In-depth polarisation resolved SHG microscopy in biological tissues using iterative wavefront optimisation.

Polarised nonlinear microscopy has been extensively developed to study molecular organisation in biological tissues, quantifying the response of nonlinear signals to a varying incident linear polarisation. Polarisation Second harmonic Generation (PSHG) in particular is a powerful tool to decipher sub-microscopic modifications of fibrillar collagen organisation in type I and III collagen-rich tissues. The quality of SHG imaging is however limited to about one scattering mean free path in depth (typically 100 micrometres in biological tissues), due to the loss of focus quality, induced by wavefront aberrations and scattering at even larger depths. In this work, we study how optical depth penetration in biological tissues affects the quality of polarisation control, a crucial parameter for quantitative assessment of PSHG measurements. We apply wavefront shaping to correct for SHG signal quality in two regimes, adaptive optics for smooth aberration modes corrections at shallow depth, and wavefront shaping of higher spatial frequencies for optical focus correction at larger depths. Using nonlinear SHG active nanocrystals as guide stars, we quantify the capabilities of such optimisation methods to recover a high-quality linear polarisation and investigate how this approach can be applied to in-depth PSHG imaging in tissues, namely tendon and mouse cranial bone.

Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells.

Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

Using fluorescent beads to emulate single fluorophores.

We study the conditions under which fluorescent beads can be used to emulate single fluorescent molecules in the calibration of optical microscopes. Although beads are widely used due to their brightness and easy manipulation, there can be notable differences between the point spread functions (PSFs) they produce and those for single-molecule fluorophores, caused by their different emission patterns and sizes. We study theoretically these differences for various scenarios, e.g., with or without polarization channel splitting, to determine the conditions under which the use of beads as a model for single molecules is valid. We also propose methods to model the blurring due to the size difference and compensate for it to produce PSFs that are more similar to those for single molecules.

Fluorescence polarization modulation super-resolution imaging provides refined dynamics orientation processes in biological samples.

Combining polarization modulation Fourier analysis and spatial information in a joint reconstruction algorithm for polarization-resolved fluorescence imaging provides not only a gain in spatial resolution but also a sensitive readout of anisotropy in cell samples.

Energy-Efficient Iodine Uptake by a Molecular Host⋠Guest Crystal.

Recently, porous organic crystals (POC) based on macrocycles have shown exceptional sorption and separation properties. Yet, the impact of guest presence inside a macrocycle prior to adsorption has not been studied. Here we show that the inclusion of trimethoxybenzyl-azaphosphatrane in the macrocycle cucurbit[8]uril (CB[8]) affords molecular porous host⋅guest crystals (PHGC-1) with radically new properties. Unactivated hydrated PHGC-1 adsorbed iodine spontaneously and selectively at room temperature and atmospheric pressure. The absence of (i) heat for material synthesis, (ii) moisture sensitivity, and (iii) energy-intensive steps for pore activation are attractive attributes for decreasing the energy costs. 1 H NMR and DOSY were instrumental for monitoring the H2 O/I2 exchange. PHGC-1 crystals are non-centrosymmetric and I2 -doped crystals showed markedly different second harmonic generation (SHG), which suggests that iodine doping could be used to modulate the non-linear optical properties of porous organic crystals.

DNA Self-Assembly of Single Molecules with Deterministic Position and Orientation.

An ideal nanofabrication method should allow the organization of nanoparticles and molecules with nanometric positional precision, stoichiometric control, and well-defined orientation. The DNA origami technique has evolved into a highly versatile bottom-up nanofabrication methodology that fulfils almost all of these features. It enables the nanometric positioning of molecules and nanoparticles with stoichiometric control, and even the orientation of asymmetrical nanoparticles along predefined directions. However, orienting individual molecules has been a standing challenge. Here, we show how single molecules, namely, Cy5 and Cy3 fluorophores, can be incorporated in a DNA origami with controlled orientation by doubly linking them to oligonucleotide strands that are hybridized while leaving unpaired bases in the scaffold. Increasing the number of bases unpaired induces a stretching of the fluorophore linkers, reducing its mobility freedom, and leaves more space for the fluorophore to accommodate and find different sites for interaction with the DNA. Particularly, we explore the effects of leaving 0, 2, 4, 6, and 8 bases unpaired and find extreme orientations for 0 and 8 unpaired bases, corresponding to the molecules being perpendicular and parallel to the DNA double-helix, respectively. We foresee that these results will expand the application field of DNA origami toward the fabrication of nanodevices involving a wide range of orientation-dependent molecular interactions, such as energy transfer, intermolecular electron transport, catalysis, exciton delocalization, or the electromagnetic coupling of a molecule to specific resonant nanoantenna modes.

