Bright Molecular and Nanoscale Probes for Biosensing and Bioimaging

Andrey S. Klymchenko

Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch, France

Advanced fluorescence imaging relies on the performance of fluorescent tools – probes, which light up biomolecular processes and cellular structures. They could be divided into molecular and nanoparticle systems. Molecular probes are particularly interesting for advanced fluorescence imaging and sensing at the cellular level. Here, we work on fluorescent molecular probes for imaging biomembranes of cell surface and organelles. [1] Thus, we developed a series of bright probes for cell plasma membranes (MemBright), compatible with multiple cell imaging modalities. [2] Exchangeable probes with efficient ON/OFF switching triggered by membrane binding enabled super-resolution imaging of plasma membranes and deciphering its lipid organization. [3, 4] We also developed a series of solvatochromic probes with organelle-targeting ligands that revealed specific signatures of membrane polarity of organelles and their response to oxidative and mechanical stress. [5] By genetic targeting of a solvatochromic dye, imaging nanoscale environment of proteins in different organelles was realized. [6] Finally, lipid-driven covalent labelling of membrane proteins enabled permanent labelling of cell surface and long-term tracking of cells. To go beyond the limits of brightness of organic dyes, luminescent nanoparticles are an attractive alternative. [7] In particular, it concerns dye-loaded fluorescent polymeric nanoparticles. [8] Their size can be tuned from 7 till 100 nm [9] and their brightness can be 100-fold higher compared to semiconductor quantum dots of similar size. [10] Their small size (below 23 nm) is essential for their free diffusion inside live cells, 9 while their high brightness enable single-particle tracking in mice brain. [11] Using nanoparticles of different color, a technique for long-term barcoding of living cells (chemical analogue of Brainbow) was developed. [12] Functionalization of these NPs with DNA yielded FRET-based color switching nanoprobes for amplified detection nucleic acid cancer markers, [10, 13] and RNA-FISH imaging inside cells. [14]

1. Klymchenko AS. Acc Chem Res 56, 1 (2023).

2. Collot M, et al. Cell Chem Biol 26, 600 (2019).

3. Danylchuk DI, et al. Angew Chem Int Ed 58, 14920 (2019).

4. Aparin IO, et al. J Am Chem Soc 144, 18043 (2022).

5. Danylchuk DI, et al. J Am Chem Soc 143, 912 (2021).

6. Pelletier R, et al. Analytical Chemistry 95, 8512 (2023).

7. Ashoka AH, et al. Chem Soc Rev 52, 4525 (2023).

8. Reisch A, Klymchenko AS. Small 12, 1968 (2016).

9. Reisch A, et al. Adv Funct Mater 28, (2018).

10. Melnychuk N, Klymchenko AS. J Am Chem Soc 140, 10856 (2018).

11. Khalin I, et al. ACS Nano 14, 9755 (2020).

12. Andreiuk B, et al. Small 13, 1701582 (2017).

13. Egloff S, et al. Biosens Bioelectron 179, (2021).

14. Egloff S, et al. ACS Nano 16, 1381 (2022).

Single molecule microscopy as a quantitative tool for biochemical and cell biological in vivo studies in microbiology

Ulrike Endesfelder

Bonn University, Institute for Microbiology and Biotechnology, Bonn, Germany

We are interested in how cellular life arises and is controlled by molecular processes. As an interdisciplinary group with a research focus on microbial cell biology, we use a comprehensive range ofmethods, including molecular biology, biochemistry, fluorescence microscopy, biophysics, and computational approaches. A special emphasis is our methodological focus on quantitative microscopy, particularly single-molecule microscopy, to study the cell biology of a wide variety of microorganisms from all domains of life - archaea, eukaryotes, prokaryotes. We aim to understand how the spatial organization and dynamics of individual molecular players in their cellular environment determines their function and regulates cellular life. In particular, we are interested in transient interactions between molecules and the plasticity of molecular complexes, which are essential machines underlying all cellular life. In this talk, I will discuss the potential of single-molecule approaches for studying (microbial) cell biology, using recent examples from our own research and outlining our future visions. While showcasing our current biological work, I will also put emphasis on the underlying “fuel” of all our projects - our method developments. Next to new fluorescent labels and sample preparation tricks, this includes the creation of smaller analysis packages for single-molecule microscopy data as well as the development of morecomplex software modules such as tracking and simulation software (available through our lab Github).

Physical Modeling of the Genome

Vittore Scolari

Institute Curie, Paris, France

Chromosomes, as primary carriers of genetic information, are located in the cell nucleus and exhibit highly dynamic and heterogeneous mechanical and structural properties. Understanding their function represents a significant frontier in biophysics. Polymer physics, a core discipline of statistical mechanics, plays a crucial role by offering models and methodologies to interpret experimental data. Several unique characteristics define chromosomes as polymers: they are large macromolecules situated in a crowded, confined, and complex environment, interact with histones and other DNA-binding molecules to form structures across multiple scales, and fulfil diverse biological functions through interactions with molecular motors that shape their conformation. This presentation explores how these elements seamlessly integrate into a very general and elegant theoretical framework that is rooted in polymer physics.