Multi-foci parallelised RESOLFT nanoscopy in an extended field-of-view.

Live-cell imaging of biological structures at high resolution poses challenges in the microscope throughput regarding area and speed. For this reason, different parallelisation strategies have been implemented in coordinate- and stochastic-targeted switching super-resolution microscopy techniques. In this line, the molecular nanoscale live imaging with sectioning ability (MoNaLISA), based on reversible saturable optical fluorescence transitions (RESOLFT), offers 45 - 65 nm $45 - 65\;{\rm{nm}}$ resolution of large fields of view in a few seconds. In MoNaLISA, engineered light patterns strategically confine the fluorescence to sub-diffracted volumes in a large area and provide optical sectioning, thus enabling volumetric imaging at high speeds. The optical setup presented in this paper extends the degree of parallelisation of the MoNaLISA microscope by more than four times, reaching a field-of-view of ( 100 - 130 µ m ) 2 ${( {100 - 130\;{\rm{\mu m}}} )^2}$ . We set up the periodicity and the optical scheme of the illumination patterns to be power-efficient and homogeneous. In a single recording, this new configuration enables super-resolution imaging of an extended population of the post-synaptic density protein Homer1c in living hippocampal neurons.

A fluorescence nanoscopy marker for corticotropin-releasing hormone type 1 receptor: computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and in situ affinity constant in cells.

Class B G protein-coupled receptors (GPCRs) are involved in a variety of human pathophysiological states. These groups of membrane receptors are less studied than class A GPCRs due to the lack of structural information, delayed small molecule drug discovery, and scarce fluorescence detection tools available. The class B corticotropin-releasing hormone type 1 receptor (CRHR1) is a key player in the stress response whose dysregulation is critically involved in stress-related disorders: psychiatric conditions (i.e. depression, anxiety, and addictions), neuroendocrinological alterations, and neurodegenerative diseases. Here, we present a strategy to label GPCRs with a small fluorescent antagonist that permits the observation of the receptor in live cells through stochastic optical reconstruction microscopy (STORM) with 23 nm resolution. The marker, an aza-BODIPY derivative, was designed based on computational docking studies, then synthesized, and finally tested in biological cells. Experiments on hippocampal neurons demonstrate antagonist effects in similar concentrations as the well-established antagonist CP-376395. A quantitative analysis of two color STORM images enabled the determination of the binding affinity of the new marker in the cellular environment.

Author Correction: Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.

Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models.

ErbB2 is a member of the ErbB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ErbB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ErbB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ErbB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ErbB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ErbB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ErbB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ErbB2: a major population located in large clusters and a minor population outside these structures. Upon ErbB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ErbB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ErbB2 and beta1 integrin activity in breast cancer cells.

Automated quantification of protein periodic nanostructures in fluorescence nanoscopy images: abundance and regularity of neuronal spectrin membrane-associated skeleton.

Fluorescence nanoscopy imaging permits the observation of periodic supramolecular protein structures in their natural environment, as well as the unveiling of previously unknown protein periodic structures. Deciphering the biological functions of such protein nanostructures requires systematic and quantitative analysis of large number of images under different experimental conditions and specific stimuli. Here we present a method and an open source software for the automated quantification of protein periodic structures in super-resolved images. Its performance is demonstrated by analyzing the abundance and regularity of the spectrin membrane-associated periodic skeleton (MPS) in hippocampal neurons of 2 to 40 days in vitro, imaged by STED and STORM nanoscopy. The automated analysis reveals that both the abundance and the regularity of the MPS increase over time and reach maximum plateau values after 14 DIV. A detailed analysis of the distributions of correlation coefficients provides indication of dynamical assembly and disassembly of the MPS.

Note: Tormenta: An open source Python-powered control software for camera based optical microscopy.

Until recently, PC control and synchronization of scientific instruments was only possible through closed-source expensive frameworks like National Instruments' LabVIEW. Nowadays, efficient cost-free alternatives are available in the context of a continuously growing community of open-source software developers. Here, we report on Tormenta, a modular open-source software for the control of camera-based optical microscopes. Tormenta is built on Python, works on multiple operating systems, and includes some key features for fluorescence nanoscopy based on single molecule localization.