Indeed, it has been shown that the diffusion properties of Qdots, dextrans, or other nanoparticles introduced in the brain extracellular space are related to its nanoscale architecture [ 8 , 9 ]. The analysis of their trajectories should thus allow critical biophysical information about this important brain compartment [ 45 ] such as tortuosity, viscosity and dynamics of endogenous extracellular molecules to be retrieved.
Because Qdots are surface passivated without targeting-functionalization, they diffuse around the cells exploring their environment at high speed with limited specific interaction with the sample. The trajectories analyzed in Figures 4b—d,f—h are indicated in Figures 4a,e with the red dashed boxes. This value is consistent with earlier 2D studies [ 8 ]. Interestingly, the 3D shapes of these trajectories indicate diffusion around what appears as spherical volumetric structures within the tissue, which would have been indiscernible in 2D.
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Figure 4. Single fluorescent QDot tracking in live organotypic brain slices.
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It extends these methods to 3D quantification in complex live samples. Since SELFI obtains the 3D information without causing significant photon loss, it is possible to apply this concept to single emitters having limited brilliance such as fluorescent proteins as used in PALM, or nanoparticles deep inside thick living tissues. SELFI also allows 3D single particle tracking at high imaging frames here, 50 frames per second to follow fast diffusion dynamics within a tissue. With SELFI, 3D localization is obtained solely by structuring the PSF with interference patterns in the image plane, which provide the freedom to use any fluorescence excitation scheme.
Note that this independence from illumination schemes is key for tissue imaging applications because intrinsic light scattering by the sample might disturb specific illumination configurations that are needed to obtain the 3D information with other methods. In this work, we have also chosen to image quasi-metrological living biological samples by studying axial molecular structuration within cellular adhesion sites. The nanoscale layering of proteins measured with SELFI is in agreement with the data obtained in fixed cells in the literature.
Finally, we have demonstrated fast 3D SPT in thick samples. This paves the way to the unambiguous study of molecular nano-organization and dynamics in complex samples where 2D imaging-only can lead to biased results. To conclude, we have demonstrated that SELFI is an efficient method for live super-resolution studies allowing 3D localization even at high depth up to few tens of microns on a regular epi-fluorescence based microscope. As long as a single molecule image can be formed in the imaging plane of the microscope, SELFI can retrieve its 3D localization well-beyond the diffraction limits, by sensing simultaneously the intensity and the phase of the light.
All authors discussed the data and commented on the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Imaging intracellular fluorescent proteins at nanometer resolution. Nat Methods. Sharonov A, Hochstrasser R. Wide-field subdiffraction imaging by accumulated binding of diffusing probes.
Breaking the diffraction barrier: super-resolution imaging of cells. High-density three-dimensional localization microscopy across large volumes. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chemie Int Ed. Identification and super-resolution imaging of ligand-activated receptor dimers in live cells. Sci Rep.
CLF Multidimensional single molecule microscopy
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Nat Commun. Nat Cell Biol. Photometry unlocks 3D information from 2D localization microscopy data. Direct optical nanoscopy with axially localized detection. Nat Photon. Axial super-localisation using rotating point spread functions shaped by polarisation-dependent phase modulation. Opt Express. Multicolour localization microscopy by point-spread-function engineering. Rosen J, Brooker G. Non-scanning motionless fluorescence three-dimensional holographic microscopy.
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Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.
Three dimensional tracking of fluorescent microparticles using a photon-limited double-helix response system. Isotropic three-dimensional super-resolution imaging with a self-bending point spread function. Self-interference 3D super-resolution microscopy for deep tissue investigations. Extended hartmann test based on the pseudoguiding property of a hartmann mask completed by a phase chessboard. Appl Opt. Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells.
A bisected pupil for studying single-molecule orientational dynamics and its application to three-dimensional super-resolution microscopy. Appl Phys Lett.
Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods. Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane.
Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis. Single-molecule imaging of EGFR signalling on the surface of living cells. Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high-and low-affinity epidermal growth factor receptors in A cells. Spatial control of EGF receptor activation by reversible dimerization on living cells.
Methods Mol Biol. Nanoscale architecture of integrin-based cell adhesions. Multi-color single particle tracking with quantum dots. Robust single-particle tracking in live-cell time-lapse sequences. Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells.
PLoS Pathog. Imaging of molecular surface dynamics in brain slices using single-particle tracking. Light sheet microscopy for single molecule tracking in living tissue. Deep and high-resolution three-dimensional tracking of single particles using nonlinear and multiplexed illumination. A single-molecule experiment is an experiment that investigates the properties of individual molecules. Single-molecule studies may be contrasted with measurements on an ensemble or bulk collection of molecules, where the individual behavior of molecules cannot be distinguished, and only average characteristics can be measured.
Since many measurement techniques in biology, chemistry and physics are not sensitive enough to observe single molecules, single-molecule fluorescence techniques that have emerged since the s for probing various processes on the level of individual molecules caused a lot of excitement, since these supplied many new details on the measured processes that were not accessible in the past.
Indeed, since the s, many techniques for probing individual molecules have been developed.
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