3D super-resolution deep-tissue imaging in living mice (original) (raw)
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We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 µm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.
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We present a parallel stimulated emission depletion (STED) nanoscope with no mechanical moving parts and submillisecond pixel dwell times, relying on electro-optical (EO) phase modulators. The nanoscope offers 1225-fold parallelization over single-doughnut-scanning STED and achieves a spatial resolution of 35 nm. We imaged immunostained nuclear pore complexes of zebrafish within their natural biological environment, demonstrating spatial and temporal resolutions of 56 nm and 0.2 s, respectively. Furthermore, we show parallel EO-STED sub-second imaging of microtubules inside living cells. Finally, we reveal the nanodomain organization of a eukaryotic initiation factor within the processing bodies of fixed cells. The potential of parallel EO-STED to offer microsecond pixel dwell times over large fields of view promises millisecond STED imaging.
Journal of Innovative Optical Health Sciences, 2014
Optical microscopy has become an indispensable tool for visualizing sub-cellular structures and biological processes. However, scattering in biological tissues is a major obstacle that prevents high-resolution images from being obtained from deep regions of tissue. We review common techniques, such as multiphoton microscopy (MPM) and optical coherence microscopy (OCM), for di®raction limited imaging beyond an imaging depth of 0.5 mm. Novel implementations have been emerging in recent years giving higher imaging speed, deeper penetration, and better image quality. Focal modulation microscopy (FMM) is a novel method that combines confocal spatial ̄ltering with focal modulation to reject out-of-focus background. FMM has demonstrated an imaging depth comparable to those of MPM and OCM, near-real-time image acquisition, and the capability for multiple contrast mechanisms.
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ABSTRACTUnderstanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at unprecedented depths in vivo.
Computational adaptive optics for live three-dimensional biological imaging
Proceedings of The National Academy of Sciences - PNAS, 2001
Light microscopy of thick biological samples, such as tissues, is often limited by aberrations caused by refractive index variations within the sample itself. This problem is particularly severe for live imaging, a field of great current excitement due to the development of inherently fluorescent proteins. We describe a method of removing such aberrations computationally by mapping the refractive index of the sample using differential interference contrast microscopy, modeling the aberrations by ray tracing through this index map, and using space-variant deconvolution to remove aberrations. This approach will open possibilities to study weakly labeled molecules in difficult-to-image live specimens. www.pnas.org͞cgi͞doi͞10.1073͞pnas.071275698
Adaptive optics enables 3D STED microscopy in aberrating specimens
Optics Express, 2012
Stimulated emission depletion (STED) microscopy allows fluorescence far-field imaging with diffraction-unlimited resolution. Unfortunately, extending this technique to three-dimensional (3D) imaging of thick specimens has been inhibited by sample-induced aberrations. Here we present the first implementation of adaptive optics in STED microscopy to allow 3D super-resolution imaging in strongly aberrated imaging conditions, such as those introduced by thick biological tissue.
Self-interference 3D super-resolution microscopy for deep tissue investigations
Nature methods, 2018
Fluorescence localization microscopy has achieved near-molecular resolution capable of revealing ultra-structures, with a broad range of applications, especially in cellular biology. However, it remains challenging to attain such resolution in three dimensions and inside biological tissues beyond the first cell layer. Here we introduce SELFI, a framework for 3D single-molecule localization within multicellular specimens and tissues. The approach relies on self-interference generated within the microscope's point spread function (PSF) to simultaneously encode equiphase and intensity fluorescence signals, which together provide the 3D position of an emitter. We combined SELFI with conventional localization microscopy to visualize F-actin 3D filament networks and reveal the spatial distribution of the transcription factor OCT4 in human induced pluripotent stem cells at depths up to 50 µm inside uncleared tissue spheroids. SELFI paves the way to nanoscale investigations of native ce...
Super-resolved live-cell imaging using Random Illumination Microscopy
SummarySuper-resolution fluorescence microscopy has been instrumental to progress in biology. Yet, the photo-induced toxicity, the loss of resolution into scattering samples or the complexity of the experimental setups curtail its general use for functional cell imaging. Here, we describe a new technology for tissue imaging reaching a 114nm/8Hz resolution at 30 µm depth. Random Illumination Microscopy (RIM) consists in shining the sample with uncontrolled speckles and extracting a high-fidelity super-resolved image from the variance of the data using a reconstruction scheme accounting for the spatial correlation of the illuminations. Super-resolution unaffected by optical aberrations, undetectable phototoxicity, fast image acquisition rate and ease of use, altogether, make RIM ideally suited for functional live cell imaging in situ. RIM ability to image molecular and cellular processes in three dimensions and at high resolution is demonstrated in a wide range of biological situation...