Fluorescent probes for biological processes and interactions

Jean-Maurice Mallet (DR CNRS), Blaise Dumat (CR CNRS)

Because of their high spatial and temporal resolutions, their relative ease of implementation and biological safety, fluorescence spectroscopies and microscopies have become a widespread mean of observing biological processes in cellulo and in vivo. Recent developments in fluorescence microscopy have led to new advanced techniques like super-resolution imaging or two-photon-excited fluorescence. The latter, using non-linear excitation with near-infrared light, is of particular interest for in vivo imaging with better light penetration in tissues, lower photodamages, and very low excitation volume (fL) allowing for 3D in-depth imaging of biological tissues. The tremendous development of fluorescence imaging techniques has been concomitant with the design of new fluorescent probes tailored for each application.
With that in mind, we are pursuing in the lab several projects aiming at developing new fluorescent probes for biological imaging with two interdependent objectives:

– Optimizing the fluorescence properties for in vivo imaging: Deep-red or near-infrared emitting biphotonic probes
– Developing smart fluorogenic probes whose emission can be triggered by a biological interaction (protein or calcium probes) or reaction (enzymatic probes).

        • Fluorescent probes for intracellular ions sensing
          • Dual-Input calcium imaging

Calcium is a ubiquitous secondary messenger involved in numerous signaling pathways in the brain and muscles for instance. The diversity of its functions originates from a precise control of its concentration in space and time at the sub-cellular level. Imaging calcium levels in the brain is of particular interest in neurosciences in order to understand this regulation but also as an indirect measure of the electrical activity of neurons. We are currently developing red-emitting biphotonic fluorescent probes able to simultaneously detect a protein of interest and Ca2+ ions in order to perform localized or targeted calcium  imaging in vivo. The use of two-photon microscopy in neurosciences enables in-depth imagin of neuronal networks and the development of new probes with optimized properties is expected to improve the penetration depth and sensitivity during imaging experiments.

Funding: ANR JCJC DIPimaging (2019-2022, PI: Blaise Dumat)

          • Calcium-Rubies

In an earlier project, we have designed red-emitting calcium probes (called Calcium-Ruby) based on a chelating moiety (tetraacid BAPTA) and X-Rhodamine. Without calcium bound, the fluorescence of the  X-rhodamine  is quenched, but  the complexed form is fluorescent during calcium spikes in neurons (in Example X = H, Y = Cl: Kd = 30 µM – The Kd is adjusted by the substituents X or Y).
The lateral side chain with an azide function allows the  immobilization on a quantum dot or  on a modiflied  dextan
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    • Gaillard, S.; Yakovlev, A.; Luccardini, C.; Oheim, M.; Feltz, A.; Mallet, J. Org. Lett. 2007, 9 (14), 2629–2632.
    • Collot, M.; Loukou, C.; Yakovlev, A. V.; Wilms, C. D.; Li, D.; Evrard, A.; Zamaleeva, A.; Bourdieu, L.; Léger, J. F.; Ropert, N.; Eilers, J.; Oheim, M.; Feltz, A.; Mallet, J. M. J. Am. Chem. Soc. 2012, 134 (36), 14923–14931.
    • Oheim, M.; van ’t Hoff, M.; Feltz, A.; Zamaleeva, A.; Mallet, J.-M.; Collot, M. Biochim. Biophys. Acta – Mol. Cell Res. 2014, 1843 (10), 2284–2306.
    • Collot, M.; Wilms, C. D.; Bentkhayet, A.; Marcaggi, P.; Couchman, K.; Charpak, S.; Dieudonné, S.; Häusser, M.; Feltz, A.; Mallet, J.-M. Elife 2015, 4, 1–18.

 

          • H-Rubies (pH probes)

In a similar fashion, we have also designed pH probes based on the association of X-Rhodamine with pH-sensitive moities.


    • Despras, G.; Zamaleeva, A. I.; Dardevet, L.; Tisseyre, C.; Magalhaes, J. G.; Garner, C.; De Waard, M.; Amigorena, S.; Feltz, A.; Mallet, J.-M.; Collot, M. Chem. Sci. 2015, 6 (10), 5928–5937.
    • Zamaleeva, A. I.; Despras, G.; Luccardini, C.; Collot, M.; De Waard, M.; Oheim, M.; Mallet, J. M.; Feltz, A. Sensors (Switzerland) 2015, 15 (9), 24662–24680.

 

        • Fluorescent lipid particles for imaging phagocytosis
        • Enzymatic probes