A versatile multi‐specimen holder for processing and critical point drying of materials for examination in the scanning electron microscope (SEM)
Authors: Crossley A. 
Published: August 2011

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 ⅛th 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.