Methods for Precisely Trimming Block Faces for Ultramicrotomy

Two devices are described to aid in trimming block faces of embedded tissue for ultramicrotomy. The first, a reticle to fit the ocular of a stereomicroscope, can be manufactured by the ultramicrotomist and is designed to outline the edges of the block face so that it can be trimmed to a standard size and shape with the area of interest centered in it. The second, a rectangular “trim-align” block mounted in the knife holder of the uitramicrotome, is, with the block face, aligned to the plane of sectioning, and aids in retrimming the top and bottom edges of the block face. This is the simplest trimming device yet described and the first which will, from any sort of embedded material, produce a block face with parallel top and bottom edges even if the block face is not perpendicular to the axis of the specimen holder. If the edge of the diamond knife used for sectioning is parallel to the axis of rotation of the knife holder, the block face has also been automatically aligned to the knife as a consequence of this aligning and trimming procedure. As a result, sectioning can begin immediately without further adjustments.     

Improved technique for electron microscopy of cultured cells.

A number of techniques are presented which precise selection and efficient preparation of individual cultured cells for electron microscopy. Techniques described include marking of the living and embedded cells, drilling and mounting cores of embedded material, and improved technique for marking of the selected area on the block face before trimming.     

An Instrument for the Controlled Trimming of Plastic Specimen Blocks for Light and Electron Microscopy

A device for the controlled trimming of plastic specimen blocks for light and electron microscopy is described. Many advantages of previously reported instruments together with 1) a rack and pinion control of knife movement, and 2) a control of rotation of the specimen block at 90°, 180°, 270°, or 360° are incorporated. In conjunction with an ocular micrometer, the device allows accurate removal of thin slices during trimming.     

Trimming Selected Areas of Embedments for Electron Microscopy—Dead Simple

Precision trimming of specific areas of plastic embedments can be accomplished without having to find the specimen detail in the block face itself. The desired area is located in a thick section of the pretrimmed block. The position of the area in the section is evaluated using an ocular micrometer. The block is then mechanically trimmed in the ultramicrotome in such a way that the amount of plastic removed from each of its sides is determined from the magnitude of the knife advance. The procedure can be used in connection with inclined blocks if the microtome head can be rotated behind the specimen orientation arc.     

A Trimming Stage for Stereomicroscopes

Scott and Thurston (1975) have described a simple, useful modification of ultratome specimen holders which permits transillumination of plastic embedded specimens for hand trimming.¹ We have refined their original concept by fabricating a Lucite chuck-mounting disc which, in addition to allowing transillumination of embedded specimens, may also be rotated 360 degrees for specimen orientation or locked in place in any desired trimming position. When used in circular stage mounts of stereomicroscopes such as the Nikon SMZ-10, the disc transforms the microscope into a highly effective trimming instrument.     

Grasping the Intentions of Others with One's Own Mirror Neuron System

Understanding the intentions of others while watching their actions is a fundamental building block of social behavior. The neural and functional mechanisms underlying this ability are still poorly understood. To investigate these mechanisms we used functional magnetic resonance imaging. Twenty-three subjects watched three kinds of stimuli: grasping hand actions without a context, context only (scenes containing objects), and grasping hand actions performed in two different contexts. In the latter condition the context suggested the intention associated with the grasping action (either drinking or cleaning). Actions embedded in contexts, compared with the other two conditions, yielded a significant signal increase in the posterior part of the inferior frontal gyrus and the adjacent sector of the ventral premotor cortex where hand actions are represented. Thus, premotor mirror neuron areas—areas active during the execution and the observation of an action—previously thought to be involved only in action recognition are actually also involved in understanding the intentions of others. To ascribe an intention is to infer a forthcoming new goal, and this is an operation that the motor system does automatically.     

Grasping the intentions of others with one's own mirror neuron system

Understanding the intentions of others while watching their actions is a fundamental building block of social behavior. The neural and functional mechanisms underlying this ability are still poorly understood. To investigate these mechanisms we used functional magnetic resonance imaging. Twenty-three subjects watched three kinds of stimuli: grasping hand actions without a context, context only (scenes containing objects), and grasping hand actions performed in two different contexts. In the latter condition the context suggested the intention associated with the grasping action (either drinking or cleaning). Actions embedded in contexts, compared with the other two conditions, yielded a significant signal increase in the posterior part of the inferior frontal gyrus and the adjacent sector of the ventral premotor cortex where hand actions are represented. Thus, premotor mirror neuron areas—areas active during the execution and the observation of an action—previously thought to be involved only in action recognition are actually also involved in understanding the intentions of others. To ascribe an intention is to infer a forthcoming new goal, and this is an operation that the motor system does automatically.     

Improved Methods for Molding, Facing and Sectioning Glycol Methacrylate Embedded Tissue Blocks

Improvements in glycol methacrylate embedding, block facing, trimming, and sectioning are described. The improvements are derived from a novel molding system, a multipurpose instrument for rapid block facing, trimming and examination, and a device for removing unwanted sections from the microtome knife while sectioning is in progress. Together, these methods facilitate specimen preparation and result in a significant reduction of the time required to prepare high resolution, very thin sections for light microscopy.     

