IBS - Institut de Biologie Structurale - Grenoble / France

Positive photoswitching in mEos4b (2025)

Bourgeois Dominique — 2025-09-08T12:19:44Z

Photo credit: IBS/Jip Wulffelé

Photoconvertible fluorescent proteins (PCFPs) like mEos4b shift their fluorescence emission from green to red upon 405 nm illumination, making them essential markers for super-resolution techniques such as Single Molecule Localization Microscopy (SMLM). However, their photophysical properties continue to reveal surprises. Besides photoconversion, PCFPs can reversibly switch between fluorescent and nonfluorescent states. In its red form, mEos4b undergoes “negative switching”: it turns off under 561 nm light and reactivates under 405 nm light due to cis-trans isomerization of the chromophore.

In this collaboration between the I2SR and NMR groups of IBS, and the SyMMES laboratory, researchers identified a new, additional, “positive switching” mode: 405 nm light turns off red mEos4b, while 561 nm light switches it back on—the inverse of negative switching. Using UV-vis spectroscopy, fluorescence imaging, NMR, and EPR, they found that this arises from light-induced conformational heterogeneity. The red form of mEos4b exists in two fluorescent states, differing in their H-bonding networks around the chromophore. Under 405 nm light, the less fluorescent state builds up, while 561 nm light or darkness favors the brighter state. Notably, the less fluorescent state has a higher pKa, making it nearly dark and long-lived at physiological or low pH.

This positive switching leads to blinking in SMLM, highlighting how subtle light-induced conformational shifts, often undetectable in crystallography, profoundly impact single-molecule imaging.

Light-Induced Conformational Heterogeneity Induces Positive Photoswitching in Photoconvertible Fluorescent Proteins of the EosFP Family. Wulffelé J, Maity A, Ayala I, Gambarelli S, Brutscher B, Bourgeois D. J Am Chem Soc. 2025; 147(12):10357-10368. doi : 10.1021/jacs.4c17311.

Nucleoid remodeling and changes in HU protein dynamics in Deinococcus radiodurans (2024)

Bourgeois Dominique — 2025-02-10T16:42:36Z

Nucleoid remodeling is a common stress response strategy in bacteria to protect their genetic material. This process is regulated by small proteins, known as NAPs or Nucleoid-Associated Proteins, which interact with DNA and play a key role in the organisation and regulation of the bacterial genome.
Using advanced conventional and single-molecule localization microscopy approaches, the I2SR group, in collaboration with F. Confalonieri's team at I2BC, recently demonstrated that exposure to UV-C radiation or entry into stationary phase induces profound changes in the morphology and size of Deinococcus radiodurans nucleoids, as well as modifications in the mobility of the HU protein, the main NAP in this bacterium. Although both stresses cause rapid nucleoid compaction, HU diffusion decreases in stationary phase while it increases following UV-C, suggesting distinct underlying mechanisms.
In addition, they show that nucleoid remodeling following exposure to UV-C occurs in three distinct stages: rapid condensation associated with an increase in HU diffusion (most likely caused by release of HU from the genomic DNA), followed by a slower decompaction phase to restore normal nucleoid morphology accompanied by a return of HU mobility to normal values (as a result of reassembly of HU on the genomic DNA), before cell growth and division resume. These observations illustrate the diversity and complexity of nucleoid remodeling processes in bacteria and represent a first step towards understanding the mechanisms involved and notably the role of HU in this process.

Stress-induced nucleoid remodeling in Deinococcus radiodurans is associated with major changes in Heat Unstable (HU) protein dynamics. Vauclare P, Wulffelé J, Lacroix F, Servant P, Confalonieri F, Kleman JP, Bourgeois D*, Timmins J*. Nucleic Acids Res. 2024 ; 52(11):6406-6423.doi : 10.1093/nar/gkae379.

