Publications
Published Articles
Minimal reproduction and calibration of a fluorescence microscope for single-cell imaging
Aliénor Lahlou, Ian Coghill, Alec Cochard, Ali Ruyer-Thompson, David Colliaux, Thierry Legou, Thomas Le Saux, Peter Hanappe, Ludovic Jullien
Journal of Open Hardware: https://doi.org/10.5206/joh.v10i1.22683
Open Access link: https://zenodo.org/records/22145441
Published on March 2026
Microscopy is a critical experimental tool in life sciences. However, when attempting to reproduce or scale-up a published method, researchers often meet difficulties in implementing specific protocols, often because they do not have full control over the hardware and software. We illustrate these challenges by demonstrating how we reproduced a single-cell fluorescence experiment from the literature. We describe how we leveraged Open-Source hardware and software tools to adapt the design of the original fluorescence microscope and the choices we made. Our modular approach gives researcher affordable tools to reproduce experimental setups and implement new protocols. It also gives a simple way to develop experiments with a persistent record of their prototype’s evolution, even at the developmental stage. We provide comprehensive guidance on building and calibrating the microscope hardware, implementing automation, and data analysis and storage methods. The highly modular Python software we introduce facilitate customization and reusability beyond microscopy, empowering researchers and engineers to adapt components for other scientific applications.
Interplay between high-energy quenching and state transitions in Chlamydomonas reinhardtii: a single-cell approach
Aliénor Lahlou, Marcelo Orlando, Sandrine Bujaldon, William Gaultier, Eliora Israelievitch, Peter Hanappe, Thomas Le Saux, Ludovic Jullien, David Colliaux, Benjamin Bailleul
New Phytologist: https://doi.org/10.1111/nph.71001
Open Access link: https://zenodo.org/records/22145648
Published on March 2026
- Studying cell-to-cell heterogeneity is essential to understand how unicellular organisms respond to stresses. We introduce a single-cell analysis framework that enables the study of intercellular heterogeneity of photosynthetic traits, particularly their interactions within individual cells that have identical genotypes, cellular contexts and histories.
- Our approach combines single-cell imaging of Chla fluorescence with machine learning, and we study light stress responses in Chlamydomonas reinhardtii as a proof of concept.
- This framework allows us to score the extent of high-light responses such as state transitions (qT) and high-energy quenching (qE). We reveal significant cell-to-cell heterogeneity and a strong correlation between qT and qE, undetectable in bulk measurements.
- This study highlights the value of single-cell phenotypic analysis for investigating light stress responses in unicellular organisms. We detail the key aspects that come into play to generalize the method to other complex stress responses involving multiple traits.
Versatile luminescence macroscope with dynamic illumination for photoactive systems
Ian Coghill, Alienor Lahlou, Andrea Lodetti, Shizue Matsubara, Johann Boucle, Thomas Le Saux, and Ludovic Jullien
Optics Express: https://doi.org/10.1364/OE.570450
Open Access link: https://doi.org/10.34734/FZJ-2025-04396
Published on November 2025
Luminescence imaging is invaluable for studying biological and material systems, particularly when advanced protocols that exploit temporal dynamics are employed. However, implementing such protocols often requires custom instrumentation, either modified commercial systems or fully bespoke setups, which poses a barrier for researchers without expertise in optics, electronics, or software. To address this, we present a versatile macroscopic fluorescence imaging system capable of supporting a wide range of protocols, and provide detailed build instructions along with open-source software to enable replication with minimal prior experience. We demonstrate its broad utility through applications to plants, reversibly photoswitchable fluorescent proteins, and optoelectronic devices.
