The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes.

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)-linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M1 and anti-green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M1-mEGFP was expressed at levels equal to the M1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M1-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M1-mEGFP-expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M1-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

The efficacy of receptor tyrosine kinase EphA2 autophosphorylation increases with EphA2 oligomer size.

The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell-cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms high-order oligomers upon ligand binding. Here, we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used fluorescence intensity fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Our comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, an autophosphorylation response contributing to EphA2 activation, correlates with EphA2 mean oligomer size. However, we found that other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. Taken together, this work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features.

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t.

CXCR4, a member of the family of chemokine-activated G protein-coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested that the receptor may exist as a dimer or an oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means, we confirm that CXCR4 can exist as a mixture of monomers, dimers, and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t-a small drug-like isothiourea derivative-rapidly destabilizes the oligomeric structure, whereas AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state.

A general method to quantify ligand-driven oligomerization from fluorescence-based images.

Here, we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands. This method is fast and is scalable for high-throughput screening of drugs targeting protein-protein interactions.

Fluorescence intensity fluctuation analysis of receptor oligomerization in membrane domains.

Fluorescence micrographs of the plasma membrane of cells expressing fluorescently labeled G protein-coupled receptors (GPCRs) often exhibit small clusters of pixels (or puncta) with intensities that are higher than those of the surrounding pixels. Although studies of GPCR interactions in uniform membrane areas abound, understanding the details of the GPCR interactions within such puncta as well as the nature of the membrane formations underlying the puncta is hampered by the lack of adequate experimental techniques. Here, we introduce an enhancement of a recently developed method termed fluorescence intensity fluctuation spectrometry, which permits analysis of protein-protein interactions within the puncta in live cell membranes. We applied the novel fluorescence intensity fluctuation data analysis protocol to previously published data from cells expressing human secretin receptors and determined that the oligomer size increases with receptor concentration and duration of treatment with cognate ligand, not only within uniform regions of the membrane (in agreement with previous publications) but also within the puncta. In addition, we found that the number density and fractional area of the puncta increased after treatment with ligand. This method could be applied for probing the evolution in the time of the chain of events that begins with ligand binding and continues with coated pits formation and receptor internalization for other GPCRs and, indeed, other membrane receptors in living cells.

Carbonic Anhydrases Function in Anther Cell Differentiation Downstream of the Receptor-Like Kinase EMS1.

Plants extensively employ leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest family of RLKs, to control a wide range of growth and developmental processes as well as defense responses. To date, only a few direct downstream effectors for LRR-RLKs have been identified. We previously showed that the LRR-RLK EMS1 (EXCESS MICROSPOROCYTES1) and its ligand TPD1 (TAPETUM DETERMINANT1) are required for the differentiation of somatic tapetal cells and reproductive microsporocytes during early anther development in Arabidopsis thaliana Here, we report the identification of ß-carbonic anhydrases (ßCAs) as the direct downstream targets of EMS1. EMS1 biochemically interacts with ßCA proteins. Loss of function of ßCA genes caused defective tapetal cell differentiation, while overexpression of ßCA1 led to the formation of extra tapetal cells. EMS1 phosphorylates ßCA1 at four sites, resulting in increased ßCA1 activity. Furthermore, phosphorylation-blocking mutations impaired the function of ßCA1 in tapetal cell differentiation; however, a phosphorylation mimic mutation promoted the formation of tapetal cells. ßCAs are also involved in pH regulation in tapetal cells. Our findings highlight the role of ßCA in controlling cell differentiation and provide insights into the posttranslational modification of carbonic anhydrases via receptor-like kinase-mediated phosphorylation.

Quantitative microspectroscopic imaging reveals viral and cellular RNA helicase interactions in live cells.

