Fluorescence microscopy has succeeded in attaining super-resolution localization of single emitters in cellular biology. However, 3D localization deep inside tissue is still challenging. A few years ago, we developed SELFI: self-interference 3D super-resolution microscopy, a framework for 3D single-molecule localization within multicellular specimens and tissues. Here, we extend the capability of SELFI to the near-infrared (NIR) region where carbon nanotubes (CNTs) are strong emitters. The aim of this work is to develop NIR SELFI for single-particle tracking applications of CNTs in live brain tissues or NIR quantum dots. SELFI uses a diffraction grating placed on the optical path of the sample image, generating an interference pattern within diffraction limited images of point emitters. A single image obtained with NIR SELFI contains two independent variables: the intensity distribution to extract the intensity centroid to determine the lateral localization, and the wavefront curvature (provided by the interfringes) to get the axial super-localization. SELFI was first developed to localize red emitting dyes and quantum dots. The performance of the system is examined by means of the standard deviation and root mean square error of the localizations. The experiments performed show that the 3D-precision and accuracy achieved with NIR SELFI are both below 100 nm for emission around 1000 nm and high photon budget. Therefore, we can now achieve 3D localization in the NIR, permitting 3D single-particle tracking of CNTs at video rate in complex environments.
In localization microscopy, the position of isolated fluorescent emitters are estimated with a resolution better than the diffraction limit. In order to image thick samples, which are common in biological applications, there is considerable interest in extending the depth-of-field of such microscopes in order to make their accuracy as invariant as possible to defocus. For that purpose, we propose to optimize annular binary phase masks placed in the pupil of the microscope in order to generate a point spread function for which the localization accuracy is almost invariant along the optical axis. The optimization criterion is defined as the localization accuracy in the plane expressed in terms of the Cram´er-Rao bound. We show that the optimal masks significantly increase the depth-of-field of single-molecule imaging techniques relatively to an usual microscope objective.
Luminescent nanoparticles are becoming fundamental tools to the field of bioimaging. The optimization of their size, brightness and stability is key for applications ranging from contrast agent assisted surgery to diagnosis and therapeutics. A plethora of formulations have been documented which can be split into inorganic, organic and hybrid categories. While each class has their own advantages and limitations, controlling the interactions occurring between nanoparticles and cellular membranes is of the utmost importance. In particular, a major challenge for various applications, especially molecular imaging of membrane receptors, is to prevent non-specific interactions. Towards this goal, popular strategies based on coating nanoparticles with PEG or zwitterionic moieties have been developed to yield stealth nanoparticles. In this study, we present a series of spontaneously water-soluble and stealth organic nanoparticles. These fluorescent nanoparticles, made from original articulated bis-dipolar dyes, show vanishing interactions with living cells as bare nanoparticles. Moreover, thanks to their brightness and stability, they can be tracked as isolated single emitters in aqueous environments. These stealth nanoparticles thus hold promise for molecular imaging of specific membrane receptors, such as neuronal receptors, after bioconjugation with dedicated targeting agents.
The brain extracellular space (ECS) is a complex network that constitutes a key microenvironment for cellular communication, homeostasis, and clearance of toxic metabolites1. Signaling molecules, neuromodulators, and nutrients transit via the ECS, therefore mediating the communication between cells. Despite the relevance of this important part of the brain, its dynamics and structural organization at the nanoscale is still mostly unknown2. We have recently demonstrated that single-walled carbon nanotubes (SWCNTs) can be used to image and probe live brain tissue, providing super-resolved maps of the brain ECS and quantitative information on the local diffusion environment3,4. Here, we propose an important refinement of this approach by implementing a structured illumination technique (named HiLo microscopy5) to image fluorescently labelled neuronal structures in parallel to SWCNT NIR imaging. This technique is based on speckle illumination and relies on the acquisition of one structured and one uniform illumination image to obtain images deep into tissues with good optical sectioning. Having access to spatially resolved SWCNT diffusivity around specific neuronal structures will provide more precise insights about the heterogeneity of the brain environment.
