DS0305 -

Small and ultrabright fluorescent stealth polymer nanoparticles for fast intracellular superresolution tracking – supertrack

Submission summary

Single molecule imaging of biomolecules in living cells using fluorescence microscopy has become of key importance for understanding biological processes at the molecular level. Following directly their dynamics requires localizing single emitters inside cells with a spatial and temporal resolution at the level of the molecular events. The achievable resolution depends strongly on the fluorescent markers used for labeling. The brighter the emitter the higher its imaging speed and the better its localization in space. Furthermore, fluorescence intermittency of the labels due to blinking or photoactivation allows resolving emitters at distances below the diffraction limit using, e.g., direct stochastic optical reconstruction microscopy (dSTORM). This makes, however, tracking of the emitters more cumbersome. The limited brightness of organic fluorophores and fluorescent proteins can be overcome by using fluorescent nanoparticles (NPs). Fluorescent polymer NPs are currently attracting increasing interest due to their versatility, biocompatibility and their potential to overcome the limitations of quantum dots in terms of brightness and control of blinking. We recently showed that encapsulating a charged dye with a bulky counterion in polymer NPs led to increased brightness and a strong cooperativity of the dyes resulting in whole NP blinking.
The main objective of this project is to design the smallest possible ultrabright fluorescent polymer NPs with controlled blinking required for resolving and tracking single molecules with superresolution and their adaptation to the intracellular environment.
The most versatile and straightforward approach to fluorescent polymer NPs is loading with organic fluorophores. In these systems the first challenge is to achieve extreme brightness as the dyes usually undergo aggregation self-quenching at the high concentrations needed for high brightness. A second challenge is that the on-off-switching of the entire NP necessary for superresolution imaging requires a collective behavior of hundreds of fluorophores. In this project we will use encapsulation of salts of rhodamine B derivatives with bulky hydrophobic counterions in polymer NPs to create ultrabright NPs with controlled blinking. For this we will engineer the organization of dyes within the NPs by varying hydrophobicity and glass transition of the polymer, hydrophobicity of the dye salt, and assembly conditions.
When using NPs as labels two further aspects have to be considered, in order to obtain optimum resolution and perturb as little as possible the observed system. First, their size should be of the order of the proteins they label. Second, aggregation and nonspecific interactions with intracellular proteins and cellular components have to be avoided. In this project we will introduce multiple charged and zwitterionic groups in polymers. Nanoprecipitation will lead to very small NPs with the thinnest possible noninteracting shells. Studying the interactions of these NPs inside cells and optimization of their surface chemistry will enable us to obtain <10 nm intracellular stealth NPs.
We will validate our NPs for intracellular tracking by directly tracking cytoplasmic dynein, a molecular motor, for the first time in mammalian cells – a challenge due to the speed and resolution needed. Individual dynein motors will be labeled with our small stealth NPs. For this, NPs bearing benzyl-guanine groups will be introduced in cells expressing dynein-SNAP-tag. The NPs will be imaged using videomicroscopy and 3D-dSTORM. Adjusting the blinking of these ultrabright NPs will make it possible to track single dyneins in living cells, resolve individual dyneins at sites of high concentration, and thus gain information on their colocalization and cooperativity.
The NPs developed here will, due to their size, surface properties, brightness, and controlled blinking, open the way to unprecedented single molecule superresolution tracking in living cells.

Project coordination

Andreas REISCH (Laboratoire de Biophotonique et Pharmacologie - Université de Strasbourg)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LBP - Unistra Laboratoire de Biophotonique et Pharmacologie - Université de Strasbourg

Help of the ANR 243,000 euros
Beginning and duration of the scientific project: September 2016 - 36 Months

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