Embedding Thin Plant Specimens for Oriented Sectioning

Small plant structures such as small primary roots, filamentous mosses and algae are difficult to orient for sectioning since they become wavy and curl during embedding. A method is described for embedding and orienting tiny plant specimens in a glycol methacrylate resin using self-constructed flat molds. Prior to sectioning, small samples can be oriented in both the longitudinal and the transverse plane. As several samples can be sectioned simultaneously, time-consuming trimming of the blocks is reduced substantially. The efficiency of this technique has been demonstrated using the tiny roots of the model plant Arabidopsis thaliana (L.) Heynh.     

Optimized Transection: A Prelude to Oriented Sectioning

A technique is described which permits blocks of tissue to be flat-embedded in euhedral plastic castings and then to be transected along a plane so that sections may be cut which are optimally oriented to the internal ultrastructure of the block. In the transection procedure a hollow plastic cylinder is placed on the specimen trimming block. The cylinder's top prescribes a plane to which the tissue block is accurately oriented and clamped at a predetermined level. Two hand files and a burnisher are worked across the cylinder's top to 1) remove extraneous material above the plane of transection, 2) expose the tissue for sectioning and 3) smooth the block face. The clear plastic at the periphery of the exposed tissue is then easily trimmed away with a razor blade. The result is a block face with a flat, reflective surface which may be quickly aligned to the knife on the ultramicrotome. The effort needed to transect, align and face the block is minimal and 1-micron or semithin sections produced will be precisely parallel to, and at, the plane of transection. Dust produced by the transection procedure is easily eliminated from the work area by use of a small disposable vacuum cleaner. The technique of producing optimally oriented light microscope sections, using the transector, is enhanced by application of solvents to the block face which cause it to develop a temporary low relief, exactly matching the structural detail of sections cut from the block face. Areas of interest can be accurately located and isolated on the block face, using only a hand-held razor blade, so that oriented ultrathin sections of important regions can be routinely cut and examined in the electron microscope.     

Optimized transection: a prelude to oriented sectioning

A technique is described which permits blocks of tissue to be flat-embedded in euhedral plastic castings and then to be transected along a plane so that sections may be cut which are optimally oriented to the internal ultrastructure of the block. In the transection procedure a hollow plastic cylinder is placed on the specimen trimming block. The cylinder's top prescribes a plane to which the tissue block is accurately oriented and clamped at a predetermined level. Two hand files and a burnisher are worked across the cylinder's top to 1) remove extraneous material above the plane of transection, 2) expose the tissue for sectioning and 3) smooth the block face. The clear plastic at the periphery of the exposed tissue is then easily trimmed away with a razor blade. The result is a block face with a flat, reflective surface which may be quickly aligned to the knife on the ultramicrotome. The effort needed to transect, align and face the block is minimal and 1-micron or semithin sections produced will be precisely parallel to, and at, the plane of transection. Dust produced by the transection procedure is easily eliminated from the work area by use of a small disposable vacuum cleaner. The technique of producing optimally oriented light microscope sections, using the transector, is enhanced by application of solvents to the block face which cause it to develop a temporary low relief, exactly matching the structural detail of sections cut from the block face.(ABSTRACT TRUNCATED AT 250 WORDS)     

Holding Plastic Embedded Specimens During Dissection: Two Improvements

Sectioning of tissue specimens of aligned cells (e.g., muscle, cochlea, retina) for micrographs, often requires the capsule containing the tissue to be positioned at a precise angle during sectioning. The correct angle can be set by trial and error, but the process can be shortened if the gross anatomy of the cell system is used as a guide to orient the embedded sections as closely as possible in the optimal plane. Thick sections are then cut in this plane with a razor blade, and these sections are re-embedded in preparation for thin sectioning. This technique eliminates the large angles of the capsule in the microtome which occur when the gross tissue is poorly aligned in the first embedding.     

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.     

A new method for studying human oocytes by light and electron microscopy

Since ovarian follicles appear to be randomly oriented with respect to the plane of the section, the method of sectioning and examining follicles at their maximum diameter described here allows direct comparison between oocyte populations of women and small differences can be detected. Re-sectioning for EM allows selected follicles of interest to be examined at a higher resolution.     

A New Method for Studying Human Oocytes by Light and Electron Microscopy

Since ovarian follicles appear to be randomly oriented with respect to the plane of the section, the method of sectioning and examining follicles at their raimm diameter described here allows direct comparison between oocyte populations of women and small differences can be detected. Re-sectioning for EM allows selected follicles of interest to be examined at a higher resolution.     