Constitutive and regulatory responses of Arabidopsis thaliana to harmonically oscillating light
Yuxi Niu, David Fuente, Shizue Matsubara, Dušan Lazár, Ladislav Nedbal
Physiologia Plantarum: https://doi.org/10.1111/ppl.70421
Open Access link: https://pmc.ncbi.nlm.nih.gov/articles/PMC12312291/
Published on July 2025
The rate of net CO2 uptake is proportional to dim light and saturates when the light exceeds the plant assimilation capacity. This simple relationship between constant light and photosynthesis becomes intriguingly complex when the light oscillates. The rates of photosynthesis may differ between the descending and ascending phases of light oscillation. This hysteresis changes with the frequency and amplitude of the light and reports on the dynamics of the photosynthetic reactions and their regulation. Here, we investigated the chlorophyll fluorescence response of Arabidopsis thaliana to light oscillating with three different amplitudes: 100–200, 100–400, and 100–800 μmol photons·m-2·s-1, each with periods ranging from 1 s to 8 min. The light amplitudes and periods were chosen to represent light patterns often appearing in nature. Three genotypes were compared: wild-type Col-0 and npq1 and npq4 mutants that are incapacitated in the rapidly reversible energy-dependent non-photochemical quenching (qE). The experiments identified two major dynamic patterns. One was found in oscillation periods shorter than 30 s, characterized by constitutive non-linearity and hysteresis. The other was mainly formed by regulatory non-linearity and hysteresis, occurring when the oscillation periods were longer than 30 s. The mathematical model simulating the chlorophyll fluorescence dynamics qualitatively reproduced the constitutive and regulatory dynamic patterns observed in the experiments. The model simulations illustrated the dynamics of non-photochemical quencher activation, plastoquinone pool reduction, and lumen pH that form the constitutive and regulatory non-linearities. The model simulations provided mechanistic insight into molecular processes forming the plant response to oscillating light.
A Reaction-Diffusion Frame for Accessing Metabolic O2 Fluxes in Single Microalgal Cells with Low-Cost Wide-Field Imaging of Nanosensor Luminescence Lifetime
Hélène Merceron, Eliora Israelievitch, Valentine Rollot, Théo Villarubias, Xiaojiang Xie, Thomas Le Saux, Karim Benzerara, François Guyot, Alix Boulouis, Emmanuelle Marie-Bègue, Laurent Thouin, Ludovic Jullien
Advanced Science: https://doi.org/10.1002/advs.202510903
Open Access link: https://pmc.ncbi.nlm.nih.gov/articles/PMC12622426/
Published: August 2025
In cells, many small molecules are membrane-permeant. This feature opens a road to analyze their flux of production or consumption by quantitatively interpreting the map of their extracellular concentration within a reaction-diffusion frame. Here, this approach is implemented with a new wide-field lifetime imaging protocol applied to single microalgae cells sparsely deposited on an agarose pad loaded with a luminescent dioxygen (O2) nanosensor. The resulting maps are processed to access the spatial distribution of the O2 concentration in the plane of the cells. After fitting the data, the cellular O2 flux is extracted, evidencing a span of magnitudes and angular dependencies in the balance between photosynthesis and respiration. Beyond pointing to the disparity of individual behavior within the same colony, this work validates a simple approach for characterizing metabolic fluxes of membrane-permeant molecules down to the single-cell level.
A Modular Low-Cost 3D Uniformly Illuminated Mini-Photoreactor with Versatile In-Situ Analysis
Advanced Optical Materials: https://doi.org/10.1002/adom.202403466
Open Access link: https://zenodo.org/records/22145782
Published: February 2025
Photons are widely used for producing and degrading matter, yet current approaches for their delivery to unstirred light-absorbing media give poor uniformity and energetic efficiency. Here, a cheap modular Do-It-Yourself (DIY)-realizable mini-photoreactor is introduced that addresses these issues, and additionally allows for in situ absorption, fluorescence, and Nuclear Magnetic Resonance (NMR) spectroscopy. Its first configuration features an internal light-scattering cylindrical liquid-filled waveguide to scatter light outward into the medium, while its second configuration, meant for more strongly absorbing media, adds an external coaxial light-scattering liquid-filled envelope, allowing balanced light injection from both sides of the medium. Extensive NMR- and fluorescence-based characterizations with new actinometers, together with optical simulations, are performed; the latter allowing the key factors that govern photon flux density and its distribution to be unraveled. The insights go beyond the reactor design, and have relevance in analytical photochemistry and photocatalysis, where poor characterization of light is widespread.
