A Motor-Controlled Device for Slow-Motion Cutting with the Ultramicrotome

The preparation of ultrathin sections of high quality with a Porter-Blum microtome requires that the drive wheel be rotated at a slow, uniform rate during actual cutting. A mechanical device to control the motion of the microtome drive wheel consists of a synchronous motor connected to a drive shaft (4 rev/min), which can be intermittently coupled to the rim of the microtome wheel by a friction drive of 1/2-inch diameter. The microtome drive wheel is rotated manually at a rapid rate until the specimen is about to come into contact with the knife; then the mechanical drive is engaged for slow movement during cutting. When the section has been cut, the drive is disengaged by a lever operated with the left hand; after which, the microtome wheel is turned quickly back to the cutting position with the right hand.     

A Microtome Drive That Gives Either A Fast Cutting Stroke Or A Slow Stroke with Fast Return

The construction comprises a 0.3 hp electric motor, a drive mechanism which transforms the speed of the motor to the two actual cutting speeds, and a clutch mechanism which transmits the slow or the fast movement to the microtome. When the slow cutting speed is used the cam (on the microtome flywheel) and lever mechanism actuates the drive to give a quick return to the cutting position after a slow cutting act. This device, which needs little supervision, has given excellent serial sections, uniform both individually and with respect to others in the ribbon, with all the material on which it has been tested.     

An electrically controlled drive with a slow-cutting, fast-return cycle for rotary microtomes

A microtome drive, which gives a low cutting speed for the specimen and a fast return to the cutting position, was constructed. To this end, a low speed and a high speed shaft, from an electric motor with a reducing gear, are alternately coupled to the final drive shaft. This coupling is obtained by means of a magnetic clutch and a specially designed pawl clutch. Alternation of low and high speed of the drive shaft depends on the action of the magnetic clutch which is governed by a specimen position contact through a relay system. The specimen position contact is actuated by the microtome itself, which leads to its fully automatic operation. The combination of a magnetic and a pawl clutch in the drive results in a vibration free two-speed cycle of the microtome.     

Microtome Knife Sharpeners Operating on the Abrasive-ground Glass Principle

Developments in the abrasive-ground glass type of microtome knife sharpener have been traced from 1857 to the present. Merits and disadvantages of each apparatus are critically discussed, particularly of the two practical power sharpeners, namely, the horizontal revolving disc and the vertical wheel. A new vertical wheel model, designed to facilitate the sharpening of long, sliding microtome knives, either with a chisel-shaped or a symmetrical bevel, is described. Notable mechanical features include: shaft rotation V-slots as sole adjustable part, thereby assuring accuracy of alignment; steel guide bar 45.7 cm. long; glass wheel mounted on its own axis, separate from motor; simple and sturdy construction with single casting constituting entire framework.     

Cryostat Techniques: Methods for Improving Conservation and Sectioning of Tissue

Tissues have been conserved for satisfactory enzymatic histochemical assay for up to 12 mo by low temperature storage (in a dry ice chest or at -40° C) and by measures designed to offset the deleterious effects of sublimation of H₂O by using the following modifications of standard procedures (which include steps to give a mechanical support to otherwise fragmenting sections): 1. tissue blocks are coated with a polystyrene solution between each storage period; 2. modified bolts are used as tissue holders and types of bolt holders have been designed to fit on standard microtomes which permit manipulation of each tissue block independently of its mates on the same bolt holder, or the simultaneous cutting of all blocks on any one holder with each advance of the microtome feed; 3. tissues are coated with 20% polystyrene in methylene chloride prior to cutting and rubber cement painted on the slide as an adhesive. Lillie's 20% polystyrene diethylbenzene is used as a mounting medium. Other details of practical importance include the technique of freezing, control of moisture within a cryostat and on the microtome, and tests on histochemical procedures.     

A Thermoelectrically Cooled Microtome Table and Knife

Thermoelectric cooling units (Frigistor thermoelements) have been used to replace CO₂ gas and solid CO₂ for microtome stage and knife cooling. These units consist of assemblies of series-connected thermoelements, functioning by the Peltier effect. Cooling is controlled by the direct current supply to the units. Current supply is from a double power pack giving 15 amp at 4.8 v for the knife cooling units, and 15 amp at 1 v for the stage. By varying the current flow, the optimum cutting temperature can be obtained and held indefinitely. An 8-couple Frigistor unit replaces the CO₂ stage of a Lipshaw freezing microtome. The stage temperature may be lowered to -36 C in 40-60 sec and at the optimum cutting temperature, 5 μ serial sections of fixed frozen tissue are obtainable. Four 12-couple units are used to cool a 160 mm Jung plane wedge microtome knife fitted to a Reichert sledge microtome, with the stage cooled by one 8-couple unit. The knife temperature can be lowered to -20 C in 5 min; the stage in 1 min. The apparatus has been used to cut a variety of unfixed rat and mouse tissues. The optimum sectioning temperature for such unfixed liver, kidney, spleen, lymph glands, heart, testes, small intestine, pancreas, skin and lung was -20 to -22 C.     

FROZEN THIN SECTIONS OF FRESH TISSUE FOR ELECTRON MICROSCOPY, WITH A DESCRIPTION OF PANCREAS AND LIVER

A simple method has been developed that allows frozen thin sections of fresh-frozen tissue to be cut on a virtually unmodified ultramicrotome kept at room temperature. A bowl-shaped Dewar flask with a knifeholder in its depths replaces the stage of the microtome; a bar extends down into the bowl from the microtome's cutting arm and bears the frozen tissue near its lower end. When the microtome is operated, the tissue passes a glass or diamond knife in the depths of the bowl as in normal cutting. The cutting temperature is maintained by flushing the bowl with cold nitrogen gas, and can be set anywhere from about -160°C up to about -30°C. The microtome is set for a cutting thickness of 540–1000 A. Sections are picked up from the dry knife edge, and are placed on membrane-coated grids, flattened with the polished end of a copper rod, and either dried in nitrogen gas or freeze-dried. Throughout the entire process the tissue is kept cold and does not come in contact with any solvent. The morphology seen in frozen thin sections of rat pancreas and liver generally resembles that in conventional preparations, although freezing damage and low contrast limit the detail that can be discerned. Among unusual findings is a frequent abundance of mitochondrial granules in material prepared by this method.     

Preferred elbow position in confined wheelchair configuration

High mechanical load leads to pain and damage in the upper extremities of wheelchair users. Wheelchair users suffer a limited range of motion of the upper extremities due to the confining wheelchair configuration. This is a key factor affecting the efficiency of wheelchair propulsion and upper extremity loading. With a view toward further understanding the interaction between the user and wheelchair, this study identifies the accessible workspace of the elbow under conventional wheelchair design and identifies the actual location and range of motion of the elbow during wheelchair propulsion. An eight-camera motion analysis system recorded the kinematics of 14 non-experienced wheelchair users. Users under standardized conservative wheelchair-sitting position moved their right elbow as widely as possible at five different wheel angles while elbow positions were recorded, thereby establishing the maximum possible elbow workspace. Actual positions of the right elbow were recorded during wheelchair propulsion. The arc angles of the elbow workspace range from 68.8° to 83.4° and are located at the lateral and posterior quadrant of the circle on which the elbow trajectories located. Reachable workspace is smaller when the hand holds the hand rim at a larger wheel angle. The preferred positions for propulsion are located approximately 2/3's of the way through the total workspace. The obtained data will be useful for improved wheelchair design and biomechanical modeling of the wheelchair/user system.     

An Inexpensive Trimmer for Paraffin Blocks

One of the minor difficulties in cutting serial sections with the rotary microtome is the accurate trimming of the block of paraffin so that the upper and lower edges facing the knife are parallel to each other and to the knife edge; this is necessary to ensure a straight ribbon. Several block trimmers have been described,¹ but they are all rather complicated and expensive to make. The device described below can be made in a short time at little or no expense in any laboratory.     

