Expanding the Palette of SWIR Emitting Nanoparticles Based on Au Nanoclusters for Single-Particle Tracking Microscopy.

Single-molecule localization microscopy has proved promising to unravel the dynamics and molecular architecture of thin biological samples down to nanoscales. For applications in complex, thick biological tissues shifting single-particle emission wavelengths to the shortwave infrared (SWIR also called NIR II) region between 900 to 2100 nm, where biological tissues are more transparent is key. To date, mainly single-walled carbon nanotubes (SWCNTs) enable such applications, but they are inherently 1D objects. Here, 0D ultra-small luminescent gold nanoclusters (AuNCs, <3 nm) and ≈25 nm AuNC-loaded-polymeric particles that can be detected at the single-particle level in the SWIR are presented. Thanks to high brightness and excellent photostability, it is shown that the dynamics of the spherical polymeric particles can be followed at the single-particle level in solution at video rates for minutes. We compared single particle tracking of AuNC-loaded-polymeric particles with that of SWCNT diffusing in agarose gels demonstrating the specificity and complementarity of diffusion properties of these SWIR-emitting nano-objects when exploring a complex environment. This extends the library of photostable SWIR emitting nanomaterials to 0D nano-objects of variable size for single-molecule localization microscopy in the second biological window, opening unprecedented possibilities for mapping the structure and dynamics of complex biological systems.

Identification of Early Stage Liver Fibrosis by Modifications in the Interstitial Space Diffusive Microenvironment Using Fluorescent Single-Walled Carbon Nanotubes.

During liver fibrosis, recurrent hepatic injuries lead to the accumulation of collagen and other extracellular matrix components in the interstitial space, ultimately disrupting liver functions. Early stages of liver fibrosis may be reversible, but opportunities for diagnosis at these stages are currently limited. Here, we show that the alterations of the interstitial space associated with fibrosis can be probed by tracking individual fluorescent single-walled carbon nanotubes (SWCNTs) diffusing in that space. In a mouse model of early liver fibrosis, we find that nanotubes generally explore elongated areas, whose lengths decrease as the disease progresses, even in regions where histopathological examination does not reveal fibrosis yet. Furthermore, this decrease in nanotube mobility is a purely geometrical effect as the instantaneous nanotube diffusivity stays unmodified. This work establishes the promise of SWCNTs both for diagnosing liver fibrosis at an early stage and for more in-depth studies of the biophysical effects of the disease.

On the equivalence of binary phase masks optimized for localization or detection in extended depth-of-field localization microscopy.

Binary annular masks have recently been proposed to extend the depth of field (DoF) of single-molecule localization microscopy. A strategy for designing optimal masks has been introduced based on maximizing the emitter localization accuracy, expressed in terms of Fisher information, over a targeted DoF range. However, the complete post-processing pipeline to localize a single emitter consists of two successive steps: detection, where the regions containing emitters are determined, and localization, where the sub-pixel position of each detected emitter is estimated. Phase masks usually optimize only this second step. The presence of a phase mask also affecting detection, the purpose of this paper is to quantify and mitigate this effect. Using a rigorous framework built from a detection-oriented information theoretical criterion (Bhattacharyya distance), we demonstrate that in most cases of practical significance, annular binary phase masks maximizing Fisher information also maximize the detection probability. This result supports the common design practice consisting of optimizing a phase mask by maximizing Fisher information only.

Reuniting philosophy and science to advance cancer research.

Cancers rely on multiple, heterogeneous processes at different scales, pertaining to many biomedical fields. Therefore, understanding cancer is necessarily an interdisciplinary task that requires placing specialised experimental and clinical research into a broader conceptual, theoretical, and methodological framework. Without such a framework, oncology will collect piecemeal results, with scant dialogue between the different scientific communities studying cancer. We argue that one important way forward in service of a more successful dialogue is through greater integration of applied sciences (experimental and clinical) with conceptual and theoretical approaches, informed by philosophical methods. By way of illustration, we explore six central themes: (i) the role of mutations in cancer; (ii) the clonal evolution of cancer cells; (iii) the relationship between cancer and multicellularity; (iv) the tumour microenvironment; (v) the immune system; and (vi) stem cells. In each case, we examine open questions in the scientific literature through a philosophical methodology and show the benefit of such a synergy for the scientific and medical understanding of cancer.