Periodic Light Modulations for Low-Cost Wide-Field Imaging of Luminescence Kinetics Under Ambient Light
Advanced Science: https://doi.org/10.1002/advs.202413291
Open Access link: https://pmc.ncbi.nlm.nih.gov/articles/PMC11904971/
Published: January 2025
Imaging luminescence kinetics is invaluable in many fields, including biology and chemistry. However, the luminescence lifetime of most photo-activated states is in the low ns-µs range and its measurement requires adding costly image intensifiers to cameras to access the fast phenomena present. Here, the Rectified Imaging under Optical Modulation (RIOM) and Heterodyne Imaging under Optical Modulation (HIOM) protocols make this possible with standard low-cost cameras only, even under ambient light. RIOM and HIOM originate from a thorough theoretical analysis, which showed that modulated illumination of any reversibly photoactivable luminophore probed at high frequency generates components of the luminescence response that are detectable at low frequency: RIOM harnesses the mean luminescence response to modulated light at a single frequency, whereas HIOM exploits the luminescence response to modulated light at two close frequencies. When the luminophore behavior obeys a two-state model, the frequency dependence of the RIOM and HIOM observables can be used to extract a photoactivation time. In this work, their ability to retrieve maps of the luminescence lifetime of a reversibly photoswitchable protein, and phosphorescent microsensors are demonstrated. Where the luminophore does not obey a two-state model, RIOM and HIOM can still retrieve rich information, as here exemplified by the recovery of kinetic fingerprints of the physiological state of a plant.
Leaves to Measure Light Intensity
Advanced Science | https://doi.org/10.1002/advs.202304420
Open Access link: https://pmc.ncbi.nlm.nih.gov/articles/PMC11423135/
Published: September 2024
Quantitative measurement of light intensity is a key step in ensuring the reliability and the reproducibility of scientific results in many fields of physics, biology, and chemistry. The protocols presented so far use various photoactive properties of manufactured materials. Here, leaves are introduced as an easily accessible green material to calibrate light intensity. The measurement protocol consists in monitoring the chlorophyll fluorescence of a leaf while it is exposed to a jump of constant light. The inverse of the characteristic time of the initial chlorophyll fluorescence rise is shown to be proportional to the light intensity received by the leaf over a wide range of wavelengths and intensities. Moreover, the proportionality factor is stable across a wide collection of plant species, which makes the measurement protocol accessible to users without prior calibration. This favorable feature is finally harnessed to calibrate a source of white light from exploiting simple leaves collected from a garden.
A mathematical model to simulate the dynamics of photosynthetic light reactions under harmonically oscillating light
David Fuente, Marcelo Orlando, Benjamin Bailleul, Ludovic Jullien, Dušan Lazár, Ladislav Nedbal
Plant Physiology and Biochemistry | https://dx.doi.org/10.2139/ssrn.4810070
Open Access link: https://zenodo.org/records/22098257
Published: September 2024
Alternating electric current and alternating electromagnetic fields revolutionized physics and engineering and led to many technologies that shape modern life. Despite these undisputable achievements that have been reached using stimulation by harmonic oscillations over centuries, applications in biology remain rare.
Photosynthesis research is uniquely suited to unleash this potential because light can be modulated as a harmonic function, here sinus. Understanding the response of photosynthetic organisms to sinusoidal light is hindered by the complexity of dynamics that such light elicits, and by the mathematical apparatus required for understanding the signals in the frequency domain which, although well-established and simple, is outside typical curricula in biology.
Here, we approach these challenges by presenting a mathematical model that was designed specifically to simulate the response of photosynthetic light reactions to light which oscillates with periods that often occur in nature. The independent variables of the model are the plastoquinone pool, the photosystem I donors, lumen pH, ATP, and the chlorophyll fluorescence (ChlF) quencher that is responsible for the qE non-photochemical quenching. Dynamics of ChlF emission, rate of oxygen evolution, and non-photochemical quenching are approximated by dependent model variables.
The model is used to explain the essentials of the frequency-domain approaches up to the level of presenting Bode plots of frequency-dependence of ChlF. The model simulations were found satisfactory when compared with the Bode plots of ChlF response of the green alga Chlamydomonas reinhardtii to light that was oscillating with a small amplitude and frequencies between 7.8 mHz and 64 Hz.