Notes and queries

An instrument for the precise trimming of flat or cylindrical embedded specimens to be sectioned in an ultramicrotome is described. It will produce the required truncated pyramid accurately by hand slicing. A simple modification to the LKB specimen holder allows it to be used with the Reichert ultramicrotome as well.     

Do Mirror Glasses Have the Same Effect on Brain Activity as a Mirror Box? Evidence from a Functional Magnetic Resonance Imaging Study with Healthy Subjects

Since its original proposal, mirror therapy has been established as a successful neurorehabilitative intervention in several neurological disorders to recover motor function or to relieve pain. Mirror therapy seems to operate by reactivating the contralesional representation of the non-mirrored limb in primary motor- and somatosensory cortex. However, mirror boxes have some limitations which prompted the use of additional mirror visual feedback devices. The present study evaluated the utility of mirror glasses compared to a mirror box. We also tested the hypothesis that increased interhemispheric communication between the motor hand areas is the mechanism by which mirror visual feedback recruits the representation of the non-mirrored limb. Therefore, mirror illusion capacity and brain activations were measured in a within-subject design during both mirror visual feedback conditions in counterbalanced order with 20 healthy subjects inside a magnetic resonance imaging scanner. Furthermore, we analyzed task-dependent functional connectivity between motor hand representations using psychophysiological interaction analysis during both mirror tasks. Neither the subjective quality of mirror illusions nor the patterns of functional brain activation differed between the mirror tasks. The sensorimotor representation of the non-mirrored hand was recruited in both mirror tasks. However, a significant increase in interhemispheric connectivity between the hand areas was only observed in the mirror glasses condition, suggesting different mechanisms for the recruitment of the representation of the non-mirrored hand in the two mirror tasks. We conclude that the mirror glasses might be a promising alternative to the mirror box, as they induce similar patterns of brain activation. Moreover, the mirror glasses can be easy applied in therapy and research. We want to emphasize that the neuronal mechanisms for the recruitment of the affected limb representation might differ depending on conceptual differences between MVF devices. However, our findings need to be validated within specific patient groups.     

Simple and Rapid Block Face Alignment Methods for the Ultramicrotome

Light from the fluorescent lamp on an ultramicrotome can be reflected from a mirror located beneath the knife face 50 that the knife and edge can he imaged on the block face. It is well known that this image can be used to accurately align the block face to the knife edge and cutting direction. A method is described of pre-aligning the lamp, stereomicroscope, knife, and the mirror, which is fixed with respect to the knife face, 80 that a bright reflection of the knife face on the block face is obtained only when the block face is brought close to alignment. This initial alignment is an extremely rapid procedure, and is followed by slower, more accurate manipulation of the block and knife for precise alignment.

The mirror, easily mounted to a Porter-Blum MT-2 ultramicrotome knife holder, is very simple in design and readily adaptable to any ultramicrotome. Methods to permit small movements of the block for the MT-1 and MT-P ultramicrotomes are also descrihed.     

Motor learning in man: A review of functional and clinical studies

This chapter reviews results of clinical and functional imaging studies which investigated the time-course of cortical and subcortical activation during the acquisition of motor a skill. During the early phases of learning by trial and error, activation in prefrontal areas, especially in the dorsolateral prefrontal cortex, is has been reported. The role of these areas is presumably related to explicit working memory and the establishment of a novel association between visual cues and motor commands. Furthermore, motor associated areas of the right hemisphere and distributed cerebellar areas reveal strong activation during the early motor learning. Activation in superior-posterior parietal cortex presumably arises from visuospatial processes, while sensory feedback is coded in the anterior-inferior parietal cortex and the neocerebellar structures. With practice, motor associated areas of the left-hemisphere reveal increased activity. This shift to the left hemisphere has been observed regardless of the hand used during training, indicating a left-hemispheric dominance in the storage of visuomotor skills. Concerning frontal areas, learned actions of sequential character are represented in the caudal part of the supplementary motor area (SMA proper), whereas the lateral premotor cortex appears to be responsible for the coding of the association between visuo-spatial information and motor commands. Functional imaging studies which investigated the activation patterns of motor learning under implicit conditions identified for the first, a motor circuit which includes lateral premotor cortex and SMA proper of the left hemisphere and primary motor cortex, for the second, a cognitive loop which consists of basal ganglia structures of the right hemisphere. Finally, activity patterns of intermanual transfer are discussed. After right-handed training, activity in motor associated areas maintains during performance of the mirror version, but is increased during the performance of the original-oriented version with the left hand. In contrary, increased activity during the mirror reversed action, but not during the original-oriented performance of the untrained right hand is observed after left-handed training. These results indicate the transfer of acquired right-handed information which reflects the mirror symmetry of the body, whereas spatial information is mainly transferred after left-handed training. Taken together, a combined approach of clinical lesion studies and functional imaging is a promising tool for identifying the cerebral regions involved in the process of motor learning and provides insight into the mechanisms underlying the generalisation of actions.     