Nanoscale and functional heterogeneity of the hippocampal extracellular space.

The extracellular space (ECS) and its constituents play a crucial role in brain development, plasticity, circadian rhythm, and behavior, as well as brain diseases. Yet, since this compartment has an intricate geometry and nanoscale dimensions, its detailed exploration in live tissue has remained an unmet challenge. Here, we used a combination of single-nanoparticle tracking and super-resolution microscopy approaches to map the nanoscale dimensions of the ECS across the rodent hippocampus. We report that these dimensions are heterogeneous between hippocampal areas. Notably, stratum radiatum CA1 and CA3 ECS differ in several characteristics, a difference that gets abolished after digestion of the extracellular matrix. The dynamics of extracellular immunoglobulins vary within these areas, consistent with their distinct ECS characteristics. Altogether, we demonstrate that ECS nanoscale anatomy and diffusion properties are widely heterogeneous across hippocampal areas, impacting the dynamics and distribution of extracellular molecules.

On Some Current Challenges in High-Resolution Optical Bioimaging.

In this Perspective we propose our current point of view and a suggestive roadmap on the field of high-resolution optical microscopy dedicated to bioimaging. Motivated by biological applications, researchers have indeed devised an impressive amount of strategies to address the diverse constraints of imaging and studying biological matter down to the molecular scale, making this interdisciplinary research field a vibrant forum for creativity. Throughout the discussion, we highlight several striking recent successes in this quest. We also identify some next challenges still ahead to apprehend biological questions in increasingly complex living organisms for integrative studies in a minimally invasive manner.

Near-Infrared Carbon Nanotube Tracking Reveals the Nanoscale Extracellular Space around Synapses.

We provide evidence of a local synaptic nanoenvironment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nanoenvironment might play a role in the regulation of brain activity.

Single-walled carbon nanotube reptation dynamics in submicron sized pores from randomly packed mono-sized colloids.

Studying the Brownian motion of fibers and semi-flexible filaments in porous media is the key to understanding the transport and mechanical properties in a variety of systems. The motion of semi-flexible filaments in gel-like porous media including polymer networks and cell cytoskeleton has been studied theoretically and experimentally, whereas the motion of these materials in packed-colloid porous media, advanced foams, and rock-like systems has not been thoroughly studied. Here we use video microscopy to directly visualize the reptation and transport of intrinsically fluorescent, semiflexible, semiconducting single-walled carbon nanotubes (SWCNTs) in the sub-micron pores of packed colloids as fixed obstacles of packed-colloid porous media. By visualizing the filament motion and Brownian diffusion at different locations in the pore structures, we study how the properties of the environment, like the pore shape and pore structure of the porous media, affect SWCNT mobility. These results show that the porous media structure controls SWCNT reorientation during Brownian diffusion. In packed-colloid pores, SWCNTs diffuse along straight pores and bend across pores; conversely, in gel pores, SWCNTs consistently diffuse into curved pores, displaying a faster parallel motion. In both gel and packed-colloid porous media, SWCNT finite stiffness enhances SWCNT rotational diffusion and prevents jamming, allowing for inter-pore diffusion.

When Super-Resolution Localization Microscopy Meets Carbon Nanotubes.