Oarii Vahirua

Bourgeois Dominique — 2025-02-10T16:23:22Z

Oarii VAHIRUA
CNRS Engineer
Institut de Biologie Structurale CNRS (UMR 5075)/CEA/UGA
71 AVENUE DES MARTYRS
CS 10090
38044 GRENOBLE CEDEX 9

Tel: 33-(0)4 57 42 87 10

Ia orana ! I'm Oarii VAHIRUA, a research engineer in the PIXEL team of the I2SR group.

I work on photoconvertible fluorescent proteins of the mEos family, expressed following a CRISPR strategy in the nuclear pores of U2OS cells.

My role is to optimize the acquisition parameters of super-resolution PALM (Photo-activated localization microscopy) images.

Jérémy Néri

Bourgeois Dominique — 2025-02-10T16:11:12Z

Jérémy Néri
CEA phD student
Institut de Biologie Structurale CNRS (UMR 5075)/CEA/UGA
71 AVENUE DES MARTYRS
CS 10090
38044 GRENOBLE CEDEX 9

Tel: 33-(0)4 57 42 57 42

I spent the first 4 years of my career as an engineer in various laboratories. During these years, I focused on fluorescence microscopy and image analysis. In particular, I worked on the development and imaging characterization of organoid models generated from diseased patient livers.
My thesis project focuses on reversibly photoswitchable red fluorescent proteins (RSFPs). The aim is to gain a deeper understanding of the fundamental functioning of these proteins, with the ultimate goal of enabling their use in advanced microscopy techniques. To achieve this, I use techniques such as spectroscopy, fluorescence microscopy and in silico simulation.

Conformational heterogeneity of mEos4b revealed by NMR (2023)

Bourgeois Dominique — 2024-03-04T13:38:59Z

NMR REVEALS NEW SECRETS OF FLUORESCENT PROTEINS USED IN SUPER-RESOLUTION MICROSCOPY

Photo credit: Jip Wulffelé (IBS/I2SR)

Photoconvertible fluorescent proteins (PCFPs) such as mEos4b change their fluorescence color from green to red upon illumination with UV light. They are popular markers for super-resolution imaging modalities such as quantitative and single particle tracking Single Molecule Localization Microscopy (SMLM). The photophysical properties of these proteins, however, are exceedingly complex. In this collaborative work involving the NMR and I2SR groups of the IBS, researchers observed by multidimensional NMR spectroscopy that mEos4b exhibits two distinct conformations in the green state that slowly interconvert. NMR also revealed that these conformations differ in the protonation states of two amino-acid side chains in the chromophore pocket, resulting in an altered hydrogen bond network. These subtle rearrangements were not visible in high-resolution x-ray structures of mEos4b. Importantly, only one of these conformations photoconverts efficiently to the red state, while the other one appears to be more susceptible to photobleaching. This study helps to explain the observed complex photophysical behavior of mEos4b and related PCFPs. More generally, it reveals how conformational dynamics of fluorescent proteins affect their photophysics, and in particular the photoconversion mechanisms of mEos-derived PCFPs. Finally, their results open the door for designing new PCFP variants with superior photoconversion efficiency.

Structural Heterogeneity in a Phototransformable Fluorescent Protein impacts its Photochemical Properties. Arijit Maity, Jip Wulffelé, Isabel Ayala, Adrien Favier, Virgile Adam, Dominique Bourgeois*, and Bernhard Brutscher*. Adv. Sci. (2023), 2306272 Doi: 10.1002/advs.202306272

Quentin Depeyre

Bourgeois Dominique — 2023-12-05T15:10:17Z

Quentin Depeyre
UGA phD student
Institut de Biologie Structurale CNRS (UMR 5075)/CEA/UGA
71 AVENUE DES MARTYRS
CS 10090
38044 GRENOBLE CEDEX 9

Tel: 33-(0)4 57 42 57 42

I'm passionate about biology by nature, and understanding how the body works and defends itself when something goes wrong has always been something that has aroused my curiosity. During my studies at Grenoble Alpes University, I became very interested in cancer research. During a placement at the Institute of Biology and Pathology in Grenoble, I had the opportunity to study the role of the microenvironment in chemoprotection of myelodysplastic syndromes. This first experience in a research laboratory was very rewarding. Now, my thesis project is to study the role of macrophage cholesterol metabolism in the formation of the CD47/SIRP-α pathway-dependent phagocytic synapse in the tumour context.