Human cells detect RNA viruses through a set of helicases called RIG-I-like receptors (RLRs) that initiate the interferon response via a mitochondrial signaling complex. Many RNA viruses also encode helicases, which are sometimes covalently linked to proteases that cleave signaling proteins. One unresolved question is how RLRs interact with each other and with viral proteins in cells. This study examined the interactions among the hepatitis C virus (HCV) helicase and RLR helicases in live cells with quantitative microspectroscopic imaging (Q-MSI), a technique that determines FRET efficiency and subcellular donor and acceptor concentrations. HEK293T cells were transfected with various vector combinations to express cyan fluorescent protein (CFP) or YFP fused to either biologically active HCV helicase or one RLR (i.e. RIG-I, MDA5, or LGP2), expressed in the presence or absence of polyinosinic-polycytidylic acid (poly(I:C)), which elicits RLR accumulation at mitochondria. Q-MSI confirmed previously reported RLR interactions and revealed an interaction between HCV helicase and LGP2. Mitochondria in CFP-RIG-I:YFP-RIG-I cells, CFP-MDA5:YFP-MDA5 cells, and CFP-MDA5:YFP-LGP2 cells had higher FRET efficiencies in the presence of poly(I:C), indicating that RNA causes these proteins to accumulate at mitochondria in higher-order complexes than those formed in the absence of poly(I:C). However, mitochondria in CFP-LGP2:YFP-LGP2 cells had lower FRET signal in the presence of poly(I:C), suggesting that LGP2 oligomers disperse so that LGP2 can bind MDA5. Data support a new model where an LGP2-MDA5 oligomer shuttles NS3 to the mitochondria to block antiviral signaling.

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination.

Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLKs) are essential for plant growth, development, and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as coregulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as coregulatory and physical partners of EMS1. The SERK1/2 genes function in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation, Förster resonance energy transfer, and coimmunoprecipitation approaches revealed that SERK1 interacted biochemically with EMS1. Transphosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among 12 in vitro autophosphorylation and transphosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 autophosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence of the interaction and phosphorylation between SERK1/2 and EMS1 in anther development.

Quaternary structure of the yeast pheromone receptor Ste2 in living cells.

Transmembrane proteins known as G protein-coupled receptors (GPCRs) have been shown to form functional homo- or hetero-oligomeric complexes, although agreement has been slow to emerge on whether homo-oligomerization plays functional roles. Here we introduce a platform to determine the identity and abundance of differing quaternary structures formed by GPCRs in living cells following changes in environmental conditions, such as changes in concentrations. The method capitalizes on the intrinsic capability of FRET spectrometry to extract oligomer geometrical information from distributions of FRET efficiencies (or FRET spectrograms) determined from pixel-level imaging of cells, combined with the ability of the statistical ensemble approaches to FRET to probe the proportion of different quaternary structures (such as dimers, rhombus or parallelogram shaped tetramers, etc.) from averages over entire cells. Our approach revealed that the yeast pheromone receptor Ste2 forms predominantly tetramers at average expression levels of 2 to 25 molecules per pixel (2.8·10-6 to 3.5·10-5molecules/nm2), and a mixture of tetramers and octamers at expression levels of 25-100 molecules per pixel (3.5·10-5 to 1.4·10-4molecules/nm2). Ste2 is a class D GPCR found in the yeast Saccharomyces cerevisiae of the mating type a, and binds the pheromone α-factor secreted by cells of the mating type α. Such investigations may inform development of antifungal therapies targeting oligomers of pheromone receptors. The proposed FRET imaging platform may be used to determine the quaternary structure sub-states and stoichiometry of any GPCR and, indeed, any membrane protein in living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Quaternary structures of opsin in live cells revealed by FRET spectrometry.

Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that initiates phototransduction in the retina. The receptor consists of the apoprotein opsin covalently linked to the inverse agonist 11-cis retinal. Rhodopsin and opsin have been shown to form oligomers within the outer segment disc membranes of rod photoreceptor cells. However, the physiological relevance of the observed oligomers has been questioned since observations were made on samples prepared from the retina at low temperatures. To investigate the oligomeric status of opsin in live cells at body temperatures, we utilized a novel approach called Förster resonance energy transfer spectrometry, which previously has allowed the determination of the stoichiometry and geometry (i.e. quaternary structure) of various GPCRs. In the current study, we have extended the method to additionally determine whether or not a mixture of oligomeric forms of opsin exists and in what proportion. The application of this improved method revealed that opsin expressed in live Chinese hamster ovary (CHO) cells at 37°C exists as oligomers of various sizes. At lower concentrations, opsin existed in an equilibrium of dimers and tetramers. The tetramers were in the shape of a near-rhombus. At higher concentrations of the receptor, higher-order oligomers began to form. Thus, a mixture of different oligomeric forms of opsin is present in the membrane of live CHO cells and oligomerization occurs in a concentration-dependent manner. The general principles underlying the concentration-dependent oligomerization of opsin may be universal and apply to other GPCRs as well.

The sigma-1 receptors are present in monomeric and oligomeric forms in living cells in the presence and absence of ligands.