Monitoring of Epoxy Block Trimming by the Use of a Laterally Mounted Mirror

Mounting a small mirror on the side of the knife holder of an ultramicrotome permits the monitoring of selected areas during epoxy block trimming without removing the block from the chuck. This technique is particularly useful for trimming toward preselected areas of in situ embedded cell cultures for subsequent sectioning. When the visibility of the embedded cells is improved by staining with Paragon-1301 or toluidine blue before embedment, such mirror monitoring can be carried out at magnifications up to 40 times during the trimming procedure.     

Anisotropy of photosynthetic membranes and the degree of fluorescence polarization

The degree of fluoresence polarization, P, of unoriented and magnetically oriented spinach chloroplasts as a function of excitation (400–680 nm) and emission wavelengths (675–750 nm) is reported. For unoriented chloroplasts P can be divided into two contributions, P_IN and P_AN. The latter arises from the optical anisotropy of the membranes which is due to the orientation with respect to the membrane plane of pigment molecules in vivo. The intrinsic polarization P_IN, which reflects the energy transfer between different pigment molecules and their degree of mutual orientation, can be measured unambiguously only if (1) oriented membranes are used and the fluorescence is viewed along a direction normal to the membrane planes, and (2) the excitation is confined to the Q_y (≈ 660−680 nm) absorption band of chlorophyll in vivo. With 670–680 nm excitation, values of P using unoriented chloroplasts can be as high as +14%, mostly reflecting the orientational anisotropy of the pigments. Using oriented chloroplasts, P_IN is shown to be +5±1%. The excitation wavelength dependence studies of P_IN indicate that the carotenoid and chlorophyll Q_y transition moments tend to be partially oriented with respect to each other on a local level (within a given photosynthetic unit or its immediate neighbors).     

Orientation of the hemes of high potential cytochromes relative to photosynthetic membranes, as shown by the linear dichroism of oriented preparations

The orientations of high potential cytochromes with respect to photosynthetic membranes was investigated in spinach chloroplasts and in Rhodopseudomonas viridis. The general approach consists in detection with polarized light of photoinduced absorbance changes related to the oxidation of the cytochromes. The orientation of cytochrome c-558 was measured at room temperature in chromatophores and whole cells of Rps. viridis, oriented on glass slides and in a magnetic field, respectively. The orientation of cytochrome b-559 of green plants was detected at 77 K in magnetically oriented chloroplasts. In both cases the dichroic ratio for the band shows that the heme plane makes an angle greater than 35°C with the membrane plane. Moreover, the dichroic ratio is not constant throughout the and β bands, for both cytochrome c-558 and b-559. Linear dichroism spectra of oriented pure horse heart cytochrome c and cytochrome c₂ of Rhodopseudomonas sphaeroides in stretched polyvinyl alcohol films show that the variations of the dichroic ratio in the and β bands can be explained by the occurrence of x- and y-polarized transitions absorbing at slightly different wavelengths.     

A Method to Identify Microinjected Nephrons of Rat

This paper describes a technique for identifying individual nephrons that have been subjected to micropuncture. The general location of the nephron is marked on the surface of the kidney by implanting two micropipette tips on opposite sides of it two or three tubule diameters away. The tubule itself is marked by the injection into the lumen of a tracer material, for purposes of this account, a suspension of E. coli. After perfusion fixation the kidneys are removed and a block of tissue containing the extrapapillary portion of the nephron is excised. This block is cut into thin slices parallel to the surface of the kidney; these are embedded in plastic for subsequent sectioning. On sectioning, the marker material makes the nephron in question readily discernible under the microscope. A major advantage of this technique is that it allows the tubule of interest to be located as much as 48 hours later.     

