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1 This article was downloaded by: [Saúl Blanco] On: 26 May 2012, At: 09:38 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Diatom Research Publication details, including instructions for authors and subscription information: Universal coordinate method for locating lightmicroscope specimens Frithjof A.S. Sterrenburg a, Paul Hamilton b & David Williams c a Stationsweg 158, Heiloo, The Netherlands b Research & Collections, Canadian Museum of Nature, Ottawa, Canada c Botany Department, The Natural History Museum, London, UK Available online: 09 May 2012 To cite this article: Frithjof A.S. Sterrenburg, Paul Hamilton & David Williams (2012): Universal coordinate method for locating light-microscope specimens, Diatom Research, 27:2, To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

2 Diatom Research Vol. 27, No. 2, June 2012, Universal coordinate method for locating light-microscope specimens FRITHJOF A.S. STERRENBURG 1,, PAUL HAMILTON 2 & DAVID WILLIAMS 3 1 Stationsweg 158, Heiloo, The Netherlands 2 Research & Collections, Canadian Museum of Nature, Ottawa, Canada 3 Botany Department, The Natural History Museum, London, UK A description is provided of a universally applicable method of locating specimens under the light microscope, which is precisely reproducible, regardless of the brand or type of instrument used. For this method, two conditions must be met: (1) the instrument should have a graduated mechanical stage, and (2) the procedure must be rigorously standardised, as described. Keywords: coordinate assignment, light-microscope specimens, types, vouchered specimens, location of specimens Downloaded by [Saúl Blanco] at 09:38 26 May 2012 Introduction A method for the coordinate assignment of specimens is especially necessary in the case of taxon types and specimens validating an identification (vouchered specimens), where future investigators must be able to locate the type specimen referred to in the protologue in the case of the former, and specimens from the materials in the case of the latter. Typically, the approaches used to date include: (1) Scribing a ring on the cover slip around the specimen, using a diamond marker. This is a permanent mark, but it may be regarded as unacceptable damage to the preparation by curators. Damage would certainly result in the case of dry mounts, or fluid mounts like the many Victorian slides mounted in monobromonaphthaline, as the cover slip will immediately fracture. Finally, such a diamond marker is expensive and, therefore, not available to all microscopists. (2) Drawing an ink ring, for instance, with a fine waterproof felt pen. This is not permanent as it will wear off if the slide is examined repeatedly with an oil immersion objective and cleaned with solvents. Also, the pressure exerted while the ring is drawn will again entail the risk of the cover slip fracturing with dry or fluid mounts. Finally, a slide may well contain type or vouchered specimens of so many species that drawing of separate ink rings may not be feasible. (3) The England Finder. This is a separate slide divided into small, individually numbered fields. Slides manufactured by a single company are identical with respect to finding position location, but the England Finders produced by different companies will produce different location results. With appropriate precautions, an England Finder permits the location of a specimen regardless of the type of microscope used, but because of its high cost it has not been widely adopted by microscopists (refer to Droop 1996, p. 405). Independently, the authors had been considering methods for precisely locating specimens on microscope slides in particular, type or vouchered specimens that do not depend on a particular brand or model of the instrument and could thus be applied with reproducible results by any investigator. A joint effort led to the procedure presented here, which was found to consistently lead to reliable specimen location using several entirely different instruments. Method All research-type microscopes have a graduated mechanical stage with vernier scales, or recently, digital readouts. Our examination of such stages, from modern instruments to stands from almost a century ago, showed them all to be graduated in millimetres with a high degree of accuracy. In addition, the movement along the y-axis (north south) and x-axis (west east) was found to be accurately perpendicular. In principle, this would permit coordinate assignment with an accuracy of 0.1 mm, but there has never been any standardisation in stage position and scale numbering between different manufacturers, so that readings will be totally different for different microscopes. Corresponding author. fass@wxs.nl (Received 23 April 2012) ISSN X print/issn online 2012 The International Society for Diatom Research