The sigma-1 receptor (S1R) is a 223-amino-acid membrane protein that resides in the endoplasmic reticulum and the plasma membrane of some mammalian cells. The S1R is regulated by various synthetic molecules including (+)-pentazocine, cocaine and haloperidol and endogenous molecules such as sphingosine, dimethyltryptamine and dehydroepiandrosterone. Ligand-regulated protein chaperone functions linked to oxidative stress and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and neuropathic pain have been attributed to the S1R. Several client proteins that interact with S1R have been identified including various types of ion channels and G-protein coupled receptors (GPCRs). When S1R constructs containing C-terminal monomeric GFP2 and YFP fusions were co-expressed in COS-7 cells and subjected to FRET spectrometry analysis, monomers, dimers and higher oligomeric forms of S1R were identified under non-liganded conditions. In the presence of the prototypic S1R agonist, (+)-pentazocine, however, monomers and dimers were the prevailing forms of S1R. The prototypic antagonist, haloperidol, on the other hand, favoured higher order S1R oligomers. These data, in sum, indicate that heterologously expressed S1Rs occur in vivo in COS-7 cells in multiple oligomeric forms and that S1R ligands alter these oligomeric structures. We suggest that the S1R oligomerization states may regulate its function(s).

Experimental verification of the kinetic theory of FRET using optical microspectroscopy and obligate oligomers.

Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photo-insensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.

Cross-talk between a regulatory small RNA, cyclic-di-GMP signalling and flagellar regulator FlhDC for virulence and bacterial behaviours.

Dickeya dadantii is a globally dispersed phytopathogen which causes diseases on a wide range of host plants. This pathogen utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to degrade the plant cell wall. Although the regulatory small RNA (sRNA) RsmB, cyclic diguanylate monophosphate (c-di-GMP) and flagellar regulator have been reported to affect the regulation of these two virulence factors or multiple cell behaviours such as motility and biofilm formation, the linkage between these regulatory components that coordinate the cell behaviours remain unclear. Here, we revealed a sophisticated regulatory network that connects the sRNA, c-di-GMP signalling and flagellar master regulator FlhDC. We propose multi-tiered regulatory mechanisms that link the FlhDC to the T3SS through three distinct pathways including the FlhDC-FliA-YcgR3937 pathway; the FlhDC-EcpC-RpoN-HrpL pathway; and the FlhDC-rsmB-RsmA-HrpL pathway. Among these, EcpC is the most dominant factor for FlhDC to positively regulate T3SS expression.

The relative antimicrobial effect of blue 405 nm LED and blue 405 nm laser on methicillin-resistant Staphylococcus aureus in vitro.

It has long been argued that light from a laser diode is superior to light from a light-emitting diode (LED) in terms of its effect on biological tissues. In order to shed light on this ongoing debate, we compared the antimicrobial effect of light emitted from a 405-nm LED with that of a 405-nm laser on methicillin-resistant Staphylococcus aureus (MRSA) at comparable fluences. We cultured 5 × 10(6) CFU/ml MRSA on tryptic soy agar and then irradiated culture plates once, twice, or thrice with either LED or laser light using 40, 54, 81, or 121 J/cm(2) fluence at 15-, 30-, or 240-min time interval between irradiation. Cultures were incubated immediately after irradiation at 37 °C for 24 h before imaging and counting remnant bacterial colonies. Regardless of the device used, LED or laser, irradiation at each fluence resulted in statistically significant bacterial growth suppression compared to non-irradiated controls (p < 0.0001). The antimicrobial effect of both light sources, LED and laser, was not statistically different at each fluence in 35 of the 36 experimental trials. Bacterial growth suppression achieved with either source of light increased with repeated irradiation, particularly at the 15- or 30-min treatment time interval. Thus, we conclude that the antimicrobial effect of 405-nm laser and 405-nm LED on MRSA is similar; neither has a superior antimicrobial effect when compared to the other.

KINNTREX: a neural network to unveil protein mechanisms from time-resolved X-ray crystallography.

Here, a machine-learning method based on a kinetically informed neural network (NN) is introduced. The proposed method is designed to analyze a time series of difference electron-density maps from a time-resolved X-ray crystallographic experiment. The method is named KINNTREX (kinetics-informed NN for time-resolved X-ray crystallography). To validate KINNTREX, multiple realistic scenarios were simulated with increasing levels of complexity. For the simulations, time-resolved X-ray data were generated that mimic data collected from the photocycle of the photoactive yellow protein. KINNTREX only requires the number of intermediates and approximate relaxation times (both obtained from a singular valued decomposition) and does not require an assumption of a candidate mechanism. It successfully predicts a consistent chemical kinetic mechanism, together with difference electron-density maps of the intermediates that appear during the reaction. These features make KINNTREX attractive for tackling a wide range of biomolecular questions. In addition, the versatility of KINNTREX can inspire more NN-based applications to time-resolved data from biological macromolecules obtained by other methods.

