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| (Database for the nematode branch of Assembling the Tree Of Life) | ||||
Our image capturing system includes a IX81 motorized, inverted Olympus microscope connected to a Retiga-1300, Mono, 12-bit cooled camera with RGB Liquid Crystal Color Filter Module (Slider), both the camera and the microscope controlled by a basic IP Lab software with additional script designed for our own specific needs. To keep the system isolated from any vibration from the surrounding both the microscope and the camera are mounted on a TMC Anti-vibration isolation table. It should be noted however that we are not here endorsing any particular type of equipment for imaging purposes.
The whole DICS is a flexible system in that it can either be controlled manually
or can also be controlled through a computer, and one can shift from manual to
computer control or vice-versa at anytime. The motorized control is in all
the X, Y and Z-axes. The system is designed to work both with a 96-well plate
as well as with glass slides, this increases the working distance and enables
to study transparent but larger organisms or those in containers that are difficult
to study in an upright system. The stages are custom designed for each system.
In the 96-well system, once the microscope is calibrated, a specified well can
be brought into focus at the lowest power (X-Y motorized control). At this step
the worm needs to be marked for the microscope by a person so that it can bring
the worm to the center of the field of vision. Then any objective can be selected
and the microscope brings the worm into focus (Z-control). The steps from here on
are similar in both the 96-well plate system and for slides. In both cases where
the worm is straight and where there is the need to collect images along the
entire body at a uniform magnification, it is possible to collect the different
digitized slices or Z-stacks of images automatically. But, as most nematodes
are not straight in shape when dead, collecting images automatically along the
whole worm may only waste time. Furthermore, different nematode body parts
have varying perceived degree of importance for taxonomic purposes and the
objective to be used and the distance between each slice for each body part
would be variable.
The digitized Z-stacks of images are collected and saved as IP Lab files at
1200 x 1400 pixels at 8 bits. When high power objectives are used only a certain
portion of the body can be looked at in a given field of vision. As a result,
we mostly collect images of only a limited part of the body (e.g. lip, pharynx,
tail etc.) starting from one lateral side of the body and ending on the other
lateral side. When these images are viewed in a sequence one can replicate the
act of focusing through a worm body without actually looking at it. To that
effect the images are exported to and saved in a QuickTime format so that the
whole sequence can be viewed as a movie.
These are deposited in our open database ready to be downloaded and viewed for free by end users who have access to the Internet. We also keep a copy of each stack of images in its original IP Lab format for future use (http://Nematol.unh.edu/how_to_use_ics.php). This gives us the flexibility of the IP Lab files in case we need at any time to work with individual slices which can potentially be used for various purposes in electronic and printed version e.g. for describing a species, preparing edited versions as teaching aid, depicting a particular structure as illustration, detail study of parts of the nematode body, for comparative work, etc.
The different body parts of an entire worm can usually be covered in 7 to 10
video clips at varying magnifications. By opening these movie clips one can
navigate forward or backward to see through the worm body at any desired position.
Simply said, one can see very high quality images of detailed internal and external
nematode structures at his/her convenience without the need for an expensive
and powerful microscope.
| Funded by NSF |  |
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