Summary
Introduction
Results
Discussion
Applications
Conclusions
References
Glossary
In accordance with the financial limitations imposed on the research
budget of most nematologists (including our own) we assembled a first basic
VCE configuration that was comparatively low-priced, not restricted for
use with any particular brand of microscope, and easily customized for
different laboratory environments and for a wide range of applications.
This setup is further referred to as Configuration 1 (Fig.
1, Table
1), and was used to establish the basic protocol outlined below and
summarized in Fig.
2.
To compare the quality of the captured video images with still photography on 35 mm film, multifocal series of pictures of some of the specimens captured with Configuration 1 were also taken with an Olympus SC35 single-lens reflex camera, mounted on the same microscope.
Next, on the basis of our experience with Configuration 1, we assembled a second version of the above system, including various updated components allowing greater flexibility in magnification and more versatility in various other aspects. Changes in this more expensive second setup, Configuration 2, included a separate video monitor, an optical magnification changer, exchangeable 1x and 0.35x camera adapters, and a video capture card equipped for both analog and digital video input (Table 2).
Also, in this second system we replaced the semi-apochromat 40x and oil 100x objectives of Configuration 1 with a single apochromat oil 60x objective, and compensated for lower on-screen magnification by utilising a video camera with 1/3" CCD chip instead of a 1/2" CCD chip. The higher numerical aperture of this 60x objective, in combination with the magnification changer and exchangeable camera adapters, allowed capture of images that were comparable in quality to those obtained with the first setup at highest magnifications, but significantly better than those obtained with Configuration 1 at mid-range magnifications. We also added a water immersion 100x objective of lower numerical aperture, to allow us to capture unfixed specimens on slides without coverslip. Finally, the computer was equipped with two hard drives, one fixed disk for program files and one removable disk for captured files, to allow swapping with other computers for e.g. file backup.
Except for the microscopes and optics of either system, we did not opt for any video or computer components designed specifically for microscopy. Instead, we chose general-purpose components that are aimed at more advanced home users, readily available from many different electronics suppliers, manufactured by companies that are among the market leaders in their respective areas, and compatible with many models and brands of other components.
We have not tested alternative brands for the optics, electronics and
software we haven chosen, and do not wish to claim or imply any preference
for the tested brands. Also, although our configurations were both based
on IBM-compatible computer components, various Macintosh-compatible hardware
and software counterparts exist and operate along identical principles.
As to our own configurations, the respective manufacturers of the different
components are listed in Table 6.
Experimentation with the two hardware and software configurations
led us to a series of general steps for obtaining edited video clips (Fig.
2, Table
3, Table
4).
A first major stage consist of the capturing of multifocal series of images, which actually involves two separate steps. First, the equipment and software must be initialized in such a way that microscope images will be transmitted correctly to the capture card, converted by the capture software into acceptable real-time video images displayed on the computer monitor, and stored in the proper folders of the computer's hard disk(s) during actual capture.
The next step is the capturing itself, of properly positioned parts of a single specimen at a time. Capturing and specimen positioning can be repeated as many times as necessary to record an entire body part at highest magnification (e.g. pharynx, genital system, male tail region, etc.), or even to record a complete specimen.
The second stage consists of editing and compression of the captured video clips. Although each source clip contains all the morphological information provided by the combination of specimen, microscope and camera, it often uses much more disk space and memory than strictly necessary, because usually at least one of five kinds of redundancy (see glossary) occurs. Also, the clips can still be compressed by various algorithms, without visible loss of image quality or extension of playback speed.
Pixel redundancy is actually minimized before capture by selecting the appropriate software settings in the initialization step. The four other kinds of data redundancy must be minimised or removed after capture, using video editing software included with the video capture card. The clip is therefore edited more or less extensively to optimize its final size. As an optional feature it is also possible to add text, scale bars and arrows to some or all of its frames, so as to highlight e.g. structures that are particularly important or hard to see.
In the last stage, the edited clip is tested and verified on various video player programs, to ensure that the end users will be able to play it correctly, regardless of their particular hardware and software facilities.
Optionally, if complete specimens were captured as a large series of clips, then it is also possible to index the clips by means of an overview of their position along the body of the specimen. We have done this by constructing "image maps" in World Wide Web pages written in HTML and accessible online or on disk through any WWW browser. On such an image map, a specimen is depicted as a low-magnification image with clickable boxes or bars delineating the different body parts captured at different magnifications, and providing embedded HTML links to the corresponding video clips. When a given box or bar is clicked, the appropriate clip will open automatically in the video player software installed as plug-in of the browser used. We refer to the aforementioned website for more details and examples.