Dual-Color Photoswitchable Fluorescent Probes for Advanced Bioimaging
Photoconvertible fluorescent molecules are powerful tools in bioimaging to unambiguously track labeled biomolecules over large spatiotemporal scales. In this field photoconvertible fluorescent proteins are predominant but suffer from several drawbacks. Conversely molecular probes are characterized by their small size and ease of use. Surprisingly, dual-color photoswitchable fluorophores (DCPSF) able to switch from a bright color form to another, and thus that can be advantageously detected prior to conversion, are only poorly developed and remain extremely rare. This project aims at developing new and bright DCPSFs for applications in advanced bioimaging. It will provide an extensive comprehension in the development of molecular DCPSF and the developed probes will be of wide use in the field of bioimaging.<br />The main objectives are:<br />- Establishing the mechanism involved in the photoconversion process. Based on our preliminary results with BODIPY-based DCPSF, we will determine the photoconverison mechanism by mean of spectroscopic analysis.<br />- Designing and synthesizing new DCPSF based on this mechanism with controlled photoswitching properties (conversion kinetic).<br />- Developing DCPSFs that will fulfill as many as possible requirements for bioimaging like: high photostability, extensive palette of available colors.<br />- The developed DCPSF will all bear a clickable moiety allowing post functionalization in order to target not only proteins (by SNAP or Halo tag) but also specific organelles by use of specific targeting moieties.<br />- Finally, the probes will be evaluated in cells using conventional microscopy and then used in bioimaging with advanced microscopy techniques (Tracking, FRAP, super resolution).
To prove the specificity of our switching moiety, we first conjugate various moieties to a coumarin fluorophore by Wittig reaction to obtain styryl-coumarin dyes. This approach confirmed that we identified a defined switching moiety able to provide DCPSF and helped in establishing a photoconversion mechanism. Once synthesized, the dyes are analyzed by spectroscopy to determine their absorption and emission spectra in various conditions as well as their extinction coefficient and quantum yield. In a second time, the probes are photoconverted under laser irradiation and the fluorescence spectra is instantaneously monitored and recorded to evaluate the properties of the switched form as well as to establish the kinetic rate. Thereafter, the switched form is analyzed by steady state spectroscopy, time-resolved fluorescence spectroscopy and HPLC coupled to mass spectroscopy.
Once the mechanism established, the switching properties has been tuned by chemical modifications on the switching moiety. Electronic and steric effect are expected to modulate the kinetic rate of the conversion. Studies are ongoing. Then, the switching moiety has been coupled to various fluorophores to exemplify our technology and to provide a wide palette of probes with different colors. Once again chemical modification will help in tuning the properties of the non-switched and switched forms. Once the DCPSF with the best properties obtained, they will be functionalized by click chemistry to label tagged proteins (SNAP, Clip, Halo, etc.) and to target specific organelles (mitochondria, actin fibers, Golgi etc.). The final probes will be evaluated in conventional fluorescence microscopy before being involved in advanced microscopy techniques (Tracking, FRAP, super resolution).
First of all, by conjugating styryl-coumarins with various structurally close moieties we demonstrated the special ability of our switching moiety to provide DCPSF. Then, spectroscopic studies with laser irradiations and HPLC/Mass analysis allowed to unambiguously establish the photoconversion mechanism. The switching moiety undergo a photooxidation followed by a solvolysis leading to the discontinuation of the conjugation between the switching moiety and the fluorophore thus provoking a significant hypsochromic shift in emission. We also established that this mechanism operates only if the coupled fluorophore was sufficiently electron-rich. Alternatively, we discovered a fluorogenic photoisomerization process leading to photocaged probes which is not related to our switching moiety. Then by chemical modifications on the switching moiety to induce electronical and steric effects, we try to modulate and control the conversion rate.
To obtain brighter probes, we then applied our technology to styryl-BODIPYs. The latter are efficient DCPSF with emission shift of 100 nm between the original form and the switched form. Moreover, these new DCPSF have absorption and emission spectra perfectly adapted to the microscopy. In parallel, we discovered that when the switching moiety is placed at a certain position of the BODIPY core, the fluorescence of the latter is quenched. However, under irradiation, the bright fluorescence of the BODIPY is turned on, thus constituting a new mechanism of fluorogenic photocaging. The first cellular studies showed that the non-targeted probes were cell permeant and were able to photoconvert using a laser scanning confocal microscope. Our preliminary results showed that the conversion was too efficient and needed to be slowed down for an optimal use in bioimaging.