3 Downloaded by [Saúl Blanco] at 09:38 26 May Sterrenburg et al. This can be solved by introducing a new aid: creating a benchmark on the slide in question, a 0:0 reference that represents the origin of an x, y coordinate system. The standard we propose for this benchmark is to scribe a tiny (<50 μm) ring on the slide next to the cover slip with a diamond marker, as illustrated in Fig. 1. The centre of this ring constitutes the permanent benchmark. Because the slide is much more robust than the cover slip, this does not entail the hazard of damage. If such an expensive diamond marker is not available, a small cross can be made freehand (not using the microscope) with a diamond scribe, the centre of the cross becomes the benchmark. The position of the benchmark can be ringed or otherwise indicated with oil-based paint so that its position is easily visible. If scribing the benchmark with a diamond is impossible or is not permitted by a curator, there are some alternatives. In these cases, the benchmark is also made freehand, without using the microscope. The first two options do not create a truly permanent benchmark, however. (1) Place a minute dot of oil-based paint on the slide with a toothpick, ring or mark as above. (2) Print a fine cross on clear acetate, punch out and gum onto the slide. (3) For square cover slips, assign and mark (e.g., with paint dot) a corner of the slip as the benchmark. In this scenario, the 0:0 position is tied to the cover slip. The position and nature of the benchmark can be standardised by the curator of a collection and must be entered in the database recording details of the slides. The best position for the benchmark is to the side and slightly higher than the edge of the cover slip, as in Fig. 2, when all coordinates will become south and east. Using the mechanical stage, north south (y-axis) and west east (x-axis) offsets can then be specified as coordinates for the specimen in question (Fig. 2). If the microscope has a rotating stage, this must be accurately centred before coordinates are read. One cardinal point in our method is to standardise the orientation of the slide on the mechanical stage of the microscope. The user will intuitively tend to place the slide on the stage in such a way that the text on the label(s) will be legible. However, there are two different models of mechanical stage: one (Fig. 3) has the slide holder (arrow) on the opposite side of the eyepieces in the binocular head, while the other (Fig. 4) has the slide holder (arrow) on the same side as the eyepieces. If the slide is placed on the stage so that the text on the label is legible, the slide edge supported by the slide holder will be the opposite for these two different models of microscope. The problem is that especially for Victorian slides, the two long edges of the slide may not be exactly parallel, which will skew the x, y-axis system (Fig. 5). The slide edge that forms the baseline must therefore be standardised as follows: the north edge of the slide shall form the baseline. To define this north edge, the slide is viewed (as in Fig. 2) so that the label(s) is (are) legible if the text is written horizontally; if the text is written vertically it shall read upwards. The north, south, west, east orientation is then as in Fig. 2. The north edge could be so marked (e.g., with oil-based paint) to aid future users. The slide is placed in the slide holder so that the north edge is supported. This means that the text will be upside Fig. 1. Diamond marker. These are available in different models. Fig. 2. Coordinate assignment of a specimen. Benchmark forms origin of coordinate system drawn and is here so placed that all coordinates are south and east for convenience.

4 Method for locating light-microscope specimens 93 Downloaded by [Saúl Blanco] at 09:38 26 May 2012 Fig. 3. Microscope stand with slide support (arrow) opposite the eyepieces. down if the slide holder is at the near end of the stage, as in Fig. 4. The second cardinal point in our method is the use of north south, west east. In this manner, the coordinates are related directly to the geometry of the slide itself. The use of describers like up, left is to be explicitly avoided as these words would refer to the observer instead of the slide and would cause complete confusion for the two different stage models of Fig. 3 and Fig. 4, or with inverted microscopes. For quick reference, the procedure is summarised in a brief protocol at the end of this paper. Results and discussion The procedure was given its ultimate test by circulating a slide with specimens whose coordinates had been assigned in this manner, which was then examined by each of us using about half a dozen microscopes of different manufacture and vintage. The accuracy obtained was c. 0.1 mm, the specimens were found within the field of view of even a 40 objective. An electronic stage allows the observer to centre the benchmark on the slide and zero the stage, like taring an electronic balance. Then locating the specimen Fig. 4. Microscope stand with slide support (arrow) directly below eyepieces. Fig. 5. edges. Skewed coordinates resulting from non-parallel slide simply amounts to moving the stage to the appropriate x, y position. In contrast to the techniques for finding individual specimens on a slide that have been used, the standard procedure we describe here uses a simple coordinate location system permitting location of a specimen using any microscope with a graduated mechanical or digital stage. When the method is used in publications or for depositing types or vouchered specimens, it should be specified with reference to this paper. Application of this procedure extends from taxonomic work to the vouchering of diatom identifications

5 Downloaded by [Saúl Blanco] at 09:38 26 May Sterrenburg et al. in ecological studies. Although taxonomic identifications are often supported by drawings and photomicrographs, actual specified specimens provide the closest realistic documentation of the living organism. Protocol Assigning specimen coordinates Create benchmark. Centre rotating stage if present. Place slide in holder with north edge supported. Note stage settings for the benchmark, which then correspond to x, y = 0, 0. Note stage settings for the specimen and specify the north south (y-axis) and west east (x-axis) offsets from the benchmark as specimen coordinates. Locating specimens Centre rotating stage if present. Place slide in holder with north edge supported. Note stage settings for the benchmark. Adjust the mechanical stage for correct north south (y-axis) and west east (x-axis) offsets as specified for the specimen in question. Acknowledgement Dr Peter Webber is thanked for helpful comments. Reference Droop S.J.M The identity of Diploneis splendida (Bacillariophyta) and some related species. Phycologia 35: