Quantitative super-resolution microscopy: pitfalls and strategies for image analysis

Super-resolution microscopy is an enabling technology that allows biologists to visualize cellular structures at nanometer length scales using far-field optics. To break the diffraction barrier, it is necessary to leverage the distinct molecular states of fluorescent probes. At the same time, the existence of these different molecular states and the photophysical properties of the fluorescent probes can complicate data quantification and interpretation. Here, we review the pitfalls in super-resolution data analysis that must be avoided for proper interpretation of images.Peer ReviewedPostprint (author's final draft

Inhibitory synapse deficits caused by familial α1 GABAA receptor mutations in epilepsy

Epilepsy is a spectrum of neurological disorders with many causal factors. The GABA type-A receptor (GABA(A)R) is a major genetic target for heritable human epilepsies. Here we examine the functional effects of three epilepsy causing mutations to the alpha 1 subunit (alpha 1(T10T), alpha 1(D192N) and alpha 1(A295D)) on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic GABA(A)R isoform, alpha 1 eta 2 gamma 2L. We employed a neuron - HEK293 cell heterosynapse preparation to record IPSCs mediated by mutant-containing GABA(A)Rs in isolation from other GABA(A)R isoforms. IPSCs were recorded in the presence of the anticonvulsant drugs, carbamazepine and midazolam, and at elevated temperatures (22, 37 and 40 degrees C) to gain insight into mechanisms of febrile seizures. The mutant subunits were also transfected into cultured cortical neurons to investigate changes in synapse formation and neuronal morphology using fluorescence microscopy. We found that IPSCs mediated by alpha 1(T10T)beta 2 gamma 2L, alpha 1(D192N)beta 2 gamma 2L GABA(A)Rs decayed faster than those mediated by alpha 1 beta 2 gamma 2L receptors. IPSCs mediated by alpha 1(D192N)beta 2 gamma 2L and alpha 1(A295D) beta 2 gamma 2L receptors also exhibited a heightened temperature sensitivity. In addition, the alpha 1(T10T)beta 2 gamma 2L GABA(A)Rs were refractory to modulation by carbamazepine or midazolam. In agreement with previous studies, we found that alpha 1(A295D)beta 2 gamma 2L GABA(A)Rs were retained intracellularly in HEK293 cells and neurons. However, pre-incubation with 100 nM suberanilohydroxamic acid (SAHA) induced alpha 1(A295D)beta 2 gamma 2L GABA(A)Rs to mediate IPSCs that were indistinguishable in magnitude and waveform from those mediated by alpha 1 beta 2 gamma 2L receptors. Finally, mutation specific changes to synaptic bouton size, synapse number and neurite branching were also observed. These results provide new insights into the mechanisms of epileptogenesis of alpha 1 epilepsy mutations and suggest possible leads for improving treatments for patients harbouring these mutations

Single molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.Peer ReviewedPostprint (author's final draft

Can we understand, control and use blinking of quantum dots in biological surroundings?