We recently assisted in a revolution in the realm of fluorescence microscopy triggered by the advent of super-resolution techniques that surpass the classic diffraction limit barrier. By providing optical images with nanometer resolution in the far field, super-resolution microscopy (SRM) is currently accelerating our understanding of the molecular organization of bio-specimens, bridging the gap between cellular observations and molecular structural knowledge, which was previously only accessible using electron microscopy. SRM mainly finds its roots in progress made in the control and manipulation of the optical properties of (single) fluorescent molecules. The flourishing development of novel fluorescent nanostructures has recently opened the possibility of associating super-resolution imaging strategies with nanomaterials' design and applications. In this review article, we discuss some of the recent developments in the field of super-resolution imaging explicitly based on the use of nanomaterials. As an archetypal class of fluorescent nanomaterial, we mainly focus on single-walled carbon nanotubes (SWCNTs), which are photoluminescent emitters at near-infrared (NIR) wavelengths bearing great interest for biological imaging and for information optical transmission. Whether for fundamental applications in nanomaterial science or in biology, we show how super-resolution techniques can be applied to create nanoscale images "in", "of" and "with" SWCNTs.

On the validity domain of maximum likelihood estimators for depth-of-field extension in single-molecule localization microscopy.

Localization microscopy approaches with enhanced depth-of-field (EDoF) are commonly optimized using the Cramér-Rao bound (CRB) as a criterion. It is widely believed that the CRB can be attained in practice by using the maximum-likelihood estimator (MLE). This is, however, an approximation, of which we define in this paper the precise domain of validity. Exploring a wide range of settings and noise levels, we show that the MLE is efficient when the signal-to-noise ratio (SNR) is such that the localization standard deviation of a single molecule is less than 20 nm. Thus, our results provide an explicit and quantitative validity boundary for the use of the MLE in EDoF localization microscopy setups optimized with the CRB.

A Bottom-Up Approach to Red-Emitting Molecular-Based Nanoparticles with Natural Stealth Properties and their Use for Single-Particle Tracking Deep in Brain Tissue.

Fluorescent nanoparticles dedicated to bioimaging applications should possess specific properties that have to be maintained in the aqueous, reactive, and crowded biological environment. These include chemical and photostability, small size (on the scale of subcellular structures), biocompatibility, high brightness, and good solubility. The latter is a major challenge for inorganic nanoparticles, which require surface coating to be made water soluble. Molecular-based fluorescent organic nanoparticles (FONs) may prove a promising, spontaneously water-soluble alternative, whose bottom-up design allows for the fine-tuning of individual properties. Here, the critical challenge of controlling the interaction of nanoparticles with cellular membranes is addressed. This is a report on bright, size-tunable, red-emitting, naturally stealthy FONs that do not require the use of antifouling agents to impede interactions with cellular membranes. As a proof of concept, single FONs diffusing up to 150 µm deep in brain tissue are imaged and tracked.

Near-infrared nanoscopy with carbon-based nanoparticles for the exploration of the brain extracellular space.

Understanding the physiology and pathology of the brain requires detailed knowledge of its complex structures as well as dynamic internal processes at very different scales from the macro down to the molecular dimensions. A major yet poorly described brain compartment is the brain extracellular space (ECS). Signalling molecules rapidly diffuse through the brain ECS which is complex and dynamic structure at numerous lengths and time scales. In recent years, characterization of the ECS using nanomaterials has made remarkable progress, including local analysis of nanoscopic dimensions and diffusivity as well as local chemical sensing. In particular, carbon nanomaterials combined with advanced optical technologies, biochemical and biophysical analysis, offer novel promises for understanding the ECS morphology as well as neuron connectivity and neurochemistry. In this review, we present the state-of-the-art in this quest, which mainly focuses on a type of carbon nanomaterial, single walled carbon nanotubes, as fluorescent nanoprobes to unveil the ECS features in the nanometre domain.

Co-designed annular binary phase masks for depth-of-field extension in single-molecule localization microscopy.

Single-molecule localization microscopy has become a prominent approach to study structural and dynamic arrangements of nanometric objects well beyond the diffraction limit. To maximize localization precision, high numerical aperture objectives must be used; however, this inherently strongly limits the depth-of-field (DoF) of the microscope images. In this work, we present a framework inspired by "optical co-design" to optimize and benchmark phase masks, which, when placed in the exit pupil of the microscope objective, can extend the DoF in the realistic context of single fluorescent molecule detection. Using the Cramér-Rao bound (CRB) on localization accuracy as a criterion, we optimize annular binary phase masks for various DoF ranges, compare them to Incoherently Partitioned Pupil masks and show that they significantly extend the DoF of single-molecule localization microscopes. In particular we propose different designs including a simple and easy-to-realize two-ring binary mask to extend the DoF. Moreover, we demonstrate that a simple maximum likelihood-based localization algorithm can reach the localization accuracy predicted by the CRB. The framework developed in this paper is based on an explicit and general information theoretic criterion, and can thus be used as an engineering tool to optimize and compare any type of DoF-enhancing phase mask in high resolution microscopy on a quantitative basis.

