Technological and Methodological Developments Frédéric PRZYBILLA and Ludovic RICHERT

Axis 3. Technological and Methodological Developments  Leaders: Frédéric PRZYBILLA and Ludovic RICHERT

This transversal research axe aims to meet the microscopy needs of the laboratory and is part of our participation as an R&D team in the Alsace node of France Bioimaging. Our objective is to develop cutting-edge imaging modalities and new fluorescent probes capable of providing decisive answers to the biological questions of the team and the UMR.

We have acquired strong expertise in quantitative microscopy approaches: F-Techniques (FLIM/FC(C)S)1 and characterizations at the single molecule scale (smFRET (single molecule FRET), SPT (single particle tracking ), SMLM (Single Molecule Localization Microscopy)2 for the study of biomolecular dynamics and interactions (protein-protein, nucleic acid/protein, lipid/protein). After the prototyping and validation phases of the new instruments, they are gradually integrated into the QuESt-IBiSA platform to make them accessible to the academic and industrial scientific community. We have already developed a multiphoton microscope FLIM (fluorescence lifetime microscopy)/FCCS (fluorescence cross correlation spectroscopy),3,4 2 super-resolution microscopes (3D PALM/ dSTORM and a λ-PAINT system),5,6 a wide field microscope and a confocal microscope dedicated to photon upconversion, respectively optimized for cellular SPT and PLIM imaging (phosphorescence lifetime imaging).7,8 

Based on this expertise and the needs of the other areas of the team and the laboratory, several developments will be undertaken both by optimizing existing instruments and by developing new approaches.

Biophysics of Nucleic Acids in Solution and Cellular Environments

In the context biomolecular interactions studies involving nucleic acids and based on the team expertise in characterizing and using fluorescent guanosine analogs (Axis 1), we are currently developing a single-molecule tracking system optimized for oligonucleotides labeled with these analogs. Given that these probes have absorption maxima between 300 and 400 nm, we have employed a prism-TIRF (Total Internal Reflection Fluorescence) architecture combined with plasmonic approaches to optimize the excitation of the probes and enhance their signal-to-noise ratio. Furthermore, to study RNA/DNA in a cellular context using these same analogs (axes 1 and 2), we are developing in parallel a FLIM microscope with pulsed UV excitation, taking advantage of the particularly long fluorescence lifetime (> 10 ns) of these analogues. Preliminary tests have demonstrated the proof of concept, allowing us to easily distinguish these guanosine analogs from cellular autofluorescence through temporal imaging. Our objective is to open a new era where fully functional fluorescent nucleic acids can be imaged and tracked in living cells.

Caption: Biophysics of nucleic acids using fluorescent guanosine analogs in solution and cellular environments. (a) Fluorescent guanosine analog. (b) Photograph of the smFRET UV microscope for the single-molecule-scale study of fluorescent analogues, (c) Example of time gated fluorescence image of internalization thG in live cells.

Biomolecular Interactions in Solution

Based on its expertise in the characterization of macromolecular interactions (proteins, nucleic acids) by fluorescence approaches, the team has set up a smFRET microscope to monitor the interaction dynamics and conformational changes of proteins at the single molecule scale. The instrument, based on a TIRF (total internal reflection fluorescence) microscope, allows to monitor a large number of single molecules simultaneously over an extended period of time. However, this approach requires the surface immobilization of one of the partners and can only resolve transition times between conformations greater than ms. Some molecular interactions are incompatible with these limitations, leading us to set up a smFRET microscope in confocal geometry with alternating laser excitation (ALEX) as a complementary tool. This approach allows to follow the interactions directly in solution with a temporal resolution on the order of μs.

Another development area focuses on the design and validation of fluorescent molecular rotors (FMR) as environmentally sensitive probes to characterize biomolecular interactions in solution. FMRs are flexible aromatic organic molecules that become highly emissive in a rigid environment where their intramolecular rotation is restricted. This feature of FMRs can be exploited to monitor changes in their molecular microenvironment by observing variations in their fluorescence intensity and lifetime. We will focus on i) fine tuning the physicochemical and photophysical properties of selected FMRs by chemically modifying their structure and ii) developing new chemoenzymatic approaches for the selective incorporation of FMRs into RNA. Our goal is to obtain and validate next-generation FMRs to monitor the interactions of G4 structures with their target proteins and to study the formation and internal organization of microheterogeneous systems, such as phase-separated droplets formed by RNA and RNA-binding proteins. These probes will also be used by the MPB team in the context of liquid-liquid phase separation.