In order to provide a green alternative to QDs for bioimaging purposes and aiming at designing bright nanoparticles combining both large one- and two-photon brightness, a bottom-up route based on the molecular engineering of dedicated red to NIR emitting dyes that spontaneously form fluorescent organic nanoparticles (FONs) has been implemented. These fully organic nanoparticles built from original quadrupolar dyes are prepared using a simple, expeditious and green protocol that yield very small molecular-based nanoparticles (radius ~ 7 nm) suspension in water showing a nice NIR emission (λem=710 nm). These FONs typically have absorption coefficient more than two orders larger than popular NIR-emitting dyes (such as Alexa Fluor 700, Cy5.5 ….) and much larger Stokes shift values (i.e. up to over 5500 cm-1). They also show very large two-photon absorption response in the 800-1050 nm region (up to about 106 GM) of major promise for two-photon excited fluorescence microscopy. Thanks to their brightness and enhanced photostability, these FONs could be imaged as isolated nanoparticles and tracked using wide-field imaging. As such, thanks to their size and composition (absence of heavy metals), they represent highly promising alternatives to NIR-emitting QDs for use in bioimaging and single particle tracking applications. Moreover, efficient FONs coating was achieved by using a polymeric additive built from a long hydrophobic (PPO) and a short hydrophilic (PEO) segment and having a cationic head group able to interact with the highly negative surface of FONs. This electrostatically-driven interaction promotes both photoluminescence and two-photon absorption enhancement leading to an increase of two-photon brightness of about one order of magnitude. This opens the way to wide-field single particle tracking under two-photon excitation
Using single-molecule microscopy, we present a method to quantify the number of single autofluorescent proteins when they cannot be optically resolved. This method relies on the measurement of the total intensity emitted by each aggregate until it photobleaches. This strategy overcomes the inherent problem of blinking of green fluorescent proteins. In the case of small protein aggregates, our method permits us to describe the mean composition with a precision of one protein. For aggregates containing a large number of proteins, it gives access to the average number of proteins gathered and a signature of the inhomogeneity of the aggregates' population. We applied this methodology to the quantification of small purified citrine multimers.
We applied single-molecule fluorescence microscopy (using organic dyes or semiconductor quantum dots) to study the lateral diffusion of glutamate receptors (AMPA and NMDA) in live synapses. We directly imaged glutamate receptors movements inside and outside synapses of live cultured hippocampal neurons. We could record exchanges of receptors through lateral diffusion between these different membrane compartments. In addition, our data suggest that this lateral diffusion might be regulated by neuronal activity. To overcome the photobleaching problem inherent to fluorescence techniques we recently developed new optical methods for the detection of individual metallic nanoparticles. We can now detect signatures of diffusing AMPA receptors labeled with 10 nm gold nanoparticles on live neurons.
We developed a photothermal method based on scattering around a nano-absorber that allows for the unprecedented detection of individual nano-objects such as gold nanoparticles with diameter down to 1.4 nm as well as CdSe nanocrystals. This method relies on the absorptive properties of the nano-object and does not suffer from the drawbacks of luminescence-based methods. We present here two different applications of this versatile detection method. First, we performed absorption spectroscopy of individual gold nanoparticles as small as 5nm and CdSe nanocrystals in the multiexcitonic regime. Second, we show the applicability of our method for new types of gold nanoparticles based DNA microarrays. In addition to the intrinsic signal stability due to the use of gold labelling, our technique does not require silver staining enhancement and permits to push the signal dynamics of such microarrays from the single nanoparticle detection to almost the full surface coverage.
Photothermal Heterodyne Imaging (PHI) is a highly sensitive optical detection method of individual absorptive nano-objects. It can be applied to absorption spectroscopy measurements Surface Plasmon Resonance spectra of individual gold nanoparticules with diameters down to 5nm were recorded. Intrinsic size effects which result in a broadening of the Resonance are unambiguously observed and analyzed within the frame of Mie theory. Preliminary results obtained with silver nanoparticles are also presented.
We have developed a photothermal method for far-field optical detection of nanometer-sized metal particles, combining high-frequency modulation and polarization interference contrast. We can image gold colloids down to 5 nm in diameter, with a signal-to-noise ratio higher than 10. This is a considerable improvement over commonly used optical methods based on resonance plasmon scattering which, for background reasons, are limited to particles of more than about 40 nm in diameter. We also show that in addition to its intrinsic sensitivity, our photothermal method is totally insensitive to non-absorbing scatterers as 10 nm nanoparticles can be imaged in cells.
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