Synucleinopathy alters nanoscale organization and diffusion in the brain extracellular space through hyaluronan remodeling.

In recent years, exploration of the brain extracellular space (ECS) has made remarkable progress, including nanoscopic characterizations. However, whether ECS precise conformation is altered during brain pathology remains unknown. Here we study the nanoscale organization of pathological ECS in adult mice under degenerative conditions. Using electron microscopy in cryofixed tissue and single nanotube tracking in live brain slices combined with super-resolution imaging analysis, we find enlarged ECS dimensions and increased nanoscale diffusion after α-synuclein-induced neurodegeneration. These animals display a degraded hyaluronan matrix in areas close to reactive microglia. Furthermore, experimental hyaluronan depletion in vivo reduces dopaminergic cell loss and α-synuclein load, induces microgliosis and increases ECS diffusivity, highlighting hyaluronan as diffusional barrier and local tissue organizer. These findings demonstrate the interplay of ECS, extracellular matrix and glia in pathology, unraveling ECS features relevant for the α-synuclein propagation hypothesis and suggesting matrix manipulation as a disease-modifying strategy.

Fluorescent sp3 Defect-Tailored Carbon Nanotubes Enable NIR-II Single Particle Imaging in Live Brain Slices at Ultra-Low Excitation Doses.

Cellular and tissue imaging in the second near-infrared window (NIR-II, ~1000-1350 nm) is advantageous for in vivo studies because of low light extinction by biological constituents at these wavelengths. However, deep tissue imaging at the single molecule sensitivity has not been achieved in the NIR-II window due to lack of suitable bio-probes. Single-walled carbon nanotubes have emerged as promising near-infrared luminescent molecular bio-probes; yet, their inefficient photoluminescence (quantum yield ~1%) drives requirements for sizeable excitation doses (~1-10 kW/cm2) that are significantly blue-shifted from the NIR-II region (<850 nm) and may thus ultimately compromise live tissue. Here, we show that single nanotube imaging can be achieved in live brain tissue using ultralow excitation doses (~0.1 kW/cm2), an order of magnitude lower than those currently used. To accomplish this, we synthesized fluorescent sp3-defect tailored (6,5) carbon nanotubes which, when excited at their first order excitonic transition (~985 nm) fluoresce brightly at ~1160 nm. The biocompatibility of these functionalized nanotubes, which are wrapped by encapsulation agent (phospholipid-polyethylene glycol), is demonstrated using standard cytotoxicity assays. Single molecule photophysical studies of these biocompatible nanotubes allowed us to identify the optimal luminescence properties in the context of biological imaging.

Plaque burden can be assessed using intravascular optical coherence tomography and a dedicated automated processing algorithm: a comparison study with intravascular ultrasound.