Septin filament compaction into rings requires the anillin Mid2 and contractile ring constriction.

Septin filaments assemble into high-order molecular structures that associate with membranes, acting as diffusion barriers and scaffold proteins crucial for many cellular processes. How septin filaments organize in such structures is still not understood. Here, we used fission yeast to explore septin filament organization during cell division and its cell cycle regulation. Live-imaging and polarization microscopy analysis uncovered that septin filaments are initially recruited as a diffuse meshwork surrounding the acto-myosin contractile ring (CR) in anaphase, which undergoes compaction into two rings when CR constriction is initiated. We found that the anillin-like protein Mid2 is necessary to promote this compaction step, possibly acting as a bundler for septin filaments. Moreover, Mid2-driven septin compaction requires inputs from the septation initiation network as well as CR constriction and the ß(1,3)-glucan synthase Bgs1. This work highlights that anillin-mediated septin ring assembly is under strict cell cycle control.

4polar-STORM polarized super-resolution imaging of actin filament organization in cells.

Single-molecule localization microscopy provides insights into the nanometer-scale spatial organization of proteins in cells, however it does not provide information on their conformation and orientation, which are key functional signatures. Detecting single molecules' orientation in addition to their localization in cells is still a challenging task, in particular in dense cell samples. Here, we present a polarization-splitting scheme which combines Stochastic Optical Reconstruction Microscopy (STORM) with single molecule 2D orientation and wobbling measurements, without requiring a strong deformation of the imaged point spread function. This method called 4polar-STORM allows, thanks to a control of its detection numerical aperture, to determine both single molecules' localization and orientation in 2D and to infer their 3D orientation. 4polar-STORM is compatible with relatively high densities of diffraction-limited spots in an image, and is thus ideally placed for the investigation of dense protein assemblies in cells. We demonstrate the potential of this method in dense actin filament organizations driving cell adhesion and motility.

Lipids-Fluorophores Interactions Probed by Combined Nonlinear Polarized Microscopy.

Studying the structural dynamics of lipid membranes requires methods that can address both microscopic and macroscopic characteristics. Fluorescence imaging is part of the most used techniques to study membrane properties in various systems from artificial membranes to cells: It benefits from a high sensitivity to local properties such as polarity and molecular orientational order, with a high spatial resolution down to the single-molecule level. The influence of embedded fluorescent lipid probes on the lipid membrane molecules is however poorly known and relies most often on molecular dynamics simulations, due to the challenges faced by experimental approaches to address the molecular-scale dimension of this question. In this work we develop an optical microscopy imaging method to probe the effect of fluorophores embedded in the membrane as lipid probes, on their lipid environment, with a lateral resolution of a few hundreds of nanometers. We combine polarized-nonlinear microscopy contrasts that can independently address the lipid probe, by polarized two-photon fluorescence, and the membrane lipids, by polarized coherent Raman scattering. Using trimethylamino derivative 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS) as model probes, we show that both probes tend to induce an orientational disorder of their surrounding lipid CH-bonds in 1,2-dipalmitoylphosphatidylcholine (DPPC) lipids environments, while there is no noticeable effect in more disordered 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid membranes.

Birefringent Fourier filtering for single molecule coordinate and height super-resolution imaging with dithering and orientation.

Super-resolution imaging based on single molecule localization allows accessing nanometric-scale information in biological samples with high precision. However, complete measurements including molecule orientation are still challenging. Orientation is intrinsically coupled to position in microscopy imaging, and molecular wobbling during the image integration time can bias orientation measurements. Providing 3D molecular orientation and orientational fluctuations would offer new ways to assess the degree of alignment of protein structures, which cannot be monitored by pure localization. Here we demonstrate that by adding polarization control to phase control in the Fourier plane of the imaging path, all parameters can be determined unambiguously from single molecules: 3D spatial position, 3D orientation and wobbling or dithering angle. The method, applied to fluorescent labels attached to single actin filaments, provides precisions within tens of nanometers in position and few degrees in orientation.

