Shooting Zombie Bacteria with Lasers, to See If They Are Still Alive
While obtaining my Masters degree at the Max Planck School of Matter to Life, I worked on a joint project in the groups of Prof. Dr. Victor Sourjik at the MPI for Terrestrial Microbiology in Marburg, and Prof. Dr. Ramin Golestanian at the MPI for Dynamics and Self Organization in Goettingen. In the project I used advanced forms of microscopy and numerical methods to measure and then model the diffusion of florescent molecules in cells. The goal of the study was to see if diffusion was different in living versus non living versus resurrected cells.
Images from my Masters, Structured Illumination Microscopy of E Coli.
A : Healthy Bacteria (E.Coli)
B: Cell killed with toluene
C: Nucleoid In a dead cell
Membrane in Green, Nucleoid in Orange, Scale Bar 1micrometer
The Structured Illumination Microscopy (SIM) images shown here were taken to understand the effects of toluene on the bacterial membrane. The idea was to study diffusion in the cytoplasm using fluorescence correlation spectroscopy (FCS). FCS measures the diffusion time of a florescent marker by counting the amount of particles in a nano-scale illumination volume created by the laser scanning confocal microscope. The the time autocorrelation of the concentrations gives rise to a diffusion time, which can be fit to a diffusion coefficient. This was one of many studies in the field which report the diffusion of biomolecules to be anomalous.
Overview of FCS
Here we see the workflow of an FCS experiment. In the top image, we see a cell in grey and the confocal volume created by our microscope in blue. We count how many particles are in there over time, which is shown in the middle figure. Finally, example autocorrelation functions are shown for various diffusion models and parameters.
In the group of Dr. Ramin Golestanian I studied how a molecule with switching diffusion constants, such as different conformations of proteins, affected FCS measurements. I am very thankful for being given the opportunity to do experimental work for the first time, especially as a theorist. In my first real experimental work, I learned the many nuances that come with any measurement. Though I learned a lot, due to the short nature of the project I am left yearning to collect more data, with hopes to shed light on this fascinating problem.