Dynamics and interplay of photosynthetic regulatory processes depend on the amplitudes of oscillating light
Plant, Cell & Environment | https://doi.org/10.1111/pce.14879
Open Access link: https://doi.org/10.34734/FZJ-2024-02707
Published: June 2024
Plants have evolved multiple regulatory mechanisms to cope with natural light fluctuations. The interplay between these mechanisms leads presumably to the resilience of plants in diverse light patterns. We investigated the energy-dependent nonphotochemical quenching (qE) and cyclic electron transports (CET) in light that oscillated with a 60-s period with three different amplitudes. The photosystem I (PSI) and photosystem II (PSII) function-related quantum yields and redox changes of plastocyanin and ferredoxin were measured in Arabidopsis thaliana wild types and mutants with partial defects in qE or CET. The decrease in quantum yield of qE due to the lack of either PsbS- or violaxanthin de-epoxidase was compensated by an increase in the quantum yield of the constitutive nonphotochemical quenching. The mutant lacking NAD(P)H dehydrogenase (NDH)-like-dependent CET had a transient significant PSI acceptor side limitation during the light rising phase under high amplitude of light oscillations. The mutant lacking PGR5/PGRL1-CET restricted electron flows and failed to induce effective photosynthesis control, regardless of oscillation amplitudes. This suggests that PGR5/PGRL1-CET is important for the regulation of PSI function in various amplitudes of light oscillation, while NDH-like-CET acts’ as a safety valve under fluctuating light with high amplitude. The results also bespeak interplays among multiple photosynthetic regulatory mechanisms.
From leaf to multiscale models of photosynthesis: applications and challenges for crop improvement
Alexandrina Stirbet, Ya Guo, Dušan Lazár & Govindjee Govindjee (corresponding author)
Photosynthesis Research | https://doi.org/10.1007/s11120-024-01083-9/
Open Access link: https://zenodo.org/records/14202548
Published: April 2024
To keep up with the growth of human population and to circumvent deleterious effects of global climate change, it is essential to enhance crop yield to achieve higher production. Here we review mathematical models of oxygenic photosynthesis that are extensively used, and discuss in depth a subset that accounts for diverse approaches providing solutions to our objective. These include models (1) to study different ways to enhance photosynthesis, such as fine-tuning antenna size, photoprotection and electron transport; (2) to bioengineer carbon metabolism; and (3) to evaluate the interactions between the process of photosynthesis and the seasonal crop dynamics, or those that have included statistical whole-genome prediction methods to quantify the impact of photosynthesis traits on the improvement of crop yield. We conclude by emphasizing that the results obtained in these studies clearly demonstrate that mathematical modelling is a key tool to examine different approaches to improve photosynthesis for better productivity, while effective multiscale crop models, especially those that also include remote sensing data, are indispensable to verify different strategies to obtain maximized crop yields.
Continual Learning with Deep Streaming Regularized Discriminant Analysis
Joe Khawand, Peter Hanappe, David Colliaux
Institute of Electrical and Electronics Engineers | 10.1109/ICCVW60793.2023.00370
Open Access link: https://arxiv.org/abs/2309.08353
Published: December 2023
Continual learning is increasingly sought after in real world machine learning applications, as it enables learning in a more human-like manner. Conventional machine learning approaches fail to achieve this, as incrementally updating the model with non-identically distributed data leads to catastrophic forgetting, where existing representations are overwritten. Although traditional continual learning methods have mostly focused on batch learning, which involves learning from large collections of labeled data sequentially, this approach is not well-suited for real-world applications where we would like new data to be integrated directly. This necessitates a paradigm shift towards streaming learning. In this paper, we propose a streaming version of regularized discriminant analysis as a solution to this challenge. We combine our algorithm with a convolutional neural network and demonstrate that it outperforms both batch learning and existing streaming learning algorithms on the ImageNet ILSVRC-2012 dataset.