Single-Molecule Tracking and Membrane Organization

In the context of studying lipid dynamics on the surface of living cells (Axis 2), we have already demonstrated that UCNP-based labels allow to record SPT trajectories that are not limited by the photophysics of the marker but only by the dynamics of the system (e.g., particle moving out of the field).9 To fully exploit the photostability of UCNPs, we will implement real-time and 3D tracking of a single particle using a feedback loop to keep the particle of interest within the detection volume. This approach will enable very long SPT trajectories, providing unprecedented insights into the long-term dynamics of lipids on the surface of living cells. Additionally, this method will allow simultaneous spectroscopic measurements using a λ-PAINT approach, leveraging the multiplexing capabilities of UCNPs. The UCNP spectra consist of well-separated fine emission bands whose ratios and positions depend on their composition. This unique feature will enable us to simultaneously track the dynamics of multiple types of lipids within the same cell. Ultimately, these experiments will provide an unprecedented description of the dynamics and organization of the plasma membrane and can be applied to other systems.

 

Caption: Long-Term Membrane Organization Dynamics by 3D Single Particle Tracking (SPT) (a) This image of a starry sky is actually the anti-Stokes emission of UCNPs when illuminated by an infrared laser beam. (b) Bright-field microscopy image of the studied cells, with insets showing the super-resolved trajectories of single membrane receptors obtained after video analysis.

Multicolor Super-Resolution Imaging

We developed a new super-resolved imaging modality called DRET-PAINT (Dark Resonance Energy Transfer PAINT). This technique uses a FRET donor probe with a very low fluorescence quantum yield (developed by A. Burger, University of Nice) in combination with a highly bright acceptor. By integrating this probe into an imaging oligonucleotide, we demonstrated that the recognition of a complementary strand labeled with the acceptor induces the illumination of the acceptor while maintaining minimal background noise. This technique optimizes PAINT imaging and is expected to enable high-resolution multicolor imaging (< 60 nm) with short acquisition times (< 5 min). It will be particularly applied in the themes of Axis 2.

Caption: Multicolor Super-Resolution Imaging. (a) New DFK probe used as FRET donor. (b) Principle of Dark Resonance Energy Transfer PAINT (DRET-PAINT). (c) Super-resolved image of the microtubule network in HELA cells (red TIRF image, green DRET-PAINT image). (inset) Zoom on a branch of the microtubule network.

1.            Anton, H., Taha, N., Boutant, E., Richert, L., Khatter, H., Klaholz, B., Rondé, P., Réal, E., Rocquigny, H. de & Mély, Y. Investigating the Cellular Distribution and Interactions of HIV-1 Nucleocapsid Protein by Quantitative Fluorescence Microscopy. PLOS ONE10, e0116921 (2015).

2.            Glushonkov, O., Réal, E., Boutant, E., Mély, Y. & Didier, P. Optimized protocol for combined PALM-dSTORM imaging. Scientific Reports8, (2018).

3.            El Meshri, S. E., Dujardin, D., Godet, J., Richert, L., Boudier, C., Darlix, J. L., Didier, P., Mély, Y. & de Rocquigny, H. Role of the Nucleocapsid Domain in HIV-1 Gag Oligomerization and Trafficking to the Plasma Membrane: A Fluorescence Lifetime Imaging Microscopy Investigation. Journal of Molecular Biology427, 1480–1494 (2015).

4.            Manko, H., Normant, V., Perraud, Q., Steffan, T., Gasser, V., Boutant, E., Réal, É., Schalk, I. J., Mély, Y. & Godet, J. FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria. JoVE 61602 (2020). doi:10.3791/61602

5.            Manko, H., Mély, Y. & Godet, J. Advancing Spectrally‐Resolved Single Molecule Localization Microscopy with Deep Learning. Small19, 2300728 (2023).

6.            Manko, H., Burton, M. G., Mély, Y. & Godet, J. Spectral Phasor Applied to Spectrally‐Resolved Single Molecule Localization Microscopy. ChemPhysChem25, e202400101 (2024).

7.            Dukhno, O., Przybilla, F., Muhr, V., Buchner, M., Hirsch, T. & Mély, Y. Time-dependent luminescence loss for individual upconversion nanoparticles upon dilution in aqueous solution. Nanoscale10, 15904–15910 (2018).

8.            Frenzel, F., Würth, C., Dukhno, O., Przybilla, F., Wiesholler, L. M., Muhr, V., Hirsch, T., Mély, Y. & Resch-Genger, U. Multiband emission from single β-NaYF4(Yb,Er) nanoparticles at high excitation power densities and comparison to ensemble studies. Nano Res.14, 4107–4115 (2021).

9.            Dukhno, O., Ghosh, S., Greiner, V., Bou, S., Godet, J., Muhr, V., Buchner, M., Hirsch, T., Mély, Y. & Przybilla, F. Targeted Single Particle Tracking with Upconverting Nanoparticles. ACS Appl. Mater. Interfaces16, 11217–11227 (2024).