Dynamic, super-resolution, volumetric imaging of cellular organization using reversible cryo-arrest technology2018 - B. Hajj (team Dahan, UMR168), M. Dahan (UMR168)
Biological functions are tightly linked to the spatial organization of different bio-molecules. Visualizing this organization with high resolution and following its evolution in time at the single cell level remains a challenge with regular optical techniques. The aim of this project is to develop an instrument that combines state of the art 3D single molecule microscopy with low temperature reversible cell arrest to follow the evolution of molecular organization at different stages of the cell cycle.
Description of the research project
The spatial organization of proteins at the molecular scale plays a crucial role in orchestrating and regulating cellular functions. In the cell nucleus for instance, RNA polymerase II (POLII) was shown to be organized in transient clusters involved in transcriptional initiation . Localization-based imaging methods (such as PALM and STORM) constitute powerful tools to assess the spatial organization of specific biomolecules with nanometer resolution. Yet, despite the wealth of information that these measurements already provide, following the evolution in time of the biomolecular organization is still a challenge. The limiting factors are related to: (i) the speed at which this organization can evolve and which is not compatible with the temporal resolution of PALM/STORM methods, and (ii) the limited photon budget emitted from single molecules that determines both the achievable resolution (namely the localization precision) and the time scale over which the molecular organization can be followed (due to photobleaching). Moreover, it should be noted that proteins are distributed within the 3D extent of a cell, and recording their localization in 3D poses specific challenges in terms of sensitivity and accuracy (see for more details our review ).
To address all these challenges, the project aims to combine several innovative technologies in order to follow in time 3D molecular organization with PALM/STORM super-resolution. More precisely, we will combine our multifocus microscopy and single objective light sheet excitation with reversible cryo-arrest technology. Low temperature enhanced photo-physics is expected to improve the localization precision, and consecutive cycles of freezing and reviving the cell will allow to monitor the evolution of the spatial organization of molecules.
In the past, we have shown that multifocus microscopy (MFM) is compatible with fast three- dimensional single molecule imaging . By splitting the emission of a widefield microscope into several diffraction orders using a custom-made diffraction grating it is possible to image several axial planes simultaneously on the same camera . Moreover, in order to reduce out of focus background, diminish phototoxicity and gain in sensitivity, we are now combining MFM with single objective light sheet microscopy (soSPiM) in collaboration with the group of J.-B. Sibarita in Bordeaux (figure 1.a-b) . Light sheet excitation scheme confines the excitation to a single plane and reduce the out of focus blur. The advantage of soSPIM compared to other light-sheet imaging schemes is that the same objective can be used for both the excitation and collection of the emitted photons. soSPIM is also compatible with high numerical aperture objectives. As shown with the preliminary data it is possible to control the light sheet thickness and adapt it to the imaging volume.
Despite the advantage of our approach for 3D imaging, following the spatial distribution of single molecules in the same cell over different time points remains a challenge. In this project, we propose to solve this difficulty by combining our 3D microscopy schemes with a novel cryo- arrest technology developed in the Bastiaens’ lab . The cryo-arrest method consists of cooling the sample to very low temperature in order to freeze the biological processes. The sample is placed in a chamber directly connected to a liquid-nitrogen-based cooling system (figure 1.c). In order to avoid lethal ice formation during the cooling process, the cell surrounding medium is gradually exchanged with a new medium containing DMSO. The DMSO percentage is well-defined in consistence with the imposed temperature step (See figure 1.d). As such it is possible to arrest the cell biological functions at -45 ̊C and, in reverse steps, revive it by gradual increase of temperature to +37 ̊C and medium exchange.
From a biological point of view, the advantage of low temperature reversible cryo-arrest is that cellular functions are paused, without any ice formation, giving sufficient time to perform PALM/STORM experiments. From a photo-physical point of view, low temperature imaging ensures a higher signal to noise ratio from single molecules due to increased photostability.
Cryo-arrest was shown to be compatible with common oil immersion and high NA objectives commonly used for single molecule super-resolution microscopy. Importantly, cells can subsequently be revived to physiological temperatures with minimum stress to carry on the biological process and the cycles of freezing/revival can be repeated a few times on the same cell. Note, that due to the physical constrains of the cooling system, it is not possible to perform light sheet excitation with common SPIM approaches (such as Betzig’s lattice light-sheet). soSPIM however is well adapted as it requires access to the sample from only one side.
In the project, we will adapt our current instrument by: (i) implementing the cryo-arrest technology on our microscopy set-up, (ii) designing sample chambers compatible with soSPIM experiments (iii) setup of a custom system for precise temperature control in parallel to medium exchange. In collaboration with groups in IPGG, we will automatize the medium exchange process during the cooling procedure, which will allow a faster and more reliable cryo-arrest compared to what has been reported so far. Therefore, we will implement a unique system combining different technologies in order to follow the time- evolution of biomolecular organization at the nanometer scale.
Figure 1 : a) soSPIM combined with MFM 3D microscopy. The light sheet thickness will be adjusted to fit the imaging volume. b) Preliminary data showing light thicknesses imaging fluorescent beads. c) Schematic representation of the cryo-arrest chamber with an observation window from bellow. The soSPIM mirrors will be installed inside the channel that controls the medium composition. d) the phase diagram of the medium for different concentrations of the DMSO+DMEM as function of the temperature. The blue line shows the current steps of temperature and medium exchange to avoid ice formation during the cryo-arrest. The dashed line represents the automated approach that we will develop in which temperature and medium composition will be automatically controlled thanks to a microfluidic system that we will develop in collaboration with IPGG.
Interest for the Labex:
This project is mainly oriented towards technological and instrumental development. We emphasize that the use of reversible cryo-arrest approach for super-resolution methods is still in its infancy and are extremely promising in cell biology. While it will be primarily used in the context of the study of the cell nucleus in our group (the biological applications have purposefully not been described in the current project which is essentially instrumental), we anticipate that our instrument and the associated methods will be beneficial to a large number of scientists in the Labex (and in Curie in general). The system can indeed be useful with a wide variety of systems where it is desirable to follow gradually the evolution of biomolecules distribution after external stimulation (e.g. using ligands) or after optical or chemical induction (e.g. using optogenetics or the RUSH system developed by the Perez lab) and where current super-resolution imaging technologies are lagging behind.
As we have done in the past with MFM microscopy (now available on a platform microscope in the Pasteur building), we will transfer the cryo-arrest technology to the Curie imaging platform if there is a general interest within the Curie community.
- Cisse, II, et al., Real-time dynamics of RNA polymerase II clustering in live human cells. Science, 2013. 341(6146): p. 664-7.
- Hajj, B., et al., Accessing the third dimension in localization-based super-resolution microscopy. Physical Chemistry Chemical Physics, 2014. 16(31): p. 16340-16348.
- Hajj, B., et al., Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy. Proc Natl Acad Sci U S A, 2014. 111(49): p. 17480-5.
- Abrahamsson, S., et al., Fast multicolor 3D imaging using aberration-corrected multifocus microscopy. Nat Methods, 2013. 10(1): p. 60-3.
- Galland, R., et al., 3D high- and super-resolution imaging using single-objective SPIM (vol 12, pg 641, 2015). Nature Methods, 2015. 12(7).
- Masip, M.E., et al., Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution. Nature methods, 2016. 13(8): p. 665.