Semiconductor nanocrystals, also known as quantum dots (QDs), are evoking remarkable technological and scientific interest due to their fascinating size-dependent electronic and optical properties. Many biophysical studies to date have used effectively as brighter and more photostable replacement of organic dyes. In this thesis we focus on the most prominent feature of their photophysical properties, a random switching between emitting and non-emitting states, also known as fluorescence intermittency, or "blinking," in order to better understand the mechanism of quantum dot emission and how it reflects interaction with their immediate environment. We first designed and built a total internal reflection florescent microscope (TIRFM) with single molecule detection capabilities and determined optimal conditions for single QD studies. We then used image correlation techniques to show that the change in blinking dynamics could be detected and that it could complicate interpretation of the commonly used analytical techniques that rely on intensity fluctuations as reporters of particle mobility. In particular, we demonstrated that the transport coefficients recovered from fluorescence fluctuation analysis of diffusional mobility using temporal image correlation spectroscopy (TICS) had significant systematic errors due to blinking of the nanoparticles. We then performed a thorough, systematic study of the effects of protons on QDs' photochemical stability by varying the pH of their aqueous environment and related the single particle properties to properties of an ensemble of QDs. As pH was lowered, shorter "on" times and longer "off" times were observed, brightness of single QDs decreased and the number of permanently non-emitting QDs (dark fraction) increased. Based on these results, we proposed a coupled role for H+ ions by which they first reduced the intensity of the emitting state as well as affected probabilities of the QD to switch between the "on" and "off" stateLes nanoparticules semi-conductrices, aussi connues sous le nom de particules quantiques (PQ), suscitent un grand intérêt dans les domaines technologiques et scientifiques en raison de leurs propriétés spectroscopiques uniques et avantageuses. À ce jour, plusieurs études de biophysique ont remplacé succès les sondes fluorescentes organiques traditionnelles par des PQ.Cette thèse porte principalement sur l'étude de la propriété la plus photophysique des PQ, soit leur capacité à activer ou à inactiver de manière aléatoire l'émission fluorescente (plus connue sous le nom de l'intermittence de l'émission fluorescente, ou « clignotement »), dans le but de mieux comprendre le mécanisme entourant l'émission des particules quantiques et son interaction avec son environnement immédiat.Nous avons tout d'abord conçu et construit un microscope de fluorescence par réflexion totale interne (TIRFM) qui a la capacité de détecter une seule molécule et de déterminer les conditions idéales afin d'étudier une PQ unique. Nous avons ensuite utilisé des techniques de spectroscopie de corrélation temporelle dans les images afin de montrer que l'intermittence de l'émission fluorescente pouvait être détectée et qu'elle pourrait rendre possible l'interprétation d'analyses plus complexes que les techniques d'analyse traditionnelles qui se fondent sur les clignotements fluorescents comme preuve du mouvement des particules.De façon plus précise, nous avons démontré que les coefficients de transport obtenus d'analyses utilisant la technique de spectroscopie de corrélation temporelle dans les images (TICS) présentaient une marge d'erreur significative due au « clignotement » des nanoparticules. Ensuite, nous avons procédé à une étude systématique plus approfondie des effets des protons sur la stabilité photochimique des PQ, en modifiant le pH de leur environnement aqueux et en faisant des liens avec les propriétés d'une seule particule dans un ensemble de PQ. Au fur et à mesure que le pH diminuait, nous avons observé une diminution du temps d'émission fluorescente, « ouvert », et une augmentation du temps d'absence d'émission, « éteint ». Nous avons également noté une diminution dans la clarté d'une seule PQ et une augmentation dans le nombre de PQ n'émettant aucune clarté (fraction sombre).À la lumière de ces résultats, nous avons avancé l'idée d'un rôle double des ions H+, qui réduiraient d'abord l'intensité de l'état d'émission et affecteraient ensuite la probabilité d'une PQ d'alterner entre les états d'émission et d'absence d'émission, pour éventuellement emprisonner la PQ dans un état de fraction sombre permanent. Nous avons analysé et élargi les modèles théoriques concernant le « clignotement » afin de tenir compte des effets des ions H+ et de mieux démontrer que le « clignotement » et la formation de fraction sombre se fondent sur un seul et même mécanisme.Les résultats présentés dans cette thèse sont particulièrement importants pour l'élaboration d'un modèle théorique complet du « clignotement » d'une PQ, mais peuvent également être importants pour les différents usages des PQ, en particulier dans les applications d'imagerie dans le domaine de la biologie quantique, où on retrouve des variations de pH entre les espaces cytoplasmiques et extracellulaires et dans les différents organes cellulaires

Quantitative super-resolution microscopy: pitfalls and strategies for image analysis

Super-resolution microscopy is an enabling technology that allows biologists to visualize cellular structures at nanometer length scales using far-field optics. To break the diffraction barrier, it is necessary to leverage the distinct molecular states of fluorescent probes. At the same time, the existence of these different molecular states and the photophysical properties of the fluorescent probes can complicate data quantification and interpretation. Here, we review the pitfalls in super-resolution data analysis that must be avoided for proper interpretation of images.Peer Reviewe

SAHA (Vorinostat) corrects inhibitory synaptic deficits caused by missense epilepsy mutations to the GABA(A) receptor gamma 2 subunit