Author Correction: Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pyclen-Based Ln(III) Complexes as Highly Luminescent Bioprobes for In Vitro and In Vivo One- and Two-Photon Bioimaging Applications.

In addition to the already described ligand L4a, two pyclen-based lanthanide chelators, L4b and L4c, bearing two specific picolinate two-photon antennas (tailor-made for each targeted metal) and one acetate arm arranged in a dissymmetrical manner, have been synthesized, to form a complete family of lanthanide luminescent bioprobes: [EuL4a], [SmL4a], [YbL4b], [TbL4c], and [DyL4c]. Additionally, the symmetrically arranged regioisomer L4a' was also synthesized as well as its [EuL4a'] complex to highlight the astonishing positive impact of the dissymmetrical N-distribution of the functional chelating arms. The investigation clearly shows the high performance of each bioprobe, which, depending on the complexed lanthanide, could be used in various applications. Each presents high brightness, quantum yields, and lifetimes. Staining of the complexes into living human breast cancer cells was observed. In addition, in vivo two-photon microscopy was performed for the first time on a living zebrafish model with [EuL4a]. No apparent toxicity was detected on the growth of the zebrafish, and images of high quality were obtained.

Cationic Biphotonic Lanthanide Luminescent Bioprobes Based on Functionalized Cross-Bridged Cyclam Macrocycles.

Cationic lanthanide complexes are generally able to spontaneously internalize into living cells. Following our previous works based on a diMe-cyclen framework, a second generation of cationic water-soluble lanthanide complexes based on a constrained cross-bridged cyclam macrocycle functionalized with donor-π-conjugated picolinate antennas was prepared with europium(III) and ytterbium(III). Their spectroscopic properties were thoroughly investigated in various solvents and rationalized with the help of DFT calculations. A significant improvement was observed in the case of the Eu3+ complex, while the Yb3+ analogue conserved photophysical properties in aqueous solvent. Two-photon (2P) microscopy imaging experiments on living T24 human cancer cells confirmed the spontaneous internalization of the probes and images with good signal-to-noise ratio were obtained in the classic NIR-to-visible configuration with the Eu3+ luminescent bioprobe and in the NIR-to-NIR with the Yb3+ one.

Brillouin microspectroscopy data of tissue-mimicking gelatin hydrogels.

Brillouin spectroscopy, based on the inelastic scattering of light from thermally driven acoustic waves or phonons [1], holds great promise in the field of life sciences as it provides functionally relevant micromechanical information in a contactless all-optical manner [2]. Due to the complexity of biological systems such as cells and tissues, which present spatio-temporal heterogeneities, interpretation of Brillouin spectra can be difficult. The data presented here were collected from gelatin hydrogels, used as tissue-mimicking model systems for Brillouin microspectroscopy measurements conducted using a lab-built Brillouin microscope with a dual-stage VIPA spectrometer. By varying the solute concentration in the range 4-18% (w/w), the macroscopic mechanical properties of the hydrogels can be tuned and the corresponding evolution in the Brillouin-derived longitudinal elastic modulus measured. An increase in Brillouin frequency shift with increasing solute concentration was observed, which was found to correlate with an increase in acoustic wave velocity and longitudinal modulus. The gels used here provide a viable model system for benchmarking and standardisation, and the data will be useful for spectrometer development and validation.

Focusing large spectral bandwidths through scattering media.

Wavefront shaping is a powerful method to refocus light through a scattering medium. Its application to large spectral bandwidths or multiple wavelengths refocusing for nonlinear bio-imaging in-depth is however limited by spectral decorrelations. In this work, we demonstrate ways to access a large spectral memory of a refocus in thin scattering media and thick forward-scattering biological tissues. First, we show that the accessible spectral bandwidth through a scattering medium involves an axial spatio-spectral coupling, which can be minimized when working in a confocal geometry. Second, we show that this bandwidth can be further enlarged when working in a broadband excitation regime. These results open important prospects for multispectral nonlinear imaging through scattering media.

The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover.

