raise the stage 200 mm vertically, by rotating the fine focusing knob Ž100 mmrrotation., and cut another section using the same procedures as described above. After all sectioning is completed, switch the circuit drive off, move the stage of the microtome to its lowest position, and drain and clean the tissue bath. Coverslip the slides Ž1:1 8% PFA, glycerol., then view and digitize the sections under rhodamine optics w9x.
5. Results The vibrating blade microtome we made was capable of producing a sequential series of 200 mm transverse sections through almost 3 cm of spinal cord ŽFig. 4a.. The sections were consistent in thickness and quality, and no tissue was lost as a result of blocking. Drawings of DiI labeled motor neurons made from digitized images allowed us to reconstruct a complete, discrete longitudinal column in the lateral ventral horn ŽFig. 4b. that extended through 5 adjacent spinal segments and was spatially restricted to a 0.25–0.5 mm diameter locus in the transverse plane. Control animals showed no cell-specific labelling Ždata not shown..
6. Discussion Tissue sectioning is central to many anatomical and physiological techniques used in biological research. Freezing or embedding tissue before sectioning is useful, especially when slices as thin as 1 mm are desired; however, these treatments can create artifacts, alter morphology, destroy enzyme activity and produce other deleterious effects. In addition, these methods are incompatible with the preparation of tissue for in vitro experiments. An alternative tissue slicing method Žpatented under the name Vibratome w ŽTechnical Products International, USA.., enabled the sectioning of fresh, living and unfrozen tissue by moving a vibrating blade through a specimen below the surface of a liquid bath. Although this method does not normally allow for sections as thin as 1 mm to be cut, thicker sections of soft tissue from 30–500 mm can be produced reliably. Most importantly, this method preserves ultrastructure, which is essential for experiments utilizing electron microscopy w3x, immunohistochemical w4x, cell tracing w5x, and living brain slice techniques w1x. Several companies other than TPI, including Energy Beam Sciences Žthe ‘MicroCut’., Dosaka Žthe ‘MicroSlicer’., Camden Instruments Žthe ‘Vibroslice’., and Leica Ž‘VT 1000’., manufacture excellent vibrating blade microtomes. In general, these instruments are designed to cut sections from relatively small blocks of tissue and are not compatible with procedures that require generating serial sections from a piece of tissue more than 1 cm in height. We had such a requirement when we set out to serially
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section motor neuron columns in the rat spinal cord, retrogradely labeled from individual muscles in the forelimb w7x. These columns of cells are located between C1 and T2; however, their precise location can vary in relation to vertebral landmarks in individual animals w7,8x. Because the vertical travel of the standard Vibratome is limited to 0.9 cm, a 2–3 cm length of spinal cord had to be blocked into at least 3 segments and individually mounted and cut, to produce a set of consecutive spinal cord sections. No matter how precise these blocking procedures were, 200– 300 mm of tissue, which often contained labeled cells, was invariably lost at the cut surfaces. In order to overcome this problem, we required a vibrating blade microtome with more vertical travel. Since modifications of our existing Vibratome was not mechanically or economically feasible, we decided to design and build our own instrument. The basic mechanical components of a vibrating blade microtome are: Ž1. A stationary vibrating blade; Ž2. A mechanism to move tissue horizontally toward and away from the vibrating blade in a controlled fashion; and Ž3. Micrometric control of tissue movement vertically. While searching for the equipment to fulfill these requirements, we reasoned that an upright compound microscope stand had two of these three components, Ži.e. an X–Y specimen stage and a coaxial focusing device.. This paper describes an inexpensive modification of an upright microscope to produce a vibrating blade microtome. This microtome provides precise control of horizontal and vertical specimen movements and is capable of producing a sequential series of uniformly thick sections through up to 3 cm of nervous system tissue. 6.1. Troubleshooting There are a number of parameters under the control of the operator that contribute to the quality of sections generated by the vibrating blade microtome described in this paper. First, steps must be taken to ensure stability of the tissue before sectioning. The spinal cord that we sectioned and illustrated in this paper was about 3 cm in length; however, it was only 0.25–0.4 cm in diameter. Therefore, it was necessary to encase this long, slender piece of tissue in a much larger cylinder of agar to provide sufficient support while sectioning. This meant that a sizable slice of agar surrounded each section of spinal cord and had to be removed from the tissue before the section was mounted on a slide. As the height of the block was reduced with sectioning, less support for the tissue was necessary and agar was trimmed from the edge of the column to reduce the amount of agar that surrounded each section. In general, whenever tissue of atypical shape or consistency is to be cut with this instrument, it must be adequately supported to produce consistent sections. Second, the characteristics of the blade must be matched to the physical properties of the tissue to be sectioned. Cosio w2x recommends that an angle of 158–208 is appro-
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priate for cutting fixed tissue. In our laboratory, however, we have found that an angle of 258 or more is best to produce high quality sections of neural tissue. We built a series of jigs to reliably reproduce angles of 158, 208 and 258 on our microtome, but jigs corresponding to other angles could also be easily produced. Unfixed or living tissue may section better if a 108–158 blade angle is used, but in any case, the specific cutting angle should be determined empirically. We prefer to use double edged razor blades ŽTatra. broken in half lengthwise, rather than single edged blades ŽShick., since their added thickness creates more drag through the tissue. Third, the amplitude of blade vibration must be matched to the physical properties of the tissue to be sectioned. Although the amplitude of the blade is continually adjustable on our instrument, in practice we almost always slice fixed brain tissue at high amplitude. Softer tissue may break up from over-agitation, so the amplitude of the blade should be reduced in this circumstance. Overall, we have found that blade sharpness contributes more to section quality than vibration amplitude. Fourth, the advance speed of the tissue should be altered during a cutting stroke. In most cases, the tissue can be moved quickly toward the blade and then slowed while the blade is cutting a section. We have found that well fixed, firm tissue, such as the spinal cord used in our study, can be cut at relatively high speeds; however, a reduced cutting speed will likely improve the quality of sections in softer tissue w2x. There is also an interaction between vibration amplitude and cutting speed. Rigid specimens may be sectioned with a low amplitude-to-advance speed ratio, while soft specimens are often best sectioned with a high amplitude-to-advance speed ratio. In addition, when specimen distortion occurs in the direction of advance, cutting speed should be reduced andror the amplitude setting increased. Finally, commercial vibrating blade microtomes often provide semi-automatic control over the speed of the cutting stroke; however, the manual control of the advance speed in our instrument can provide more precise manipulation over the amplitude-to-advance speed ratio throughout the entire cutting stroke, to produce superior results.
