A new live-of-life imaging method of light film of C. elegans adults

。 —The beauty of in vivo imaging studies is that specimens are alive and can record dynamics
such as cell division and embryonic development over time.

However, the frustration of in vivo imaging research is that the specimen is alive – writhing, twisting, escaping the field of view
.
Moreover, it is fragile and easily damaged or killed
by thermal damage by imaging equipment.

MBL imaging research expert Carsten Wolff said the recent emergence of technical solutions to this dilemma in MBL's embryology course is "a classic example of MBL's collaborative efforts.
"

"In the 2021 embryology course, we set out to develop a technique that would allow us to image adults Caenorhabditis elegans to visualize worms over a longer period of time and at high resolution by using light-sheet microscopy," Wolf said
.
A team of course faculty and staff worked with MBL imaging staff to fine-tune the protocol and write papers
during the 2022 course.
   

Caenorhabditis elegans is a popular organism
in biological and biomedical research.
Light sheet fluorescence microscopy (LSFM) has been very successful in capturing the embryonic process Caenorhabditis elegans as well as in
mice and zebrafish.
But once an organism hatches, LSFM has limitations
.

The difficulty lies in the installation
of the sample.
Due to its optical properties, low melting point agar works well as a sample medium for large organisms, but small roundworms tend to burrow into soft agar and disappear.

Thus, prior to this protocol, the longest LSFM imaging time for C.
elegans in adults had been 20 min
.
The new protocol extends this time to more than two hours while avoiding heat stress
in the specimen.

"The innovation we describe is essentially a combination of two known installation methods," Wolf said
.
"One is a biopolymer, which is sticky during sample preparation, but once you expose it to UV light, it hardens and keeps the sample (in this case, Caenorhabditis elegans) fixed
.
The second part is the mounting method in a plastic tube, allowing the use of a light sheet microscope
.
This combination allows one to realistically imagine adult Caenorhabditis elegans for more than 2 hours
.
It may sound like a short time, but it was not possible
due to the problem of fixing the specimen before.
In addition, imaging from different angles is not possible
due to the constant movement of the specimen's body.
”

The team used this protocol to perform time-lapse imaging
of dendritic branching and pruning of sensory neurons.
They hope it will enable better real-time imaging of other important cell and developmental processes, such as germ stem cell biology, cell migration, cell division and cell invasion
.
This protocol can be generalized to other organisms with little modification
.