Physics Professor Extends Innovative Microscopy Technique to New Group of Organisms
International Collaboration Used Single-Molecule Localization Microscopy to Study Dead Sea Archaea
By Ben Panko
In a new study, a team led by Carnegie Mellon University Associate Professor of Physics Ulrike Endesfelder and Ulm University Biology Professor Anita Marchfelder has laid the groundwork for expanding the use of single-molecule localization microscopy (SMLM) to archaea — organisms known for their ability to live in harsh, unhospitable conditions. The research was published in the journal Frontiers of Microbiology.
"The beauty of the technique we are using is we can localize single molecules in living cells," Endesfelder said of SMLM, which can image the minute structures in fluorescent cells. This represents a sharp break from previous biochemistry work on the structures and mechanisms of cells that required purifying the cellular components of interest in test tubes to study them.
While it's much more complicated and data intensive to observe live cellular structures and mechanisms, Endesfelder noted, the benefits are clear.
"It gives us the strong advantage that we really can observe the molecules in their native environment and see what they naturally do," she said.
SMLM has been widely used on bacteria and yeast for years but has not been used before in archaea, a unique group of single-cell organisms. Originally thought to be a type of bacteria, there is still much debate in the scientific community about how exactly to classify archaea, which are commonly found around the world but especially well known for surviving in conditions no other life could tolerate.
"They live in these extreme niches, and they have biology you will not find elsewhere," Endesfelder said. "They're one of the least studied domains of life."
To develop a protocol for studying them, the teams joined their expertise and forces. Marchfelder’s lab in Germany has long studied a particular species of archaea, Haloferax volcanii, which is found living in warm and extremely salty environments such as the Dead Sea, while the Endesfelder lab is specialized in SMLM studies in microorganisms.
Endesfelder and her collaborators had to balance keeping conditions ideal for both the archaea and microscope and develop fluorescent proteins that could be inserted into the archaea to image their structures. After testing various options, they found that codon-optimized versions of photoactivatable mCherry1 and photoconvertible Dendra2 worked the best in this species of archaea.
Furthermore, to get better images of the H. volcanii, the researchers got further help from colleagues in Israel and modified the archaeon's genes to stop it from producing the carotenoid pigments. Finally, building on the fluorescence work of their collaborator from Australia, the researchers ended up successfully imaging two intracellular proteins in the archaeon as proof of SMLM's possibility in studying the organism.
Endesfelder said the study opens up lots of possibilities for more closely studying archaea, which could present new opportunities to the scientific community. “We could already show in this first work that levels and organization of our two proteins strongly depends on the cellular growth conditions — so there are certainly lots of details about their biology to investigate in the future”.
Other authors on the study include Bartosz Turkowyd of Carnegie Mellon, Sandra Schreiber and Julia Wörtz of Ulm University, Ella Shtifman Segal and Moshe Mevarech of Tel Aviv University and Iain G. Duggin of the University of Technology Sydney.
Endesfelder’s lab’s work was supported by grants from the National Science Foundation (PHY-2020295), the Max Planck Society, the Fonds der Chemischen Industrie and the DFG priority program SPP 2141.