Phagocytosis seen by SMLM (2023)

Bourgeois Dominique — 2023-10-27T07:16:12Z

Super-resolution microscopy on the surface of cells in apoptosis reveals the play of molecules involved in their elimination by phagocytosis

The increased resolution obtained thanks to STORM super-resolution microscopy demonstrated that SIRPα is no longer colocalized with its CD47 ligand on the apoptotic blebs

Modifications in the exposure of molecules at surface of abnormal cells, or those dying by apoptosis, ordinarily lead to their elimination by macrophages (phagocytosis). These changes are impaired in the tumor context and this can impede phagocytosis and cause resistances to therapies.
Using super-resolution fluorescence microscopy, also known as nanoscopy, because it can achieve a resolution of a few nanometers, researchers of the IBS/I2SR group have studied the distribution, diffusion and interactions of molecules (such as CD47, calreticulin, phosphatidylserine or the SIRPα receptor) known to induce or block phagocytosis. Their experiments suggest that disorganization of the lipid bilayer at the plasma membrane, by inducing inaccessibility of CD47, may have an influence on CD47/SIRPα interaction, an immune checkpoint well known to regulate phagocytosis. In particular, the cholesterol content of the cell membrane may play a central role in the phagocytosis process (high levels may inhibit phagocytosis, by promoting CD47-SIRPα interaction).
This result opens up new perspectives for countering the mechanisms by which cancer cells escape the immune system.
Super-resolution imaging (STORM, Stochastic Optical Reconstruction Microscopy) and single-particle tracking (SPT) were carried out on instruments from the M4D platform at the Institut de Biologie structurale (member of ISBG, Integrated Structural Biology Grenoble).

Nanoscale imaging of CD47 informs how plasma membrane modifications shape apoptotic cell recognition. Samy Dufour; Pascale Tacnet-Delorme; Jean-Philippe Kleman; Oleksandr Glushonkov; Nicole Thielens; Dominique Bourgeois; Philippe Frachet. Communications Biology 2023; 6(1):207. doi: 10.1038/s42003-023-04558-y.

rsEGFP2 in the cold (2023)

Bourgeois Dominique — 2023-10-27T07:00:43Z

How does a fluorescent protein switch in the cold ?

Structural biology methods have often benefited from their cryogenic version. X-ray crystallography and electron microscopy have been revolutionized by the possibility to flash cool biological samples, reducing radiation damage or better preserving the native state of the investigated macromolecules, respectively. With the development of super-resolution microscopy (nanoscopy), the question now arises of moving towards cryo-nanoscopy. The approach has already been demonstrated by several leading labs, notably with the goal of performing cryo-correlative studies (cryo-CLEM). There is however a big obstacle to obtain good cryo-nanoscopy images when the SMLM (single molecule localization microscopy) technique is used. For SMLM to work, the fluorophores employed to label the molecule of interest need to efficiently switch between a fluorescent on-state and a nonfluorescent off-state. However, in the case of fluorescent proteins, this switching property is typically reduced or abolished at cryogenic temperature, because switching depends on protein dynamics which is mostly arrested below the glass transition temperature.

In this work, members of the Pixel team of the Integrated Imaging of Stress Response Group (I2SR/Pixel), in collaboration with the University of Göttingen in Germany, have shown that rsEGFP2, a rapidly switching fluorescent protein at room temperature, still switches at 100K, yet based on an entirely different mechanism that likely involves radical states instead of cis trans chromophore isomerization. Most importantly, the work demonstrates that the fraction of rsEGFP2 molecules that can efficiently switch before photobleaching is significantly enhanced by using UV illumination at 355 nm instead of the classically used violet illumination at 405 nm. Thus, the study opens the door to obtaining crisper cryo-SMLM images.