Mechanistic insights from the atomic-level quaternary structure of short-lived GPCR oligomers in live cells.

The functional significance of the interactions between proteins in living cells to form short-lived quaternary structures cannot be overemphasized. Yet, quaternary structure information is not captured by current methods, neither can those methods determine structure within living cells. The dynamic versatility, abundance, and functional diversity of G protein-coupled receptors (GPCRs) pose myriad challenges to existing technologies but also present these proteins as the ideal testbed for new technologies to investigate the complex inter-regulation of receptor-ligand, receptor-receptor, and receptor-downstream effector interfaces in living cells. Here, we present development and use of a novel method capable of overcoming existing challenges by combining distributions (or spectrograms) of FRET efficiencies from populations of fluorescently tagged proteins associating into oligomeric complexes in live cells with diffusion-like trajectories of FRET donors and acceptors obtained from molecular dynamics (MD) simulations. Our approach provides an atom-level picture of the binding interfaces within oligomers of the human secretin receptor (hSecR) in live cells and allows for extraction of mechanistic insights into the function of GPCRs oligomerization. This FRET-MD spectrometry approach is a robust platform for investigating protein-protein binding mechanisms and opens a new avenue for investigating stable as well as fleeting quaternary structures of any membrane proteins in living cells.

Development and experimental testing of an optical micro-spectroscopic technique incorporating true line-scan excitation.

Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects.

Fluorescence Intensity Fluctuation Analysis of Protein Oligomerization in Cell Membranes.

Fluorescence fluctuation spectroscopy (FFS) encompasses a bevy of techniques that involve analyzing fluorescence intensity fluctuations occurring due to fluorescently labeled molecules diffusing in and out of a microscope's focal region. Statistical analysis of these fluctuations may reveal the oligomerization (i.e., association) state of said molecules. We have recently developed a new FFS-based method, termed Two-Dimensional Fluorescence Intensity Fluctuation (2D FIF) spectrometry, which provides quantitative information on the size and stability of protein oligomers as a function of receptor concentration. This article describes protocols for employing FIF spectrometry to quantify the oligomerization of a membrane protein of interest, with specific instructions regarding cell preparation, image acquisition, and analysis of images given in detail. Application of the FIF Spectrometry Suite, a software package designed for applying FIF analysis on fluorescence images, is emphasized in the protocol. Also discussed in detail is the identification, removal, and/or analysis of inhomogeneous regions of the membrane that appear as bright spots. The 2D FIF approach is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of live cells expressing protein constructs Basic Protocol 2: Image acquisition and noise correction Basic Protocol 3: Drawing and segmenting regions of interest Basic Protocol 4: Calculating the molecular brightness and concentration of individual image segments Basic Protocol 5: Combining data subsets using a manual procedure (Optional) Alternate Protocol 1: Combining data subsets using the advanced FIF spectrometry suite (Optional; alternative to Basic Protocol 5) Basic Protocol 6: Performing meta-analysis of brightness spectrograms Alternate Protocol 2: Performing meta-analysis of brightness spectrograms (alternative to Basic Protocol 6) Basic Protocol 7: Spot extraction and analysis using a manual procedure or by writing a program (Optional) Alternate Protocol 3: Automated spot extraction and analysis (Optional; alternative to Protocol 7) Support Protocol: Monomeric brightness determination.

Comparative photophysical properties of some widely used fluorescent proteins under two-photon excitation conditions.

Understanding the photophysical properties of fluorescent proteins (FPs), such as emission and absorption spectra, molecular brightness, photostability, and photo-switching, is critical to the development of criteria for their selection as tags for fluorescent-based biological applications. While two-photon excitation imaging techniques have steadily gained popularity - due to comparatively deeper penetration depth, reduced out-of-focus photobleaching, and wide separation between emission spectra and two-photon excitation spectra -, most studies reporting on the photophysical properties of FPs tend to remain focused on single-photon excitation. Here, we report our investigation of the photophysical properties of several commonly used fluorescent proteins using two-photon microscopy with spectral resolution in both excitation and emission. Our measurements indicate that not only the excitation (and sometimes emission) spectra of FPs may be markedly different between single-photon and two-photon excitation, but also their relative brightness and their photo-stability. A good understanding of the photophysical properties of FPs under two-photon excitation is essential for choosing the right tag(s) for a desired experiment.