AIMS: Plaque burden (PB) measurement using intravascular optical coherence tomography (IVOCT) is currently thought to be inferior to intravascular ultrasound (IVUS). We developed an automated IVOCT image processing algorithm to enhance the external elastic lamina (EEL) contour. Thus, we investigated the accuracies of standard IVOCT and an IVOCT enhancement algorithm for measuring PB using IVUS as the reference standard. METHODS AND RESULTS: The EEL-enhancement algorithm combined adaptive attenuation compensation, exponentiation, angular registration, and image averaging using three sequential frames. In two different laboratories with intravascular imaging expertise, PB was quantified on 200 randomized, matched IVOCT and IVUS images by four independent observers. Fibroatheroma, fibrocalcific plaque, fibrous plaque, pathological intimal thickening (PIT), and mixed plaque were included in each set. Pearson's correlation coefficients between IVUS and standard IVOCT measurements of PB were 0.61, 0.67, 0.76, 0.78, and 0.87 for fibroatheromas, mixed plaques, fibrocalcific plaques, fibrous plaques, and PIT plaques, respectively. Pearson's correlation coefficients increased to 0.81, 0.83, 0.83, 0.84, and 0.90 when using the EEL-enhanced images (P = 0.003, P = 0.004, P = 0.08, P = 0.12, and P = 0.23, respectively). EEL-enhanced IVOCT analysis was associated with a lower EEL-area measurement absolute error for fibroatheromas, mixed plaques, and all pooled plaques (P = 0.006, P = 0.02, and P < 0.001, respectively). Compared with standard IVOCT, the EEL-enhanced IVOCT images had a higher sensitivity (79% vs. 28%, P < 0.001) and specificity (98% vs. 85%, P = 0.03) for plaques with an IVUS PB ≥70%. CONCLUSION: EEL-enhanced IVOCT can be used to reliably measure PB in all types of coronary atherosclerotic lesions, including fibroatheromas and mixed plaques.

Nanoscale exploration of the extracellular space in the live brain by combining single carbon nanotube tracking and super-resolution imaging analysis.

The brain extracellular space (ECS) is a system of narrow compartments whose intricate nanometric structure has remained elusive until very recently. Understanding such a complex organisation represents a technological challenge that requires a technique able to resolve these nanoscopic spaces and simultaneously characterize their rheological properties. We recently used single-walled carbon nanotubes (SWCNTs) as near-infrared fluorescent probes to map with nanoscale precision the local organization and rheology of the ECS. Here we expand our method by tracking single nanotubes through super-resolution imaging in rat organotypic hippocampal slices and acute brain slices from adult mice, pioneering the exploration of the adult brain ECS at the nanoscale. We found a highly heterogeneous ECS, where local rheological properties can change drastically within few nanometres. Our results suggest differences in local ECS diffusion environments in organotypic slices when compared to adult mouse slices. Data obtained from super-resolved maps of the SWCNT trajectories indicate that ECS widths may vary between brain tissue models, with a looser, less crowded nano-environment in organotypic cultured slices.

Photoswitchable single-walled carbon nanotubes for super-resolution microscopy in the near-infrared.

The design of single-molecule photoswitchable emitters was the first milestone toward the advent of single-molecule localization microscopy, setting a new paradigm in the field of optical imaging. Several photoswitchable emitters have been developed, but they all fluoresce in the visible or far-red ranges, missing the desirable near-infrared window where biological tissues are most transparent. Moreover, photocontrol of individual emitters in the near-infrared would be highly desirable for elementary optical molecular switches or information storage elements since most communication data transfer protocols are established in this spectral range. Here, we introduce a type of hybrid nanomaterials consisting of single-wall carbon nanotubes covalently functionalized with photoswitching molecules that are used to control the intrinsic luminescence of the single nanotubes in the near-infrared (beyond 1 µm). Through the control of photoswitching, we demonstrate super-localization imaging of nanotubes unresolved by diffraction-limited microscopy.

Unveiling the Extracellular Space of the Brain: From Super-resolved Microstructure to In Vivo Function.

The extracellular space occupies approximately one-fifth of brain volume, molding a spider web of gaps filled with interstitial fluid and extracellular matrix where neurons and glial cells perform in concert. Yet, very little is known about the spatial organization and dynamics of the extracellular space, let alone its influence on brain function, owing to a lack of appropriate techniques (and a traditional bias toward the inside of cells, not the spaces in between). At the same time, it is clear that understanding fundamental brain functions, such as synaptic transmission, memory, sleep, and recovery from disease, calls for more focused research on the extracellular space of the brain. This review article highlights several key research areas, covering recent methodological and conceptual progress that illuminates this understudied, yet critically important, brain compartment, providing insights into the opportunities and challenges of this nascent field.