Summary
Introduction
Material and Methods
Discussion
Applications
Conclusions
References
Glossary
After familiarizing ourselves with the hardware and software, both
our VCE systems proved to be versatile and easy to use. In terms of time
needed for capture and editing, the entire procedure took between five
minutes and a whole day, depending mainly on the size of the specimen,
the concomitant number of files captured, and the addition of text and
labels of varying complexity (see examples in Table
5).
Nevertheless, some limitations were evident, both in the optical and electronic components. Variability in the smoothness and speed of manual focusing meant that some parts of a specimen were sometimes captured with less multifocal resolution than others, or that different operators would consistently capture with greater or lesser duration redundancy. These problems could probably be overcome with a motorized focusing system, although this would represent a significant extra cost.
With the combination of objectives and camera adapter assembly of Configuration 1, small nematodes (body length below 2 mm) were easily recorded, but for larger nematodes or dissected organs it was more difficult to obtain a good fit and positioning in the video display area. The entire nematode could not always be covered by one frame at 4x magnification, while video capture of elongate structures (such as a long tail, a cylindrical bulb or an extended dissected gonad) was less convenient, because the 40x objective did not always provide enough resolution, and the display area obtained with 100x required too many separate files to be recorded.
If at least the width of the studied object was completely contained within the display area, then it was fairly easy to capture it as a series of separate files, and distill a reconstruction of the entire object - much as if one were to directly observe it in several steps with the LM. However, when neither the width nor the length fit within the display area, e.g. when recording larger dissected gonads from Meloidogyne spp., then it became too difficult to compile a set of files and to use these subsequently for re-assembling an overview. In that case, we were forced to compromise on resolution by choosing a lower magnification.
Such limitations were largely overcome, albeit at additional expense, by the various components of Configuration 2 providing greater flexibility in magnification and viewing area. In practice, the highest useful setting of the magnification changer was 1.6x, as the 2x setting only represented "empty magnification" without any gain in resolution on the video monitor or computer screen.
The resolution provided by both the tested video cameras was sufficient for many purposes, but nevertheless clearly limited in comparison to the actual resolution of the microscope optics. In particular, VCE images of fixed specimens did not always resolve very small or thin features such as cuticular punctations, nerve endings or minute openings, even when these were relatively distinctly visible with the LM itself. Similarly, in dissections of gonads the precise appearance of cell membranes was often resolved much more poorly in the video clips than through the eyepieces of the LM. Improvements in this respect must await the advent of digital video cameras with both high resolution and appropriate adapters for fitting to LM.
This being said, the obtained resolution was definitely good enough
for most illustrative purposes. When assembled into a figure and printed
next to photographic plates, for instance, VCE frames were somewhat poorer
than still photographs in contrast, but slightly better in resolution,
less grainy, and the contrast was easily enhanced with image editing software
(Fig. 3, Fig. 4).
Again, the magnification-changing add-ins of Configuration 2 were less
affected by resolution issues, although at reduced magnifications using
the 60x objective and the 0.35x camera adapter, there was an even greater
discrepancy between the actual quality of the microscope optics and the
obtained final on-screen resolution.
Configuration 1 was installed on an ordinary benchtop in a room
below three floor centrifuges, while configuration 2 was set up in a laboratory
with various kinds of large and noisy equipment (fume hood, flow bench,
incubators, etc.). The obtained images were in both cases affected by microvibrations,
which were reduced to a minimum by mounting the microscopes on a heavy
board resting on various kinds of elastic material (bubble wrap, packing
foam, or a rectangular lifesaver). This largely took care of the problem,
to the extent that video clips were no longer significantly affected, even
when there were still sufficient vibrations to prevent traditional photography.
On the computer side, the DC30plus video capture card is known to be prone to overheating, in which case it produces clips containing frames with green or red bands and desynchronised interlacing, resulting in playback problems. This problem occurred with Configuration 1, and was minimised by equipping the computer housing with better two ventilation fans instead of the usual single one. Also, these and other occasional errors in single frames could be edited out of the captured clip, by removing the affected frame in the Monitor window of Adobe Premiere 5.1. The DV500 card in Configuration 2 did not present such problems.
We encountered an intriguing bug with Configuration 1 in Adobe Premiere 5.1, in the "Special Processing" window of the "Export Movie" settings. At 720 x 540 frame size, the cropping rectangle control positions were not properly converted into coordinates, the latter values only being half of the correct ones (e.g. if the rectangle controls are set at 70 pixels from left, 100 pixels from right, 50 pixels from top and 124 pixels from bottom, then the coordinate boxes display the respective values 35, 50, 25 and 62). Apparently the program incorrectly assumed that frame size was 360 x 270, regardless of the actual value in "video settings".
Without compensation, this resulted in less extensive cropping of the frames, and therefore suboptimal reduction of background redundancy. Fortunately, in most cases the error could easily be compensated for by always selecting the proper rectangle first, and then typing the double of each value in each coordinate box (but never the other way round). However, if the corrected coordinate values were such that they would add up to more than 360 pixels horizontally or 270 pixels vertically, then a conflict arose and it was not possible to crop the desired area completely.
With Configuration 2, Adobe Premiere 5.1 invariably caused a complete freeze of the operating system. However, this problem disappeared after upgrading to Premiere 6.0. The latter version has a number of minor bugs affecting e.g. windows refreshing or available menu options, the most notable one being that the function for capturing still images instead of movie clips did not work, despite being listed in the appropriate menu and in the manual. This meant that still images had to be obtained instead by exporting them out of captured clips. Otherwise, none of the observed bugs significantly affected the procedure summarized in Table 4. Finally, we experienced one major crash of the removable hard drive when it was moved from the computer of Configuration 2 to another system. We therefore adopted the practice of copying sets of newly captured or edited files to CD-R immediately after capture or editing.