Summary
Introduction
Material and Methods
Results
Discussion
Conclusions
References
Glossary
Based on the developed procedures, we have tested the quality (Fig.
3, Fig. 4, Fig.
5), resulting file size and time load (Table
5) of producing VCE files for a number of different purposes.
First, using only half of the available resolution (360 x 270) but the high data transfer rate of 4000 kbps, we produced very modestly sized files after compression with the "Cinepak" codec. (see Radopholus bridgei in Table 5). Although clearly inferior to direct observation with LM, these low-resolution files nevertheless contained sufficient detail to capture and label many important characters, illustrating that a VCE file can offset lower frame resolution to an important degree by its multifocal information contents, compared to a single still image captured at high resolution. Next, we tried a limited data rate during capture, but this resulted in negligible differences in file size of the final clip (see Prionchulus punctatum, with and without limited data rate during capture,Table 5). In order to produce acceptable clips for websites, it is therefore sufficient to limit resolution during clip exporting.
Capturing the various body regions takes very little time in itself, but for maximum transfer of knowledge it is best to use the titling facilities of the video editor to their fullest, by naming all informative or interesting structures, and adding arrows to point them out in those frames where they occur only (Fig. 5). This titling step is much more time-consuming (Table 5), especially during the first few attempts to utilise the various functions. However, in our experience the result is an extremely effective demonstration tool, especially when compared to traditional line drawings, or having to point out structures through each student's LM in a full class. Not only does it allow the viewer to learn to locate and distinguish each structure, but it also greatly improves the three-dimensional perception of inexperienced microscopists, by mimicking the focusing action of the microscope, and encouraging the use of focusing as a tool for assessing the relative positions of different structures at different transverse levels in a transparent specimen.
In the case of nematodes, it is often useful to verify the appearance and identification of specimens by taking a quick look at its lip region, cardia, vulval or spicular region, and tail. VCE protocols can be used to routinely capture and optimize these five body parts for large numbers of nematode specimens, and the resulting VCE clips can then be included in, or linked to, a database. Overall body shape and size can be captured on a single still image at appropriate magnification (4x or 10x objective).
Depending on space constraints, it may be advisable to restrict file size of archive clips in the same ways as for website files, and "Sorenson" compression would then be preferable over "Cinepak" if the database will be used strictly by local access. However, we assume that this problem will largely disappear with the ever-increasing capacity and speed of hard disks, CD-writers and DVD-writers, as the download speeds allowed on a Local Area Network are usually sufficient to handle quite large VCE files.
To test feasibility of routine screening and capturing of specimens, we have therefore processed a series of 10 slides by capturing at 720 x 540 pixels and 4000 kbps, and timed the average duration per specimen at 22 minutes (Table 5). No titling or scale bars were added, as we assume that routine application would not allow for the added time required per specimen, and in that case it is essential to document each clip by including all relevant information in its file name, or in a separate text file or database record.
Furthermore, the 100x water immersion objective of Configuration 2 allows screening and capture of nematodes immediately after extraction, without any fixation or the need to place a coverslip over the specimens. We have performed trial runs of sample screening, by heat-killing extracted nematodes on a hot plate at 65° C, and then applying VCE to specimens in an open drop of water on glass slides. Click here for some examples. Although the 100x objective is inferior in resolution to its oil immersion counterparts, it nevertheless provides images of sufficient detail to allow rapid screening and archiving of nematodes, without requiring the time investment and health risks of fixation and transfer to permanent slides.
For smaller nematodes like Hemicriconemoides variodus, Panagrobelus stammeri, or Plectonchus n. sp., the required number of captured clips was fairly limited (up to about 30 files, cf. Table 5), and it therefore took little time to give all edited files a name including basic information such as taxon name, body part, slide number and magnification. Also, in order to allow subsequent on-screen measurements, we first recorded the scale on a calibration slide at each magnification, as clips or TIFF files. For H. variodus and Plectonchus n. sp., we then used the titling functions to construct a scale bar for each magnification, and paste it into the appropriate clips with captured parts of the specimen. We also titled each clip with taxon name and slide number.
As appears from Table 5, total processing time for H. variodus and Plectonchus n.sp. was moderately to significantly longer than that of Panagrobelus stammeri, where we did not apply titling. In practice, adding titles may require too much time when large numbers of type material need to be processed, and in this case it may suffice to give each clip an informative name. Nevertheless, to eliminate any confusion we would nevertheless recommend that titling always be applied to at least the holotype.
In larger nematodes like Paraxiphidorus michelluci, the number of files required for complete capture becomes much too large to be feasible without automation of the focusing and stage controls. It even becomes too time-consuming to use detailed filenames, and in this case we therefore just named the files as a numbered series for each magnification. In such cases, an acceptable compromise is to capture an incomplete series of multifocal files, by omitting only those parts of the body that are irrelevant to diagnosis. Thus, we did not record those parts of the P. michelluci holotype where the body only contained intestine, and this yielded file numbers, time and disk requirements that proved more manageable.
In order to keep track of all recorded parts of a completely or incompletely captured specimen, we constructed clickable image maps to show the magnification and display area of each VCE file. Click here for some examples.However, the construction of this type of map requires several hours more work, and we would therefore not recommend it for routine VCE processing of type material.
The most informative components were recorded of several female nematode gonads , i.e. the region around the spermatheca: end of the ovary, oviduct, spermatheca and beginning of uterus (see Fig. 4 for an example). In most cases, the region of interest could be captured within the display area, and the production of a single videoclip was sufficient to record the dissected structures. In those cases, capturing and editing times were minimal: per gonad, we timed an average duration of only 4 minutes without titles and 45 minutes with titles (Table 5).
We tested a slightly modified regressive staining technique (essentially carmine and propionic acid) based on Khrustalev & Hoberg (1996). Stained Panagrolaimus rigidus specimens were mounted directly in the destaining solution (50 % acetic acid and 70% ethanol), and we then captured a section of the intestine at several time intervals during the destaining process. Afterwards, we could easily select the clip with the best contrast between nuclei and background, thus recording the cellular structure of the intestine on a whole mount individual. The effective time spent on recording and editing of these clips was minimal (see Table 5).