The core-microtome: A new tool for surface preparation on cores and time series analysis of varying cell parameters

A microtome designed for the surface preparation of entire increment cores allows cutting plane surfaces on cores up to a length of 40 cm. Compared to the common sanding procedure, the wood cells of the annual rings remain open, not filled with swarf, and the cell walls are smooth and hence clearly visible. This article aims at describing the functionality of the microtome and the procedures needed for an accurate surface preparation to achieve a good contrast for subsequent image analysis. Possible applications for a more detailed analysis of variations in the tracheid structure of conifers and vessel sizes of oak are presented, which can be included in time series analyses.     

Effects of freezing and thawing on mammalian oocyte

In an attempt to study the deleterious effects which occur during the freezing and thawing of mammalian oocytes, we developed a cryomicroscope controlled by digital programmable equipment. The program permits any cooling rate between 0.1 and 60 degrees C/min with a precision of 0.6 degrees C. Using a precooled stage, it is possible to obtain rapid cooling (100 degrees C/min). The maximum thawing rate is about 60 degrees C/min. A copper-- constantan microthermocouple allows precise measurement of the specimen temperature. All information (specimen, temperature of the specimen, date, hour, and minutes) is recorded at the same time on photographic film by a camera fitted with a " Recordata Back" and a motor drive which allows three frames per second. Our preliminary results show that: (1) rapid cooling yields a supercooling with simultaneous extra- and intracellular crystallization; (2) slow cooling with seeding at -8 degrees C gives an extracellular crystallization which is achieved by -9 degrees C, followed by an extracellular recrystallization occurring at almost -8 degrees C which alters the morphology of the oocyte and the zona pellucida, without any visible intracellular crystallization; (3) during continued slow cooling the oocytes dehydrate without any intracellular freezing; and (4) during rewarming a partial rehydration of the cell occurs with a swelling of the oocytes to their original volumes after the thawing has been achieved.     

The Effect Of Temperature and Of Relative Humidity On Sectioning Of Tissues Embedded In Polyethylene Glycol Wax

Experiments carried out in a sealed room, where temperature and humidity could be controlled at will, showed that a high relative humidity in the atmosphere interferes with microtome cutting of tissues embedded in polyethylene glycol wax. The higher the atmospheric temperature the less became the degree of relative humidity which could be tolerated if successful sections were to be obtained.     

A Ralph Knife Holder Assembly for Use with the Sorvall “Porter-Blum” MT-1 Ultramicrotome

The superiority of plastic embedding for the production of high quality sections for light microscopy is well known, but the use of conventional glass knives with a cutting edge of approximately 4 mm has severely restricted the size of specimens in the past. Ralph knives provide a much longer cutting edge and adapters are available for certain models of microtomes and ultramicrotomes. A modified knife holder for use with the Sorvall “Porter Blum” MT-2 microtome was described by Gorycki and Sohm (1979); however, this is not suitable for the MT-1 model. We have therefore designed and made an adapter which enables Ralph knives to be used with this instrument. The design allows approximately 18 mm of cutting edge to be used on each knife, allowing larger specimens to be sectioned than with a conventional glass knife and reducing the frequency with which the knife needs to be changed when working with smaller blocks.     

Physiological Studies on Pea Tendrils VIII. The Relationship of Circumnutation to Contact Coiling. — With a Description of a Laboratory Intervalometer Using Integrated Digital Circuits

The average rate of rotation of circumnutating tendrils of Pisum sativum L. ev. Alaska, was 1.57 ± 0.29 mm/min. 53% of the tendrils rotated clockwise and 47% counterclockwise. Circumnutation is apparently dependent on the maintenance of sufficient turgor as it stopped when either the roots or all the shoot appendages except the terminal tendril were excised, but resumed when the aerial wounds were covered with petroleum jelly. Both circumnutation and contact coiling were similarly retarded when the plant was cut in the middle of the top inter-node, or by the use of either juvenile or senescent organs. As the tendril circumnutated rapidly during the sweeping portion of its circuit, it was capable of coiling at only about 57% of the rate of which it could coil if stimulated during the relatively slow moving turn, Conversely, when the tendril was mechanically stimulated to coil, its rate of circumnutation decreased markedly and remained retarded as long as the tendril continued to coil. On the basis of these observations, it is concluded that contact coiling does not seem to be simply a modified form of circumnutation, but the two modes of movement might be related through a negative feedback system.     