DNA cytofluorometry in micro-dissected paraffin embedded breast tissue: description of a method

DNA microfluorometry on smears obtained from paraffin embedded tissue has been shown to be a distinctive possibility. In this paper a simple method for the detachment of cells from mammary ducts and ductules is described. Areas of interest were selected in conventional slides stained with Haematoxylin and Eosin. The corresponding paraffin embedded block was then dewaxed. The areas under study were retraced with a stereomicroscope and the cells within ducts and ductules were scraped out with a 0.4 mm diameter fine needle. Cells were isolated with mechanical and enzymatic procedures and stained with the Feulgen reaction. The DNA content of single cells was then measured using a microfluorometer.     

Measuring the resolution of uncompressed plastic sections cut using an oscillating knife ultramicrotome

Thin sections of biological tissue embedded in plastic and cut with an ultramicrotome do not generally display useful details smaller than approximately 50 A in the electron microscope. However, there is evidence that before sectioning the embedded tissue can be substantially better preserved, which suggests that cutting is when major damage and loss of resolution occurs. We show here a striking example of such damage in embedded insect flight muscle fibres. X-ray diffraction of the embedded muscle gave patterns extending to 13A, whereas sections cut from the same block showed only approximately 50 A resolution. A possible source of this damage is the substantial compression that was imposed on sections during cutting. An oscillating knife ultramicrotome eliminates the compression and it seemed possible that sections cut with such a knife would show substantially improved preservation. We used the oscillating knife to cut sections from the embedded muscle and from embedded catalase crystals. Preservation with and without oscillation was assessed in Fourier transforms of micrographs. Sections cut with the knife oscillating did not show improved preservation over those cut without. Thus compression during cutting does not appear to be the major source of damage in plastic sections, and leaves unexplained the 50 A versus 13A discrepancy between block and section preservation. The results nevertheless suggest that improvements in ultramicrotomy will be important for bringing thin-sectioning and tomography of plastic-embedded cells and tissues to the point where macromolecule shapes can be resolved.     

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.     

BMP signaling is essential for development of skeletogenic and neurogenic cranial neural crest

BMP signaling is essential for a wide variety of developmental processes. To evaluate the role of Bmp2/4 in cranial neural crest (CNC) formation or differentiation after its migration into the branchial arches, we used Xnoggin to block their activities in specific areas of the CNC in transgenic mice. This resulted in depletion of CNC cells from the targeted areas. As a consequence, the branchial arches normally populated by the affected neural crest cells were hypomorphic and their skeletal and neural derivatives failed to develop. In further analyses, we have identified Bmp2 as the factor required for production of migratory cranial neural crest. Its spatial and temporal expression patterns mirror CNC emergence and Bmp2 mutant embryos lack both branchial arches and detectable migratory CNC cells. Our results provide functional evidence for an essential role of BMP signaling in CNC development.     

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.     

Aligning Block Faces by Reflected Light for Precise Sectioning

A microscope substage mirror is mounted by means of a channeled lucite block on the base of a Porter-Blum knife holder. The plane face of the mirror, centered on, and about 2 inches below the edge of the knife, reflects light to the front edge of the knife so that the formation of an image of the knife edge on the face of the tissue block results. Alignment of the edge of the knife with the edge of the block to parallelism is accomplished by rotating the block. Alignment of the face of the block to the cutting plane is done by tilting the block until the image of the knife neither approaches the knife nor recedes from it with up and down movement of the block. Ultrathin sections of an area within a 1-2 μ section examined by light microscopy can thus be obtained from a previously established cutting face without loss of material.     

A new technique for removal of amorphous phase tissue water without ice crystal damage: a preparative method for ultrastructural analysis and immunoelectron microscopy

An apparatus has been produced that can remove amorphous phase tissue water via molecular distillation without devitrification or rehydration. This method represents a fundamental advance in tissue preparation, making possible for the first time ultrastructural localization of soluble molecular entities without the problems of alteration, re-distribution, and loss which have plagued conventional techniques. Fresh slices of rat brain, liver, or kidney, and monkey retinal tissue were cryofixed by bounce-free, metal mirror cooling on copper bars immersed in liquid nitrogen (LN2). Tissue transferred under LN2 was then placed in a precooled copper specimen block, which was subsequently lowered into a LN2-cooled stainless steel chamber. After rough pumping at 1 X 10(-3) mbar with a mechanical pump to remove LN2, the chamber was evacuated with a cryopump or turbomolecular pump to achieve a hydrocarbon-free, ultra-high vacuum of 1 X 10(-8) mbar. Equilibrium temperature in the chamber before the drying cycle was -192 degrees C. The copper specimen block was equipped with a thermocouple and a programmable feedback-controlled heating circuit. Tissue was dried by increasing the specimen block temperature 1 degree C/hr during the critical drying phase while monitoring the rate of water removal with a partial pressure analyzer. Results obtained indicate that drying is complete below the devitrification temperature of amorphous phase tissue water. Dried tissue was fixed with osmium tetroxide vapor, vacuum-embedded in a low-viscosity epoxy resin, sectioned, stained, and viewed with the electron microscope. Processed tissue exhibits excellent morphological preservation without the use of pre-fixation or cryoprotective agents. Thin sections of this tissue are excellent for immunocytochemical staining and electron microprobe analysis.