Schedules for Sectioning Maize Kernels in Paraffin

For paraffin sectioning of maize kernels, the following technic is recommended: Use fresh, turgid kernels and utmost care in removing kernels from the cob and in subdividing into properly oriented slices. Kill and harden in a chrome-acetic-formalin formula. Rinse in water and dehydrate in four grades of dioxane to anhydrous; evacuate with an aspirator and infiltrate with paraffin. If anhydrous dioxane is excessively costly, dehydrate as above to the commercial grade and transfer by intermediate steps to one of the following two-solvent mixtures, using anhydrous ingredients, (A) dioxane and normal butyl alcohol, (B) dioxane and tertiary butyl alcohol, (C) normal butyl alcohol and chloroform, (D) tertiary butyl alcohol and chloroform. Evacuate in the final solvent. (Melted parowax floats on any of the above mixtures, affording gradual, progressive infiltration to pure parowax by periodic decantation and addition of wax.) Finally, transfer to compounded casting wax and cast in paper boats. To prepare a kernel segment for sectioning, fasten to a plastic block, shave the wax from the cutting plane and soak for 12-24 hours, at 35° C, in water containing a trace of safranin or other dye. Mordant starch grains in 1% tannic acid + 1/2% potassium metabisulphite. A wide choice of simple or multiple stains can be used. These methods are also applicable to tough old stems of corn and hemp, and possibly to many caryopses and seeds.     

Shell structure, ontogeny and affinities of the Lower Cambrian bivalved problematic fossil Mickwitzia muralensis Walcott, 1913

Exceptionally preserved carbonate- and shale-hosted Mickwitzia muralensis from the Lower Cambrian Mural Formation, southern Canadian Rocky Mountains, complement one another to yield an unusually complete account of its ontogeny, ecology and phylogenetic relationships. The shell of M. muralensis is composed of dense phosphatic layers interspersed with porous organic-rich layers. At the insertion of shell-penetrating tubes, shell layers deflect inwards to produce inwardly pointing cones. The tubes are interpreted as having hosted setae that were secreted by outer-epithelial follicles. Follicular setae also occurred at the mantle margin, where they were oriented within the plane of the shell as in modern brachiopods. During ontogeny, the initial setae oriented in the plane of the shell occurred before the first shell-penetrative setae. In the juvenile and early-mature stages of shell secretion, a posterior opening was present between both valves and was used for the protrusion of an attachment structure. In the late-mature shell, this opening became fixed in the ventral valve. Based on the posterior margin and the shell microstructure, a close relationship between Mickwitzia and the paterinids is proposed with differences interpreted as heterochronic. The shell-penetrative setal apparatus of M. muralensis is distinct from that previously described of Micrina, though both types are conceivably homologous to adult and juvenile setae of modern brachiopods.     

Mechanical fixation techniques for processing and orienting delicate samples, such as the root of Arabidopsis thaliana, for light or electron microscopy

Despite improvements in live imaging, fixation followed by embedding and sectioning for light or electron microscopy remains an indispensible approach in biology. During processing, small or delicate samples can be lost, damaged or poorly oriented. Here we present a protocol for overcoming these issues when, along with chemical fixation, the sample is fixed mechanically. The protocol features two alternatives for mechanical fixation: the sample is encased either in a rectangular block of agarose or between Formvar films suspended on a wire loop. We also provide methods for key steps all the way through to sectioning. We illustrate the method on the root of Arabidopsis thaliana, an object that is ～0.15 mm in diameter and difficult to process conventionally. With this protocol, well-oriented sections can be obtained with excellent ultrastructural preservation. The protocol takes about 1 week.     

Observations on the ultrastructure of the thickened sieve cell wall inPinus strobus L.

Summary The thickened sieve cell wall of white pine is shown to comprise a crossed-helical polylamellate structure in which the predominant microfibrillar orientation is greater than 45° with respect to the cell axis. The previously reported observation that microfibrils may be oriented other than parallel to the plane of the cell wall is disputed and it is demonstrated that such an appearance may derive from appropriately oblique sectioning of the wall.     

人类牙齿齿冠和齿根分离两种技术方法对牙釉质厚度测量的影响

在基于计算机断层扫描技术（CT）和虚拟图像处理技术的灵长类牙齿测量学研究中,经常需要分离三维虚拟模型的齿冠和齿根,再进行后续测量工作,如计算机辅助的生物力学分析、釉质厚度测量等。而分离齿冠和齿根这一步骤,目前有多种方法,如,1)根据齿颈线切分齿冠,或2)人工建立基底平面切分齿冠。为了评估这两种不同的处理方式对后续的牙齿测量学上的影响,本文使用三维方法测量了82例化石和现代人类下颌后部牙齿的釉质厚度,包括南方古猿、早期人属、尼安德特人和现代人。使用配对t检验对比发现,两种方法得到的釉质厚度数值上没有显著差别,但随后进行的种间比较发现,使用基底平面切分齿冠的方法比较费时,更依赖于测量者的人工操作,并且可能弱化了物种间前臼齿绝对釉质厚度的差异,造成系统误差。其原因是对于前臼齿和前部牙齿等齿颈线形状不规则的标本,基底平面难以建立或误差较大。在未来对釉质厚度的种间差异的研究中,特别对齿颈线形状不规则的标本（如人类前部牙齿及猩猩、黑猩猩的牙齿等）,本文推荐使用齿颈线分离齿冠和齿根,测量和计算齿颈线之上的釉质厚度。釉质厚度有一定的分类学、功能形态学和系统发育学意义。本文积累了一批可供未来对比研究的原始数据,并且发现尼安德特人前臼齿的相对釉质厚度显著小于现代人,这与前人利用臼齿、犬齿所做的对比研究结果相同,支持了尼安德特人拥有较薄的相对釉质厚度这一观点。     

