Periodic Reporting for period 1 - FluoTRAM (Fluorescence-detected Transient Absorption Microscopy)
Période du rapport: 2021-09-01 au 2023-08-31
The goal of the FluoTRAM (fluorescence-detected transient absorption microscopy) project is to combine techniques of fluorescence microscopy and ultrafast laser spectroscopy in a new method of ultrafast fluorescence microscopy applicable to biological samples.
Fluorescence microscopy is an indispensable tool in life sciences and biophysics, where it has been perfected for biological sample imaging either by its autofluorescence or using fluorescent markers such as dyes or fluorescent proteins. For example, it is possible to localize molecules in cells, obtaining information on their local environment and interactions. The fluorescence detection presents a limitation as well, as it provides by its nature information only about on the final, emissive state of the molecules after photoexcitation. Meanwhile, ultrafast nonlinear spectroscopy enables to track the initial state of the molecules after absorption and the following excitation dynamics. However, such techniques typically require volume samples and are detected coherently.
In project FluoTRAM, we implement ultrafast nonlinear transient absorption spectroscopy in the fluorescence microscope equipped with fluorescence lifetime imaging. Using the established imaging techniques and probes, Fluorescence-detected TRansient Absorption Microscopy brings the additional information on the excitation event and the dynamics towards the emissive state. This comprehensive additional information will be of great use in life sciences and beyond, with applications for example to dye probes of interaction between proteins or energy and charge transport in materials for photovoltaics.
Fluorescence microscopy is an indispensable tool in life sciences and biophysics, where it has been perfected for biological sample imaging either by its autofluorescence or using fluorescent markers such as dyes or fluorescent proteins. For example, it is possible to localize molecules in cells, obtaining information on their local environment and interactions. The fluorescence detection presents a limitation as well, as it provides by its nature information only about on the final, emissive state of the molecules after photoexcitation. Meanwhile, ultrafast nonlinear spectroscopy enables to track the initial state of the molecules after absorption and the following excitation dynamics. However, such techniques typically require volume samples and are detected coherently.
In project FluoTRAM, we implement ultrafast nonlinear transient absorption spectroscopy in the fluorescence microscope equipped with fluorescence lifetime imaging. Using the established imaging techniques and probes, Fluorescence-detected TRansient Absorption Microscopy brings the additional information on the excitation event and the dynamics towards the emissive state. This comprehensive additional information will be of great use in life sciences and beyond, with applications for example to dye probes of interaction between proteins or energy and charge transport in materials for photovoltaics.
All work within the project revolved around the combination of ultrafast spectroscopy and fluorescence microscopy. First, a method has been found for generation of spectrally very broad pulses at a high repetition rate (tens of millions of pulses per second) needed for a good signal in the fluorescence microscopy imaging. A newly developed module based on a nonlinear photonic crystal fiber (collaboration with Photonics Bretagne) is capable of creating pulses from ultraviolet to near infrared at 80 MHz repetition rate (see attached photos). Further optical parts have been built, capable of splitting these pulses into a pulse pair with a very precisely determined delay, combining these pulses with another strong excitation pulse, and guiding all these pulses into a fluorescence microscope using a free-space part that keeps the pulses short in time. These pulses were successfully used for imaging, including a method of detecting the fluorescence lifetime of the measured samples simultaneously.
This general arrangement of pulses was used for several different types of measurement. First, on a different setup, we applied such three-pulse sequence to single terylene-disimide molecules. We were able to measure the excitation-induced vibrational motion of these molecules, and wrote a paper about it: D. Fersch, P. Malý et al., “Single-Molecule Ultrafast Fluorescence-Detected Pump–Probe Microscopy”, published in J. Phys. Chem. Lett. 4, 21 (2023), and publicly available in the OPUS repository, doi: 10.25972/OPUS-31348.
The setup in Prague is very versatile and, crucially, includes the imaging microscope. Having the broadband, high-repetition pulse pairs at hand, we developed a new variant of interferometrically detected fluorescence lifetime imaging microscopy, that we call ixFLIM. ixFLIM has one more dimension than standard FLIM (the excitation spectrum), and thus contains much more information within a single measurement. As an example, ixFLIM can quantitatively measure energy transfer between molecules and use it to prove interaction of proteins, which is useful in medicine. We demonstrated ixFLIM on several increasingly complex samples, from dyes to proteins, and wrote a paper about it: P. Malý et al., “ixFLIM: Interferometric Excitation Fluorescence Lifetime Imaging”, is publicly available at the arXiv repository, doi: 10.48550/arXiv.2310.17627.
Finally, the setup with all three pulses measures ultrafast dynamics of excitation in complex molecular samples, spatially resolved in the fluorescence microscope (see attached photo). We used this setup to measure individual perylene-bisimide crystals (from F. Wuerthner group in Wuerzburg) and obtained their nonlinear spectra. However, there is no interesting dynamics, and we only now realize why. At present, we proceed to improve the signal to noise ratio in the working setup and apply it to more suitable samples.
This general arrangement of pulses was used for several different types of measurement. First, on a different setup, we applied such three-pulse sequence to single terylene-disimide molecules. We were able to measure the excitation-induced vibrational motion of these molecules, and wrote a paper about it: D. Fersch, P. Malý et al., “Single-Molecule Ultrafast Fluorescence-Detected Pump–Probe Microscopy”, published in J. Phys. Chem. Lett. 4, 21 (2023), and publicly available in the OPUS repository, doi: 10.25972/OPUS-31348.
The setup in Prague is very versatile and, crucially, includes the imaging microscope. Having the broadband, high-repetition pulse pairs at hand, we developed a new variant of interferometrically detected fluorescence lifetime imaging microscopy, that we call ixFLIM. ixFLIM has one more dimension than standard FLIM (the excitation spectrum), and thus contains much more information within a single measurement. As an example, ixFLIM can quantitatively measure energy transfer between molecules and use it to prove interaction of proteins, which is useful in medicine. We demonstrated ixFLIM on several increasingly complex samples, from dyes to proteins, and wrote a paper about it: P. Malý et al., “ixFLIM: Interferometric Excitation Fluorescence Lifetime Imaging”, is publicly available at the arXiv repository, doi: 10.48550/arXiv.2310.17627.
Finally, the setup with all three pulses measures ultrafast dynamics of excitation in complex molecular samples, spatially resolved in the fluorescence microscope (see attached photo). We used this setup to measure individual perylene-bisimide crystals (from F. Wuerthner group in Wuerzburg) and obtained their nonlinear spectra. However, there is no interesting dynamics, and we only now realize why. At present, we proceed to improve the signal to noise ratio in the working setup and apply it to more suitable samples.
The newly built, working setup is unique in the world. Already the two-pulse excitation with fluorescence lifetime imaging (ixFLIM) is novel, and provides an excellent way for researchers in biophysics and life sciences to learn more information about the samples they study. Regarding the three-pulse technique, its application to single molecules is really pushing the boundaries, showing what is possible in ultrafast spectroscopy. The lessons we learn from the combination with imaging are valuable for the growing community of researchers that apply the fluorescence detected nonlinear spectroscopy.