Focal adhesion (FA) turnover depends on microtubules and actin. Microtubule ends are captured at FAs, where they induce rapid FA disassembly. However, actin's roles are less clear. Here, we use polarization-resolved microscopy, FRAP, live cell imaging, and a mutant of Adenomatous polyposis coli (APC-m4) defective in actin nucleation to investigate the role of actin assembly in FA turnover. We show that APC-mediated actin assembly is critical for maintaining normal F-actin levels, organization, and dynamics at FAs, along with organization of FA components. In WT cells, microtubules are captured repeatedly at FAs as they mature, but once a FA reaches peak maturity, the next microtubule capture event leads to delivery of an autophagosome, triggering FA disassembly. In APC-m4 cells, microtubule capture frequency and duration are altered, and there are long delays between autophagosome delivery and FA disassembly. Thus, APC-mediated actin assembly is required for normal feedback between microtubules and FAs, and maintaining FAs in a state "primed" for microtubule-induced turnover.

Manipulating the transmission matrix of scattering media for nonlinear imaging beyond the memory effect.

The measurement of the transmission matrix (TM) of a scattering medium is of great interest for imaging. It can be acquired directly by interferometry using an internal reference wavefront. Unfortunately, internal reference fields are scattered by the medium, which results in a speckle that makes the TM measurement heterogeneous across the output field of view. We demonstrate how to correct for this effect using the intrinsic properties of the TM. For thin scattering media, we exploit the memory effect of the medium and the reference speckle to create a corrected TM. For highly scattering media where the memory effect is negligible, we use complementary reference speckles to compose a new TM, not compromised by the speckled reference anymore. Using this correction, we demonstrate large field of view second harmonic generation imaging through thick biological media.

Collagen reorganization in cartilage under strain probed by polarization sensitive second harmonic generation microscopy.

Type II collagen fibril diameters in cartilage are beneath the diffraction limit of optical microscopy, which makes the assessment of collagen organization very challenging. In this work we use polarization sensitive second harmonic generation (P-SHG) imaging to map collagen organization in articular cartilage, addressing in particular its behaviour under strain and changes which occur in osteoarthritis. P-SHG yields two parameters, molecular order and orientation, which provide measures of the degree of organization both at the molecular scale (below the diffraction limit) and above a few hundred nanometres (at the image pixel size). P-SHG clearly demonstrates the zonal collagen architecture and reveals differences in the structure of the fibrils around chondrocytes. P-SHG also reveals sub-micron scale fibril re-organization in cartilage strips exposed to tensile loading, with an increase in local organization in the superficial zone which weakly correlates with tensile modulus. Finally, P-SHG is used to investigate osteoarthritic cartilage from total knee replacement surgery, and reveals widespread heterogeneity across samples both microscale fibril orientations and their sub-micron organization. By addressing collagen fibril structure on scales intermediate between conventional light and electron microscopy, this study provides new insights into collagen micromechanics and mechanisms of degradation.

Image analysis applied to Brillouin images of tissue-mimicking collagen gelatins.

Brillouin spectroscopy is an emerging analytical tool in biomedical and biophysical sciences. It probes viscoelasticity through the propagation of thermally induced acoustic waves at gigahertz frequencies. Brillouin light scattering (BLS) measurements have traditionally been performed using multipass Fabry-Pérot interferometers, which have high contrast and resolution, however, as they are scanning spectrometers they often require long acquisition times in poorly scattering media. In the last decade, a new concept of Brillouin spectrometer has emerged, making use of highly angle-dispersive virtually imaged phase array (VIPA) etalons, which enable fast acquisition times for minimally turbid materials, when high contrast is not imperative. The ability to acquire Brillouin spectra rapidly, together with long term system stability, make this system a viable candidate for use in biomedical applications, especially to probe live cells and tissues. While various methods are being developed to improve system contrast and speed, little work has been published discussing the details of imaging data analysis and spectral processing. Here we present a method that we developed for the automated retrieval of Brillouin line shape parameters from imaging data sets acquired with a dual-stage VIPA Brillouin microscope. We applied this method for the first time to BLS measurements of collagen gelatin hydrogels at different hydration levels and cross-linker concentrations. This work demonstrates that it is possible to obtain the relevant information from Brillouin spectra using software for real-time high-accuracy analysis.