We have completed the construction of a cryotank for 12h measurements with optical microscopy. It is based on the design from Joerg Enderlein in Goettingen, but with several improvements such as longer measurement times, possibility to move and position the sample with piezo motors and image from the side on the optical table for improved vibrational stability and axial drift. We have started designed a cryo-stage together with an industrial collaborator that could allow us a similar cooling capacity as the cryo tank filled with liquid nitrogen, but is much smaller/lighter and allows directly for dip/tilt xy-stage control of the sample without complicated constructions. In addition, imaging from both sides of the sample should be possible then
We already investigated several red dyes for imaging at cryogenic temperatures.
With regards to the polarization control have we completed the calibration setup for the excitation and STED light paths. We have summarized the technicalities of the setup and calibration protocol in a manuscript that is accepted. This is the basis for the polarization controlled induced sparsity. We have completed experiments at room temperature with fixed dipole emitters. Measurements at cryogenic temperature with fixed emitters have been performed. We introduced a STED beam for depletion with the perpendicular orientation than the excitation beam, however, we found that we do shelf too many molecules too quickly in a long-lived dark state to achieve the required sparsity at the moment.
We have developed phase plate (Vortex PSF) which has the unique capability of simultaneously estimating the 3D position of the emitter, as well as the polar and azimuthal of the molecular orientation, and the degree of orientational constraint, all from a single 2D image. A manuscript is nearly finished at the time of reporting.
We have developed an information optimal algorithm for data fusion in super-resolution light microscopy. From several talks at conferences about the topic we received very positive feedback. Several leading groups are interested in collaboration with us. We have published on our data fusion pipeline in a high impact journal and given several talk on international conferences on it. The resolution achieved by the data fusion approach is ~3-4 nm in 2D.
From this we have embarked on a journey to extend the method to full 3D imaging. We have a manuscript on bioarchive, that is under review at the time of reporting. Here Nuclear Pore Complexes from three different imaging systems and methods of the same macromolecular complex have been reconstructed by our method. We have been able to infer symmetry from the data itself and use it to promote the reconstruction with it.
We have further started to investigate if and how different class can be found from the data alone before averaging. To this end we introduced a suitable dissimilarity measure between particles and used unsupervised clustering after multi-dimensional scaling. This result is again only in the early phase and a manuscript is in preparation at the time of reporting.
For dissemination we provide code on all our methods on github and the version at the time of submission on our ftp site (and journal page). The data for our publication is stored on 4TU servers (an initiative of the Dutch research community) that allows data storage in a findable manner for years to come.
