My research aims to understand the light-harvesting ability of photosynthetic organisms and their regulation mechanisms in response to the spectral fluctuation of the environment light. I develop and use microscopic spectroscopies to investigate the natural photosynthetic system (In vivo and in vitro) including cryogenic optical microscopy, single-molecule spectroscopy, time-resolved spectroscopy, super-resolution spectral microscopy, and so on (Journal of Photochemistry and Photobiology C., 2023, 100616).
Main Themes
1. The Development of Excitation Spectral Microscopy
For the pigments-protein supercomplexes (photosynthetic complexes), it is well known that the excitation energy rapidly transferred from the high-energy pigments to the low-energy pigments. The remaining energy is emitted in the form of fluorescence from the terminal pigments with the lowest energy within supercomplexes. The traditional fluorescence microscope is unsatisfactory for the measurement of high-energy pigments because these molecules within natural photosynthetic complexes in practice hardly emit fluorescence.
Thus, we developed a high-speed excitation spectral microscope (ESM) that enables the efficient acquisition of excitation spectra in the small spatial scale ~ submicron (see Biophysical Journal, 2020, 118 (1), 36-43; Plant Cell Physiology, 2021, 62 (5), 872-882). Since fluorescence excitation spectroscopy is very sensitive to the measurement of the pigments with high-lying states, this progress in the fluorescence spectral microscope could guide efforts in the spatial-resolved studies of the high-energy pigments-related mechanisms, such as state transition, non-photochemical quenching, and so on.
2. Light-Harvesting Mechanisms and Ultrastructural Changes of Thylakoid Membrane
Photosynthetic organisms adjust to fluctuating natural light under physiological ambient conditions through the flexible light-harvesting ability of light-harvesting complex II (LHCII). A process called state transition is an efficient regulation mechanism to balance the excitations between photosystem II (PSII) and photosystem I (PSI) by shuttling mobile LHCII between them (Journal of Photochemistry and Photobiology B., 2022, 236, 112584). Some studies have assumed coupling of the ultrastructural dynamics of the thylakoid membrane to the state transition (ST) mechanism, how ST is related to the ultrastructural dynamic of the thylakoid in Chlamydomonas remains elusive.
To clarify the above-mentioned relation, here we used two specialized microscope techniques, observation via the excitation spectral microscope (ESM) developed recently by us and the super-resolution imaging based on structured illumination microscopy (SIM). The ESM observation revealed changes in the intracellular arrangement of LHCII-related fluorescence. More importantly, it clarified lower ST activity in the region surrounding the pyrenoid, which is the specific subcellular compartment associated with the carbon-fixation reaction. On the other hand, the SIM observation resolved partially irreversible fine thylakoid transformations induced by the ST-inducing illumination. Fine irreversible thylakoid transformation was also observed for the STT7-kinase-lacking mutant. This result, together with the nearly equal structural changes in the less active ST regions around the pyrenoid, suggested the independence of the observed fine structural changes from the LHCII phosphorylation (Proceedings of the National Academy of Sciences, 2022, 119 (37), e2122032119).
3. Single Molecule Spectroscopy (SMS)
In the development of single-molecule spectroscopy, the simultaneous detection of the excitation and emission spectra has been limited. The fluorescence excitation spectrum based on background-free signals is compatible with the fluorescence-emission-based detection of single molecules and can provide insight into the variations in the input energy of the different terminal emitters.
The first attempt in the simultaneous acquirements of excitation-emission spectroscopy in single molecules was completed in Proceedings of the National Academy of Sciences, 2019, 116 (10) 4064-4069. Here, we updated our excitation spectral microscope (ESM) into a low-temperature-compatible mode. We developed a cryogenic single-molecule excitation-emission spectroscopy (SMEES) to study single pigment-protein complexes. The SMEES technique could acquire a 2D-excitation-emission matrix of a single molecule, thereby realizing the rapid simultaneous acquisition of excitation-emission spectra of single complexes at 80 K (The Journal of Physical Chemistry B, 2024, 128, 11, 2664-2674). This method makes the analysis of the excitation-emission correlation in a single molecule possible, in principle, it can be extended to all light-harvesting systems.
4. Super-Resolution Microscopic Spectroscopies
Stimulated emission depletion (STED), a super-resolution imaging technique was awarded the Nobel Prize in Chemistry 2014 (Optics Letters, 1994, 19 (11) 780-782). STED introduced a physical method in which two lasers are used to illuminate a sample where the ground electrons are pumped to the excitation state by an excitation laser and the excited electrons subsequently transfer to their ground state upon a donut-shaped STED laser input, causing the fluorescence spots from samples to get smaller so that the spatial resolution is increasing (Figure A). Generally, under room temperature STED laser easily leads to an anti-stokes excitation of molecules within photosynthetic organisms rather than the occurrence of stimulation radiation (Figure B). This is because the ideal wavelengths for the depletion of the electronic excitation state are probably different for different pigments and their multiple spectral forms. Therefore, it is difficult to apply the STED principle to photosynthetic organisms.
Theoretically, STED and cryogenic optical microscopy are an optimal combination because cryo-cooling reduces the electron transition from the vibrational ground state to the excited state (anti-stokes excitation effect) giving rise to the stimulated radiation more easily occurring. We are challenging the realization of super-resolution spectral microscope by integrating the STED method into our cryogenic microscope.
