In order to fully understand transcription dynamics, we need microscope technologies that can image all the players involved with sufficient spatial resolution, over time.
We are designing novel microscopes to overcome the limitations of current instruments: for instance, our Multifocus Microscope (MFM ) can visualize the entire volume of the nucleus in one shot for fast tracking of individual molecules or 3D superresolution . The MFM is now available to the scientific community worldwide through the Janelia Advanced Imaging Center (AIC).
We have also built a drift-stabilized TIRF microscope that can be used for in vitro single molecule experiments in which the position of individual molecules can be tracked with nm precision over hours , allowing the quantification of single rounds of transcription on purified systems . The TIRF setup is also open to outside users through the Janelia Advanced Imaging Center (AIC).
In addition to these systems, we are also developing other approaches that will for example allow multiplexing the dynamics of multiple biological species simultaneously.
Comparison between conventional wide-field detection and multifocus detection. (A and B) The excitation Beam (Purple) is identical in the conventional (A) and multifocal schemes (B); however, the wide filed microscope only collects lights emitted from a single plane (orange), versus multiple planes in the multifocal microscope. Adapted from (Hajj, Wisniewski et al. 2014).
Example 3D superresolution data collected with the MFM: the reconstructed 3D superresolution volume of a budding yeast shows the cell wall (red–orange) and tubulin (white–blue) (Hajj, Wisniewski et al. 2014).
Our microscopes generate large datasets in which minute biological signals are emitted by individual molecules over important fluorescent background. In order to extract valuable biological data, we continuously devise innovative data analysis algorithms and techniques. These allow us to detect molecules, measure their trajectories with nanometer resolution so we can reconstitute the nucleus structural organization and its dynamics. Beyond data analysis, we also develop theoretical models in order to relate the genome architecture to transcription regulation.
Localization map of histone H2B in the nucleus, image by 3D PALM superresolution (Recamier, Izeddin et al. 2014) (A) demonstrating chromatin clusters within the nuclear space and a density enrichment towards the nuclear envelope, compared with simulation of a spatially homogenous random distribution (B).
Another key aspect of our work is data visualization: the Dahan lab recently developed a software dedicated to 3D superresolution data visualization (ViSP (El Beheiry and Dahan 2013)), and we continue to explore new ways to seamlessly navigate the multiple dimensions of data-rich movies.
ViSP is a software developed by the Dahan lab that allows visualizing 3D superresolution data (El Beheiry and Dahan 2013).