Electrical perception of "death message" in chara: analysis of rapid component and ionic process

Electrical response upon wounding was analyzed in Chara corallina. A specimen comprising two adjoining internodal cells was prepared. One cell (victim cell) was killed by cutting and any changes in the membrane potential of the neighboring cell (the receptor cell) were measured. Upon cutting the victim cell, the receptor cell generated four kinds of depolarizations: (1) rapid component, (2) slow and long-lasting component, (3) action potential and (4) small spike. Rapid and slow components were observed in most cells. On the other hand, the action potential and small spike were not always ubiquitous among specimens. When an action potential was generated just after cutting the victim cell, the rapid component could not be observed due to masking by the action potential. It was suggested that both rapid and slow components were generated at the nodal end. On the other hand, action potentials were thought to be generated at the flank of the receptor cell. High turgor pressure of the cell was necessary for generating both rapid and slow components. Experiments under K(+)-induced depolarization unequivocally showed that the Cl(-) channel at the nodal end of the receptor cell was activated upon cutting the victim cell.     

Orienting Minute Objects for Sectioning in a Definite Plane

Minute objects can be prepared for sectioning in a definite plane by a method which reembeds them directly on the cutting block under a dissecting microscope. By melting the paraffin immediately around the specimen, the latter can be oriented with reference to the planes of the block. After trimming, the block can be oriented squarely with reference to the microtome knife. Objects as small as 0.2 mm. have been cut successfully. The material sectioned included carpel primordia of Lathyrus, and young embryos, shoot apices and young axillary buds of Pinus. The technic is simpler than most methods previously suggested and it permits good control over the plane of sectioning.     

Impaired sarcoplasmic reticulum Ca(2+) release rate after fatiguing stimulation in rat skeletal muscle.

The purpose of the study was to characterize the sarcoplasmic reticulum (SR) function and contractile properties before and during recovery from fatigue in the rat extensor digitorum longus muscle. Fatiguing contractions (60 Hz, 150 ms/s for 4 min) induced a reduction of the SR Ca(2+) release rate to 66% that persisted for 1 h, followed by a gradual recovery to 87% of prefatigue release rate at 3 h recovery. Tetanic force and rate of force development (+dF/dt) and relaxation (-dF/dt) were depressed by approximately 80% after stimulation. Recovery occurred in two phases: an initial phase, in which during the first 0.5-1 h the metabolic state recovered to resting levels, and a slow phase from 1-3 h characterized by a rather slow recovery of the mechanical properties. The recovery of SR Ca(2+) release rate was closely correlated to +dF/dt during the slow phase of recovery (r(2) = 0.51; P < 0.05). Despite a slowing of the relaxation rate, we did not find any significant alterations in the SR Ca(2+) uptake function. These data demonstrate that the Ca(2+) release mechanism of SR is sensitive to repetitive in vitro muscle contraction. Moreover, the results indicate that +dF/dt to some extent depends on the rate of Ca(2+) release during the slow phase of recovery.     

Preparing Single or Serial Sections of Calcified Bones and Teeth

An apparatus for cutting single or serial sections of calcified bone and teeth consists of a motor-driven shaft on which is mounted one saw (for single section cutting) or a gang (for serial sectioning at one cutting operation). The plastic-embedded specimen is attached to a cylindrical plastic holder which is in turn mounted on the machine and fed into the saw. Prior to cutting the specimen may be oriented in two planes, as well as rotated, with respect to the cutting edge. Single or serial sections made by means of repeated cuts with a single saw, may be 0.3 mm or more thick as determined by the setting of a micrometer screw. For serially sectioning a tooth or bone specimen at one cutting operation, the thickness of the separators between adjacent saws (0.5 mm or more) determines the section thickness. After sectioning, specimens may be ground and polished, with or without reimbedding in fresh plastic.