A series of caged fluorophores for calibrating light intensity
Mrinal Mandal, Hessam Sepasi Tehrani, Qianhua Mai, Emma Simon, Marie-Aude Plamont, Christine Rampon, Sophie Vriz, Isabelle Aujard, Thomas Le Saux, and Ludovic Jullien
Chemical Science | https://doi.org/10.1039/D3SC04183B
Open Access link: https://pmc.ncbi.nlm.nih.gov/articles/PMC10699554/
Published: November 2023
Absolute measurement of light intensity is sought for in multiple areas of chemistry, biology, physics, and engineering. It can be achieved by using an actinometer from analyzing the time-course of its reaction extent on applying constant light. However, most reported actinometers exploit the absorbance observable for reporting the reaction extent, which is not very sensitive nor relevant in imaging systems. In this work, we report a series of hydrophobic and hydrophilic caged fluorophores that overcome the preceding limitations. Based on the robust pyranine backbone, they can easily be synthesized on a large scale in one to a few steps. Their brightness increases over illumination and their uncaging cross-sections have been thoroughly characterized upon one- and two-photon excitation. As a demonstration of their use, we calibrated light intensity in various chemical and biological samples, which have been observed with epifluorescence and confocal imaging systems.
Fluorescence to measure light intensity
Aliénor Lahlou, Hessam Sepasi Tehrani, Ian Coghill, Yuriy Shpinov, Mrinal Mandal, Marie-Aude Plamont, Isabelle Aujard, Yuxi Niu, Ladislav Nedbal, Dusan Lazár, Pierre Mahou, Willy Supatto, Emmanuel Beaurepaire, Isabelle Eisenmann, Nicolas Desprat, Vincent Croquette, Raphaël Jeanneret, Thomas Le Saux ✉️ & Ludovic Jullien ✉️
Nature Methods | https://doi.org/10.1038/s41592-023-02063-y
Open Access link: https://pmc.ncbi.nlm.nih.gov/articles/PMC10703675/
Published: November 2023
Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources.
Dynamic monitoring of photosynthesis in light-acclimated organisms
Aliénor Lahlou
Biological Physics [physics.bio-ph]. Sorbonne Université, 2023. English. ⟨NNT : 2023SORUS384⟩. ⟨tel-04703169⟩
This CIFRE PhD work between ENS and Sony CSL Paris led to design a new instrument and new protocols to characterize the physiological status of photosynthetic organisms. This objective was addressed at the single-cell level to quantify 3 light-stress response mechanisms in the microalgae Chlamydomonas reinhardtii. It further lays the grounds of the European project DREAM and addresses the constraints that come with the level of generalization and applicability of DREAM. Chapter 1 describes the specifications for the epifluorescence microscope developed. A strong accent was put on modularity and automation. The long-term DREAM objective to provide an affordable portable device was also taken into account in the design and hardware choices. The calibration and quality control of the instrument is described. The calibration of light intensity led to an extended work on the use of fluorescent actinometers in the visible spectrum, in 1D, 2D and 3D. It resulted in an accepted manuscript entitled «Fluorescence to measure light intensity». Chapter 2 deals with the role of chlorophyll fluorescence (ChlF) in remote sensing of photosynthesis. The concept of de-excitation pathways of the energy harvested by the photosynthetic organisms is described, allowing to introduce the classical methods in ChlF remote sensing. The submitted manuscript «Leaves to measure light intensity» is introduced to describe the diversity of light-dependent phenomena in plants. The challenge to adapt the current protocols to an imaging system with a CMOS camera at the single-cell algae level was lifted by using a new light protocol, and validated. Chapter 3 describes in a manuscript the case study of the light-stress response in Chlamydomonas reinhardtii after having introduced key concepts in the biological process studied and the computational methods used. The experiments consisted in collecting a video of ChlF time traces, and retrieve the individual responses by image segmentation. The three light-stress responses studied are entangled and have dynamic signatures that overlap within the ChlF time trace. A machine learning framework was introduced to convert the ChlF traces into stress scores. A reference dataset was built to train the framework. Biological protocols including mutant strains and sample preparation were introduced to probe the stresses separately and build the reference populations of the dataset. The framework consisted in two parts. Dictionary learning first reduced the dimension of the ChlF traces with optimized reconstruction. It was followed by a dimension reduction using Linear Discriminant Analysis to maximize the separability of the annotated classes. This combination allowed to represent a general stress response along three dimensions, each axis representing one single stress response. This method could unmix the stress responses within the ChlF traces of wild-type strains. An emphasis was put on the reconstruction capability of the pipeline, which can be used as a criterion to validate the hypothesis made to build the latter, or to identify new stress-responses. Chapter 4 presents in a manuscript the open-source hardware reproduction of the instrument, demonstrating the reproducibility of the work but also providing instructions to allow the community to reproduce it. It also introduces tools to control in a modular way several devices and to implement a database system which can be useful when designing Data Management Plans.
