Positive displacement holder for critical point drying of small particle materials*

A specimen holder has been designed specifically to contain small particulate matter, such as microorganisms, fibres or powders, during the fluid exchange cycle and final evaporation step of the critical point drying process. The mode of fluid exchange is positive displacement rather than diffusion. The holder can accommodate up to ten different samples without loss of material or cross-contamination during the dehydration process. The samples are separated by silver membrane filters, and fluid exchange is accomplished in minimal time by only slight positive pressure. Experiments with soluble dyes have shown that any fluid can be displaced completely with another in less than 2 min, whereas diffusion requires more than 30 min. The holder and individual capsules have been tested with different materials and a standard water/ethyl alcohol/amyl acetate/carbon dioxide fluid sequence. This paper describes the design, production, and testing of the holder and capsules, and presents selected examples of results.     

A versatile multi-specimen holder for processing and critical point drying of materials for examination in the scanning electron microscope (SEM)

A specimen holder has been designed specifically for use in the Polaron E.3000 critical point drier (CPD) and is capable of drying up to twenty different specimens within a size range of 4.5 mm to 30 μm by utilization of a variable grid system. The principle, however, could be employed in designs for most other critical point dryers. A large number of designs have been produced for handling small specimens during preparation and CPD procedures prior to observation in the scanning electron microscope (SEM). In a great number of cases these have been specific to one particular organism or preparation method. Marchant (1973) suggested culturing organisms on Millipore filters which could then be used in conjunction with a plain filter as a sieve mechanism to contain the specimens, the complete unit being housed in a BEEM embedding capsule. Scott et al. (1973) produced another design, again utilizing the BEEM capsule together with 100 grade copper mesh as the specimen retaining section. Both these designs were flexible in that different grade filters and meshes could be employed. The disadvantage of using BEEM capsules was noted by Taylor (1975) in that the chemical plasticizers present in the capsule material could leak out in the presence of substitution solvents. To overcome this he produced an all metal design for a container employing a similar type of grid system as in this multispecimen holder. His design was for a single chamber specimen holder with a maximum specimen thickness of 1 mm and was such that the complete chamber could be placed directly into the SEM. The need for a multispecimen holder arose when large numbers of specimens, each specific in its preparation required critical point drying without tedious repetition of critical point drying runs. It was necessary to consider the inflexible features of the design. These were, the external dimensions of the container, which had to fit within the existing aluminium specimen-holder of the Polaron E.3000 CPD, and the dimensions of the transmission electron microscope (TEM) grids used as the specimen retainers. Transmission electron microscope grids are available in two sizes, 3.01 mm and 2.3 mm, with a large range in patterns and materials to choose from to suit most types of specimen preparation. From these fixed dimensions a design was drawn up which allowed specimens of up to 4.5 mm across to be prepared and yet still gave the large number of individual chambers required. The construction of the holder can be seen, from the drawing (Fig. 1) and the photographs (Fig. 2) to be comprised of large dimension chambers with a TEM grid housed at each end to contain the specimens and yet still allow the free flow of dehydrating and substitution agents. The complex arrangement of screws was necessary to facilitate the assembly and use of a container which has a separate lid section that can be dismantled to allow different grids to be inserted depending on the dimensions of the material under preparation. The specimens are supported on the lower grid system which can also be varied and for the ease of removal of larger specimens the chamber section divides leaving the specimens readily accessible in the lower half. Where the specimens are very small, the chamber section can be completely removed by carefully removing all screws and then screwing a stud down the centre thread and extracting the chamber section leaving all the specimens supported on their grids on the base plate. These can easily be transferred directly onto the SEM stub and secured either by double-sided sellotape or careful application of an adhesive such as Durafix. It has even been found that discs punched out of Visking tubing can be used in place of the TEM grids to provide a finer sieve mechanism. It was noted, however, that the tubing was hardened by substitution solvents but this still did not seem to impair the results as satisfactory preparations of Penicillium expansum sporangiophores and pollen of Dactylis glomeratus have been achieved (Medi-Cine, 1976). The container has been made out of high quality brass because of its good machining properties necessary when such fine work has to be carried out. The metric design utilizes standard milling cutters with the inclusion of the ?th cutter to produce satisfactory grid housings allowing free movement to ensure that they always settle on the base plate. The versatility of the design can be further increased with the production of two accessory structures (see Fig. 1). The first, simply produced by cutting brass tubing, is for the preparation of small numbers of specimens per chamber, and ensures that the specimens are deposited onto the grid. The holes drilled through the tube wall allow easy removal with fine forceps. The second structure simply partitions the large chambers increasing the capacity to eighty specimens where such a large specific separation is required. The production of artifacts resulting from specimen handling is reduced considerably with this specimen holder. Once the specimens have been loaded, all the processing stages can be carried out on all the material at once and the specimens are ready for mounting prior to observation.     

A container for processing small volumes of cell suspensions for critical point drying

The attachment of lymphocytes to glass or filters, in order to facilitate handling for processing prior to scanning electron microscopy, may introduce artefacts in surface topography. A container has therefore been adapted, from an embedding capsule, for the preparation of small volumes of cell suspensions.     

A multisample chamber for dehydration and critical point drying

The principles and methods for constructing an improved chamber for dehydration and critical point drying of multiple biological samples are described. The specimen chamber design is based on vertical positioning of the electron microscope grids or coverslips and permits minimal perturbation of laminar solvent flow past the specimens. This condition is requisite for optimal exposure of samples to solvents, which is necessary for complete dehydration and drying. Fragile samples, including chromosomes, critical point dried in the multisample chamber demonstrate crisp, well-preserved, three-dimensional morphology.     