A Simple Method for Preparing Thin (10 μm) Histological Sections of Undecalcified Plastic Embedded Bone with Implants

A simple method for preparing undecalcified thin sections of bone with implants has been developed. After exposing a surface of bone and implant in a plastic block by sawing thick sections, the surface is stained prior to making a thin section. A glass coverslip is affixed with a thin layer of cement to the stained surface to stabilize the tissue and implant during sectioning. A mixture of glycerine and water is used as a coolant and lubricant. The orientation in situ is preserved allowing demonstration of bone architecture and cells, and the tissue-implant interface.     

Orientation of Small Specimens for Cryosectioning

A simple technique is introduced to achieve symmetrically oriented frozen sections of small specimens such as young fish or frog larvae. Small samples are especially difficult to orient if they are already frozen to the chuck in a freezing microtome. Orientation of the sample in a mold filled with embedding medium prior to freezing permits sectioning as well as easy labeling and storage of the specimens. The use of a stereo microscope during orientation is optional.     

The orientation of membrane bound radicals. An EPR investigation of magnetically ordered spinach chloroplasts

The orientation of membrane-bound radicals in spinach chloroplasts is examined by electron paramagnetic resonance (EPR) spectroscopy of chloroplasts oriented by magnetic fields. Several of the membrane-bound radicals which possess g-tensor anisotropy display EPR signals with a marked dependence on the orientation of the membranes relative to the applied EPR field. The fraction of oxidized and reduced plastocyanin, P-700, iron-sulfur proteins A and B, and the X center, an early acceptor of Photosystem I, can be controlled by the light intensity during steady-state illumination and can be trapped by cooling. The X center can be photoreduced and trapped in the absence of strong reductants and high pH, conditions previously found necessary for its detection. These results confirm its role as an early electron acceptor in P-700 photo-oxidation. X is oriented with its smallest principal g-tensor axis (g_x) predominantly parallel to the normal to the thylakoid membrane, the same orientation as was found for an early electron acceptor based on time-resolved electron spin polarization studies. We propose that the X center is the first example of a high potential iron-sulfur protein which functions in electron transfer in its ‘;superreduced’; state. We present evidence which suggests that iron-sulfur proteins A and B are 4Fe-4S clusters in an 8Fe-8S protein. Center B is oriented with g_y predominantly normal to the membrane plane. The spectra of center A and plastocyanin do not show significant changes with sample orientation. In the case of plastocyanin, this may indicate a lack of molecular orientation. The absence of an orientation effect for reduced center A is reconcilable with a 4Fe-4S geometry, provided that the electron obtained upon reduction can be shared between any pair of Fe atoms in the center. Orientation of the ‘;Rieske’; iron-sulfur protein is also observed. It has axial symmetry with g close to the plane of the membrane. A model is proposed for the organization of these proteins in the thylakoid membrane.

A new EPR signal was observed in oriented chloroplasts. This broad unresolved resonance displays a g value of 3.2 when the membrane normal is parallel to the field. It shifts to g = 1.9 when the membrane normal is perpendicular to the field. The signal is sensitive to illumination and to washing of the thylakoid membranes of broken chloroplasts. We suggest that there is a relation between this signal and the water-oxidizing enzyme system.     

Sections from Double Mesas Obtained at the Same Tissue Plane

A method for obtaining sections from two areas in the face plane of a tissue block is described. It facilitates ultrathin sectioning where virtually identical planes of section are essential but where areas of interest are too far apart to be included in a single section. Two horizontally separated mesas are prepared; sections are cut from the first with the knife rotated around its vertical axis by 2-3° to provide clearance for the other. The second mesa is then sectioned with the knife rotated 4-6° in the opposite direction. Similarly, by changing the vertical inclination of the block, two additional vertically separated mesas can be cut. This procedure is of great value for comparative morphometric studies of material from opposite sides of individual specimens.