Plants cope with fluctuating light by frequency-dependent nonphotochemical quenching and cyclic electron transport
New Phytologist | https://doi.org/10.1111/nph.19083
Open Access link: https://doi.org/10.34734/FZJ-2023-02631
Published: 10 July 2023
In natural environments, plants are exposed to rapidly changing light. Maintaining photosynthetic efficiency while avoiding photodamage requires equally rapid regulation of photoprotective mechanisms. We asked what the operation frequency range of regulation is in which plants can efficiently respond to varying light.
Chlorophyll fluorescence, P700, plastocyanin, and ferredoxin responses of wild-types Arabidopsis thaliana were measured in oscillating light of various frequencies. We also investigated the npq1 mutant lacking violaxanthin de-epoxidase, the npq4 mutant lacking PsbS protein, and the mutants crr2-2, and pgrl1ab impaired in different pathways of the cyclic electron transport.
The fastest was the PsbS-regulation responding to oscillation periods longer than 10 s. Processes involving violaxanthin de-epoxidase dampened changes in chlorophyll fluorescence in oscillation periods of 2 min or longer. Knocking out the PGR5/PGRL1 pathway strongly reduced variations of all monitored parameters, probably due to congestion in the electron transport. Incapacitating the NDH-like pathway only slightly changed the photosynthetic dynamics.
Our observations are consistent with the hypothesis that nonphotochemical quenching in slow light oscillations involves violaxanthin de-epoxidase to produce, presumably, a largely stationary level of zeaxanthin. We interpret the observed dynamics of photosystem I components as being formed in slow light oscillations partially by thylakoid remodeling that modulates the redox rates.
Resonances at Fundamental and Harmonic Frequencies for Selective Imaging of Sine-Wave Illuminated Reversibly Photoactivatable Labels
ChemPhysChem | doi.org/10.1002/cphc.202200295
Open Access link: https://pmc.ncbi.nlm.nih.gov/articles/PMC10087976/
Published: 17 August 2022
We introduce HIGHLIGHT as a simple and general strategy to selectively image a reversibly photoactivatable fluorescent label associated with a given kinetics. The label is submitted to sine-wave illumination of large amplitude, which generates oscillations of its concentration and fluorescence at higher harmonic frequencies. For singularizing a label, HIGHLIGHT uses specific frequencies and mean light intensities associated with resonances of the amplitudes of concentration and fluorescence oscillations at harmonic frequencies. Several non-redundant resonant observables are simultaneously retrieved from a single experiment with phase-sensitive detection. HIGHLIGHT is used for selective imaging of four spectrally similar fluorescent proteins that had not been discriminated so far. Moreover, labels out of targeted locations can be discarded in an inhomogeneous spatial profile of illumination. HIGHLIGHT opens roads for simplified optical setups at reduced cost and easier maintenance.
Preparatory research
Correlation of fluorescence evolution for quantitative analysis of labels and sensors
Analytica Chimica Acta | doi.org/10.1016/j.aca.2022.340180
Open Access link: https://hal.sorbonne-universite.fr/hal-03775423/
Published: 8 September 2022
Titration without separation, e.g. quantification of a target species in living cells, is a challenge of analytical chemistry. We perform the selective detection of a target using the kinetics involved in a photochemical process and develop a correlation method that we illustrate by the titration of a fluorescent photoswitcher and the target of a photoswitching sensor. Correlating an input time series and a well-chosen weighting function associated with a variable characteristic time yields a spectrum of characteristic times. The upper integration limit of the correlation output can be chosen to match the argument of an extremum of the spectrum with a characteristic time of the input time series in order to quantify the target. A similar procedure is followed to optimize the signal-to-noise ratio. Selectivity and signal-to-noise ratio associated with 15 weighting functions are theoretically predicted. The results are applied to the titration of the reversibly photoswitchable fluorescent protein Dronpa-2 and the titration of calcium using a reversibly photoswitchable fluorescent sensor. The performance of the correlation method is favorably compared to the one of other dynamic contrast protocols.