75 mm. If one wanted to optimize the microtome for thinner sections, a shock absorbing interface between the vibrating blade attachment and the stand could be installed, or the blade attachment could be mounted on a separate, isolated stand. We have never cut living tissue on this instrument but we can see no reason why the cutting apparatus would not work for this purpose. When cutting living tissue, some investigators mount the tissue in a box that is surround by another box filled with ice. This allows the tissue to be cooled, without being in direct contact with the ice itself. Since our instrument has a large amount of open space on the stage, constructing and using such a cooling box should not be problematic. In addition, boxes of different shapes could be manufactured to accommodate tissue in different orientations or of dramatically different sizes. We have compared the consistency of section thickness between our instrument and a vibratome and found them to be very similar Žapproximately "1 mm.. This is not surprising given the accuracy of the coaxial focusing units on most microscopes. We have found, however, that our instrument is easier to clean than a vibratome, because the cutting box can be quickly removed from the stage and drained, and the glue holding the specimen can be quickly scraped-off of the metal plate with a razor blade. The instrument described in this paper has been remarkably free of mechanical or electrical breakdown for 2 years. We periodically lubricate the coaxial gears with lithium grease, but we have found no need to provide any other servicing. We expect that the solenoid will have to be replaced at some point, as it eventually does on all vibrating blade microtomes. However, if the vertical travel of the blade assembly is dampened as described above, the solenoid should work reliably for a long period of time. The reconstruction of motor neuron columns in spinal cord is not the only application for the instrument described in this paper. The size of many biological structures exceeds the vertical travel of commercially available vibrating blade microtomes. The accurate reconstruction of these structures would benefit from using this simple instrument.
6.2. AlternatiÕe and support protocols
7. Quick procedure
The vibrating blade microtome described in this paper was designed primarily to provide more vertical cutting capability than existing instruments; it was not optimized to cut exceptionally thin sections. On our instrument, the vibrating blade attachment is not physically isolated from the stand, so some horizontal vibrations are translated through the stand and tissue bath to the tissue. Consequently, there are undulations Ž- 1 mm. on the cut surface of the sections. This has not noticeably reduced the quality of data in our labeled spinal cord sections, but it may reduce the quality of information in sections thinner than
ŽA. Inject a muscle with DiI and allow sufficient time for it to retrogradely transport to motor neuron somata in the spinal cord. ŽB. Perfuse animals with a fixative and remove spinal cord. ŽC. Section, mount and reconstruct labeled tissue.
8. Essential literature references References w1,2,6,8,9x.
G.T. Prusky, J.E. McKennar Brain Research Protocols 3 (1999) 302–307
Acknowledgements w4x
The authors would like to thank Greg Tompkins, Frank Klassen, and Tatiana Arjannikova for their technical assistance on this project. This work was supported by a Natural Sciences and Engineering Research Council of Canada research grant to G.P.
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w7x
References w8x w1x P.G. Aitken, G.R. Breese, F. Edwards, M.T. Espanol, P.M. Larkman, P. Lipton, G.C. Newman, T.S. Nowak Jr., K.L. Panizzon et al., Preparative methods for brain slices: a discussion, J. Neurosci. Meth. 59 Ž1995. 139–149. w2x L. Cosio, Modifications to the Vibratome w , J. Histotechnology 4 Ž1981. 166–168. w3x H.D. Dellmann, R.L. Denadel, C.D. Jacobson, Preservation of fine
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structure in vibratome-cut sections of the central nervous system stained for light microscopy, Stain Technol. 58 Ž1983. 319–323. A. Herzog, C. Brosamle, ‘Semifree-floating’ treatment: a simple and fast method to process consecutive sections for immunohistochemistry and neuronal tracing, J. Neurosci. Meth. 72 Ž1997. 57–63. S.J. Hill, D.L. Oliver, Visualization of neurons filled with biotinylated-lucifer yellow following identification of efferent connectivity with retrograde transport, J. Neurosci. Meth. 46 Ž1993. 59–68. M.C. Honig, R.I. Hume, Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures, J. Cell Biol. 103 Ž1986. 171–187. J.E. McKenna, G.T. Prusky, I.Q. Whishaw, Spinal motoneuron columns related to forelimb function in the rat, Soc. Neurosci. Abstr. 23 Ž1997. 763. S. Nicolopoulos-Stornos, J.F. Iles, Motor neuron columns in the lumbar spinal cord of the rat, J. Comp. Neurol. 217 Ž1983. 75–85. D.K. Simon, D.D.M. O’Leary, Development of topographic order in the mammalian retinocollicular projection, J. Neurosci. 12 Ž1992. 1212–1232. D. Yu, F. Gordon, A simple method to improve the reliability of iontophoretic administration of tracer substances, J. Neurosci. Meth. 52 Ž1994. 161–164.