Improvement to critical point drying technique for SEM

We have developed an improved method for dehydration and critical point drying which leads to a marked reduction in morphological artefact in at least two classes of problematical specimen: Foetal enamel and avian embryonic heads. Water is replaced by ethanol and ethanol by C₂C1₃F₃ by refluxing in a Soxhlet apparatus. Containers are designed to prevent air drying on transfer to the CPD bomb. We concluded that the thorough removal of both water and ethanol prior to CPD can reduce the types of artefact associated with post-CPD shrinkage (superdrying).     

A method for processing polymeric beads for critical point drying

Polymeric beads are increasingly being used for surface studies. Processing of these beads for critical point drying (CPD) poses a problem since these beads must be handled in suspension. For this processing, a method has been described here which relies on a capsule made by an ordinary micropipette tip covered on the end by parafilm and on the other by a filter paper which permits the exchange of fluid without the loss of beads.     

Simple magnetic holders for critical point drying of microspecimen suspensions

Microspecimens can be critical point dried from suspension in holders consisting of three magnets and two filters. These holders are inexpensive, require no construction, and are easily assembled and disassembled.     

A fine-sieve processing container for use in critical point drying

A container to hold specimens during dehydration and critical point drying can be constructed from a 100 mesh screen cylinder and a BEEM embedding capsule.     

A method for handling free cells through critical point drying.

A method is described in which free cells were dehydrated in suspension, put one double-coated tape attached to a specimen stub and dried from CO2. The method provides a simple, fast means of handling free cells when using the critical point method of drying.     

A multipurpose specimen-carrier for handling small biological objects through critical point drying

A specimen-carrier for safely handling, protecting and drying minute biological specimens down to 1·0 μm in size during the various steps of the critical point drying procedure is described. Free, unattached cells or subcellular fragments can be processed without loss and without risk of unintentional air-drying.     

The preparation of small, particulate specimens by critical point drying: application for scanning electron microscopy

A procedure is described using round glass cover-slips as supports for preparing specimens by critical point drying for the scanning electron microscope. Also described is the construction of a plastic Teflon holder to facilitate handling of the cover-slips.     

Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration and critical point drying.

The critical point drying method of preparing samples for scanning electron microscopy is associated with a variable amount of specimen shrinkage. We studied the causes of this phenomenon is isolated mouse hepatocyte nuclei and in human erythrocytes and found that the critical point drying process itself caused most of the shrinkage that we observed (a 25-30% reduction in diameter in both specimens). Glutaraldehyde fixation and ethanol dehydration caused only minimal size reduction, prior to critical point drying. Substitution of an inert (ethylene glycol-ethylene glycol monethyl ether) dehydration technique did not alter the final result. Previous studies in our laboratory using high resolution SEM and correlative transmission microscopy of isolated nuclei have demonstrated that the shrinkage represents a miniaturization of the organelles in which all structural components retain their usual relationships.     

Prolonged ethanol replacement by CO2 increases splits on articular cartilage surface after critical point drying

Ethanol replacement by CO₂ of glutaraldehyde-fixed and ethanol-dehydrated rabbit articular cartilage specimens was monitored with both gas chromatograph and alcometer prior to critical point drying (CPD). The surface structure of the patellar specimens was also systematically registered with a semiquantitative scanning electron microscopic method. After a 2 h interval, when about 28 μl of ethanol/15 min CO₂ extract was removed, the articular surface was smooth, although small areas of striated surface and superficial splits were present. A long-term CO₂ treatment (16 h) removed ethanol completely, but increased superficial splitting of the articular surface after CPD. Air-drying of the specimens gave rise to inferior preservation of the cartilage: large areas with pitted and leafy surface qualities, but no superficial splits, were present on the surface. It was evident that prolonged ethanol replacement by CO₂, prior to CPD, degraded surface structure of the articular cartilage which should be taken into consideration in the planning and design of experiments. Ethanol removal by CO₂ could conveniently be monitored by an alcometer.     

Use of Peldri II (a fluorocarbon solid at room temperature) as an alternative to critical point drying for biological tissues

A new chemical, Peldri II, is evaluated as a compound for drying soft biological tissues for scanning electron microscopy. Peldri II, a fluorocarbon, is a solid at room temperature and is a liquid above 25°C. Cells or tissues are embedded in Peldri II by immersing them in the liquid form and allowing it to solidify. Once solidified, Peldri II will sublime with or without vacuum to dry tissues, probably without introducing surface tension. Several types of cells and tissues have been examined to compare preservation with Peldri II and critical point drying techniques. No differences were detected between the two techniques when normal surface structures were examined. Peldri II appears to be a significant improvement over hexamethyldisilazane as a drying agent for scanning electron microscopy. It is also very convenient for drying large numbers of samples.     

A flexure-based tool holder for sub-μm positioning of a single point cutting tool on a four-axis lathe

A tool holder was designed to facilitate the machining of precision meso-scale components with complex three-dimensional shapes with sub-μm accuracy on a four-axis lathe. A four-axis lathe incorporates a rotary table that allows the cutting tool to swivel with respect to the workpiece to enable the machining of complex workpiece forms, and accurately machining complex meso-scale parts often requires that the cutting tool be aligned precisely along the axis of rotation of the rotary table. The tool holder designed in this study has greatly simplified the process of setting the tool in the correct location with sub-μm precision. The tool holder adjusts the tool position using flexures that were designed using finite element analyses. Two flexures adjust the lateral position of the tool to align the center of the nose of the tool with the axis of rotation of the B-axis, and another flexure adjusts the height of the tool. The flexures are driven by manual micrometer adjusters, each of which provides a minimum increment of motion of 20 nm. This tool holder has simplified the process of setting a tool with sub-μm accuracy, and it has significantly reduced the time required to set a tool.