Insights on the regulation of photosynthesis in pea leaves exposed to oscillating light
Journal of Experimental Botany | doi.org/10.1093/jxb/erac283
Open Access link: https://pmc.ncbi.nlm.nih.gov/articles/PMC9578350/
Published: 29 August 2022
Plants growing in nature often experience fluctuating irradiance. However, in the laboratory, the dynamics of photosynthesis are usually explored by instantaneously exposing dark-adapted plants to constant light and examining the dark-to-light transition, which is a poor approximation of natural phenomena. With the aim creating a better approximation, we exposed leaves of pea (Pisum sativum) to oscillating light and measured changes in the functioning of PSI and PSII, and of the proton motive force at the thylakoid membrane. We found that the dynamics depended on the oscillation period, revealing information about the underlying regulatory networks. As demonstrated for a selected oscillation period of 60 s, the regulation tries to keep the reaction centers of PSI and PSII open. We present an evaluation of the data obtained, and discuss the involvement of particular processes in the regulation of photosynthesis. The forced oscillations provided an information-rich fingerprint of complex regulatory networks. We expect future progress in understanding these networks from experiments involving chemical interventions and plant mutants, and by using mathematical modeling and systems identification and control tools.
Extra kinetic dimensions for label discrimination
Nature Communications | https://doi.org/10.1038/s41467-022-29172-0
Open Access link: https://pmc.ncbi.nlm.nih.gov/articles/PMC8933551/
18 March 2022
Due to its sensitivity and versatility, fluorescence is widely used to detect specifically labeled biomolecules. However, fluorescence is currently limited by label discrimination, which suffers from the broad full width of the absorption/emission bands and the narrow lifetime distribution of the bright fluorophores. We overcome this limitation by introducing extra kinetic dimensions through illuminations of reversibly photoswitchable fluorophores (RSFs) at different light intensities. In this expanded space, each RSF is characterized by a chromatic aberration-free kinetic fingerprint of photochemical reactivity, which can be recovered with limited hardware, excellent photon budget, and minimal data processing. This fingerprint was used to identify and discriminate up to 20 among 22 spectrally similar reversibly photoswitchable fluorescent proteins (RSFPs) in less than 1s. This strategy opens promising perspectives for expanding the multiplexing capabilities of fluorescence imaging.
Photosynthesis dynamics and regulation sensed in the frequency domain
Plant Physioly | 10.1093/plphys/kiab317
Open Access link: https://doi.org/10.1093/plphys/kiab317
Foundations of photosynthesis research have been established mainly by studying the response of plants to changing light, typically to sudden exposure to a constant light intensity after dark acclimation or light flashes. This approach remains valid and powerful, but can be limited by requiring dark acclimation before time-domain measurements and often assumes that rate constants determining the photosynthetic response do not change between dark and light acclimation. We show that these limits can be overcome by measuring plant responses to sinusoidally modulated light of varying frequency. By its nature, such frequency-domain characterization is performed in light-acclimated plants with no need for prior dark acclimation. Amplitudes, phase shifts, and upper harmonic modulation extracted from the data for a wide range of frequencies can target different kinetic domains and regulatory feedbacks. The occurrence of upper harmonic modulation reflects nonlinear phenomena, including photosynthetic regulation. To support these claims, we measured chlorophyll fluorescence emission of the green alga Chlorella sorokiniana in light that was sinusoidally modulated in the frequency range 1000-0.001 Hz. Based on these experimental data and numerical as well as analytical mathematical models, we propose that frequency-domain measurements can become a versatile tool in plant sensing.