Photophysical Studies at Cryogenic Temperature Reveal a Novel Photoswitching Mechanism of rsEGFP2. Angela M. R. Mantovanelli,§ Oleksandr Glushonkov,§ Virgile Adam,§ Jip Wulffelé, Daniel Thédié, Martin Byrdin, Ingo Gregor, Oleksii Nevskyi, Jörg Enderlein, and Dominique Bourgeois. Journal of the American Chemical Society 2023; 145(27):14636-14646. doi: 10.1021/jacs.3c01500.

SMIS Simulator (2023)

Bourgeois Dominique — 2023-07-11T11:55:35Z

Fluorophore photophysics and super-resolution microscopy get married in SMIS

Single-molecule localization microscopy (SMLM) is the most popular super-resolution fluorescence imaging technique. It is also the technique providing the best resolution enhancement, therefore being most suited for integrating structural biology into the cellular context. Like all super-resolution methods, SMLM fundamentally relies on specific fluorophore photophysical properties, like photoswitching. Its limitations, however, are also largely bound to –unwanted- fluorophore photophysics.
A main goal of the Pixel team of the Integrated Imaging of Stress Response Group is to understand how fluorophore photophysics and SMLM are intertwined. To this aim, since 10 years, dominique Bourgeois has been developing a Matlab-based simulation tool named SMIS (Single Molecule Imaging Simulator). SMIS simulates a SMLM microscope and generates data sets from virtual samples while taking account arbitrarily complex photophysics of the employed fluorophores. This way, all sorts of weird behaviors and artifacts can be evaluated and understood. Taking advantage of the Covid confinement, he could finalize the work in the form of a GUI (Graphical User Interface) available to the community, also showing in a number of examples how SMIS can be used to understand what's going on. This personal project turned out to be hugely time-consuming, and kept him busy in parallel with the other projects of the I2SR group!

Single molecule imaging simulations with advanced fluorophore photophysics. Bourgeois D. Communications Biology 2023; 6, 53.

Developpement of qPALM, cryoPALM and sptPALM microscopies

Bourgeois Dominique — 2022-11-30T14:01:50Z

We develop PALM super-resolution microscopy, a single-molecule-based localization method now used worldwide to overcome the diffraction limit. PALM and its derivatives qPALM and sptPALM are almost entirely based on the proper manipulation of PTFP's photophysics, which brings coherence to our activities.

Our PALM microscope is used to study PTFPs in cellulo or in vitro at the single-molecule level. We conduct 3 main activities:

We are developing cryogenic temperature PALM microscopy (cryo-PALM), which is one of the major current advancements in the field, particularly for applications in integrated structural biology based on correlative microscopy (cryo-CLEM). For this project, we benefit from a cryo-microscope developed by Jörg Enderlein's German team (University of Göttingen). Our goal is to study the photocommutation properties of PTFPs at low temperatures, to engineer fluorescent proteins better suited for cryo-PALM, or to improve laser illumination protocols during cryo-PALM data collection. For example, we recently discovered a new photocommutation mechanism occurring at cryogenic temperatures in rsEGFP2 and showed that illumination at 355 nm increased labeling efficiency.

We study how the photophysics of photoconvertible fluorescent proteins, such as mEos variants, affect qPALM and sptPALM techniques. For example, we found a method to increase the length of traces in sptPALM with mEos variants by adding low illumination at 488 nm during data collection. We also recently showed that, due to a non-linear photobleaching mechanism, using high intensities of 405 nm light can be very detrimental to labeling efficiency in qPALM. Finally, our work in collaboration with the NMR group at IBS revealed the existence of multiple conformational states in mEos proteins like mEos4b, both in their green and red states, with consequences for photoconversion and blinking mechanisms in PALM experiments.

We are developing a single-molecule imaging simulator called SMIS, which, unlike all other simulators available to date, integrates an advanced description of the spectral and photophysical properties of fluorophores. SMIS enables complex simulations that can predict, for example, the effects of various illumination conditions in a PALM experiment or the subtle effects of crosstalk in multicolor SMLM experiments.