The GABA(A) receptor (GABA(A)R) alpha 1 subunit A295D epilepsy mutation reduces the surface expression of alpha 1(A295D)beta 2 gamma 2 GABA(A)Rs via ER-associated protein degradation. Suberanilohydroxamic acid (SAHA, also known as Vorinostat) was recently shown to correct the misfolding of alpha 1(A295D) subunits and thereby enhance the functional surface expression of alpha 1(A295D)beta 2 gamma 2 GABA(A)Rs. Here we investigated whether SAHA can also restore the surface expression of gamma 2 GABA(A)R subunits that incorporate epilepsy mutations (N40S, R43Q, P44S, R138G) known to reduce surface expression via ER-associated protein degradation. As a control, we also investigated the gamma 2(K289M) epilepsy mutation that impairs gating without reducing surface expression. Effects of mutations were evaluated on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic alpha 1 beta 2 gamma 2 GABA(A)R isoform. Recordings were performed in neuron-HEK293 cell artificial synapses to minimise contamination by GABA(A)Rs of undefined subunit composition. Transfection with alpha 1 beta 2 gamma 2(N40S), alpha 1 beta 2 gamma 2(R43Q), alpha 1 beta 2 gamma 2(P44S) and alpha 1 beta 2 gamma 2(R138G) subunits produced IPSCs with decay times slower than those of unmutated a alpha 1 beta 2 gamma 2 GABA(A)Rs due to the low expression of mutant gamma 2 subunits and the correspondingly high expression of slow-decaying alpha 1 beta 2 GABA(A)Rs. SAHA pre-treatment significantly accelerated the decay time constants of IPSCs consistent with the upregulation of mutant gamma 2 subunit expression. This increase in surface expression was confirmed by immunohistochemistry. SAHA had no effect on either the IPSC kinetics or surface expression levels of alpha 1 beta 2 gamma 2(K289M) GABA(A)Rs, confirming its specificity for ER-retained mutant gamma 2 subunits. We also found that alpha 1 beta 2 gamma 2(K289M) GABA(A)Rs and SAHA-treated alpha 1 beta 2 gamma 2(R43Q), alpha 1 beta 2 gamma 2(P44S) and alpha 1 beta 2 gamma 2(R138G) GABA(A)Rs all mediated IPSCs that decayed at significantly faster rates than wild type receptors as temperature was increased from 22 to 40 degrees C. This may help explain why these mutations cause febrile seizures (FS). Given that SAHA is approved by therapeutic regulatory agencies for human use, we propose that it may be worth investigating as a treatment for epilepsies caused by the N40S, R43Q, P44S and R138G mutations. Although SAHA has already been proposed as a therapeutic for patients harbouring the alpha 1(A295D) epilepsy mutation, the present study extends its potential utility to a new subunit and four new mutations

Exosomes taken up by neurons hijack the endosomal pathway to spread to interconnected neurons

In Alzheimer disease and related disorders, the microtubule-associated protein tau aggregates and forms cytoplasmic lesions that impair neuronal physiology at many levels. In addition to affecting the host neuron, tau aggregates also spread to neighboring, recipient cells where the misfolded tau aggregates, in a manner similar to prions, actively corrupt the proper folding of soluble tau, and thereby impair cellular functions. One vehicle for the transmission of tau aggregates are secretory nanovesicles known as exosomes. Here, we established a simple model of a neuronal circuit using a microfluidics culture system in which hippocampal neurons A and B were seeded into chambers 1 and 2, respectively, extending axons via microgrooves in both directions and thereby interconnecting. This system served to establish two models to track exosome spreading. In the first model, we labeled the exosomal membrane by coupling tetraspanin CD9 with either a green or red fluorescent tag. This allowed us to reveal that interconnected neurons exchange exosomes only when their axons extend in close proximity. In the second model, we added exosomes isolated from the brains of tau transgenic rTg4510 mice (i.e. exogenous, neuron A-derived) to neurons in chamber 1 (neuron B) interconnected with neuron C in chamber 2. This allowed us to demonstrate that a substantial fraction of the exogenous exosomes were internalized by neuron B and passed then on to neuron C. This transportation from neuron B to C was achieved by a mechanism that is consistent with the hijacking of secretory endosomes by the exogenous exosomes, as revealed by confocal, super-resolution and electron microscopy. Together, these findings suggest that fusion events involving the endogenous endosomal secretory machinery increase the pathogenic potential and the radius of action of pathogenic cargoes carried by exogenous exosomes

Probing the structural mechanism of partial agonism in glycine receptors using the fluorescent artificial amino acid, ANAP

The efficacy of an agonist at a pentameric ligand-gated ion channel is determined by the rate at which it induces a conformational change from the resting closed state to a preopen ("flip") state. If the ability of an agonist to promote this isomerization is sufficiently low, then it becomes a partial agonist. As partial agonists at pentameric ligand-gated ion channels show considerable promise as therapeutics, understanding the structural basis of the resting-flip-state isomerization may provide insight into therapeutic design. Accordingly, we sought to identify structural correlates of the resting-flip conformational change in the glycine receptor chloride channel. We used nonsense suppression to introduce the small, fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP), into specific sites in the extracellular and transmembrane domains. Then, under voltage-clamp conditions in Xenopus oocytes, we simultaneously quantified current and fluorescence responses induced by structurally similar agonists with high, medium, and low efficacies (glycine, β-alanine, and taurine, respectively). Analyzing results from nine ANAP-incorporated sites, we show that glycine receptor activation by agonists with graded efficacies manifests structurally as correspondingly graded movements of the β1β2 loop, the β8 β9 loop, and the Cys-loop from the extracellular domain and the TM2-TM3 linker in the transmembrane domain. We infer that the resting-flip transition involves an efficacy-dependent molecular reorganization at the extracellular-transmembrane domain interface that primes receptors for efficacious opening