This project has already allowed to decipher a new photoconversion mechanism based on photo oxidation of the switching moiety and applicable to two fluorophore families (BODIPY and coumarins). The control of this reaction leads to dual-color photoswitchable fluorescent probes which are expected to find their applications in bioimaging. It is planned to patent this technology. The challenge now is to control this conversion by chemical engineering and by spectroscopic study. DCPSFs displaying the most advantageous features for the bioimaging will be used to label proteins of interest (by SNAP, Halo tag) or will target specific organelles by coupling with targeting groups. Ultimately, this project should be able to provide dual-color photoswitchable fluorescent probes with conversion rates, photo-physical properties (color, photostability, brightness) and phototoxicity suitable for the study of biological processes and the monitoring of biomolecules by microscopy. To do endeavor, we plan to continue to extend our technology to several different fluorophores.
To date, there is no scientific production yet. A patent application will be made soon. The publication startegy will follow thereafter.
Photosensitive or phototransformable fluorescent molecules able of photoactivation, photoswitching or photoconverison are powerful tools in bioimaging to unambiguously track labeled biomolecules over large spatiotemporal scales. In addition to this feature these molecules have led to significant advances in biophotonics due to their ability to be used in super resolution imaging (PALM and STORM). Since the discovery of paGFP in 2002, phototransformable fluorescent proteins are predominant in the field of bioimaing. Although this approach is robust and powerful it is not universal as it is limited to proteins and not straightforward, as it requires a transfection step thus leading to heterogeneous samples and toxic effects. Conversely, molecular probes are characterized by their ease of use; they homogenously stain the cells and can be used in tissue imaging. Moreover their accessible chemical modifications offer more possibilities for improvement compared to proteins. Although photoactivatable fluorophores have drawn a notable attention, they only reveal their fluorescence upon activation thus mainly finding their use in super resolution imaging. Similarly, photoswitchable fluorophores generally switch from non-emissive state to a fluorescent form and require UV irradiation, which is phototoxic. Surprisingly, dual-color photoswitchable fluorophores (DCPSF) able to switch from a bright color to another, and thus that can be advantageously detected prior to conversion, were only poorly developed. While complex FRET pair system that can be separated by photocleavage have been proposed, the chemical development of single molecular DCPSF remains extremely rare. Indeed in most studies, dual color switching properties were evaluated on existing commercially available fluorophores like AlexaFluor 647 and cyanines thus not allowing an extensive comprehension in the development of dual-color photoswitchable fluorophores.
This project aims at developing bright and multicolor small Dual-Color Photoswitchable Fluorophores (DCPSFs) able to switch from a color to another by connecting conjugated photoactivatable moieties to the p-system of fluorescent dyes. This project is based on promising preliminary results where organelle-specific (mitochondria and plasma membrane) DCPSFs were synthesized and successfully used in photoconversion and super resolution imaging. We herein propose to develop DCPSF bearing a clickable moiety allowing post functionalization in order to target not only proteins (by SNAP or Halo tag) but also specific organelles (mitochondria, nucleus, reticulum, etc) using identified targeting moieties to provide universal tools in cellular imaging. This project also aims at understanding the mechanism leading to the photoswitching by establishing a structure/photophysical-properties relationship involving advanced photophysical caracterizations. Once fully characterized (brightness, photostability, reversibility, etc), the probes will be evaluated in cells using conventional microscopy and then used in bioimaging with advanced microscopy techniques including tracking, FRAP and super resolution. The project will provide an extensive comprehension in the development of molecular DCPSF and the developed probes will be of wide use and interest in the field of bioimaging.
Monsieur Mayeul COLLOT (Laboratoire de Bioimagerie et Pathologies (UMR 7021))
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.
LBP_UNISTRA Laboratoire de Bioimagerie et Pathologies (UMR 7021)
Help of the ANR 193,840 euros
Beginning and duration of the scientific project: September 2019 - 42 Months