RESUMEN
The axial resolution of three-dimensional structured illumination microscopy (3D SIM) is limited to â¼300 nm. Here we present two distinct, complementary methods to improve axial resolution in 3D SIM with minimal or no modification to the optical system. We show that placing a mirror directly opposite the sample enables four-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with â¼120-nm lateral and 160-nm axial resolution. We also developed a deep learning method achieving â¼120-nm isotropic resolution. This method can be combined with denoising to facilitate volumetric imaging spanning dozens of timepoints. We demonstrate the potential of these advances by imaging a variety of cellular samples, delineating the nanoscale distribution of vimentin and microtubule filaments, observing the relative positions of caveolar coat proteins and lysosomal markers and visualizing cytoskeletal dynamics within T cells in the early stages of immune synapse formation.
Asunto(s)
Imagenología Tridimensional , Iluminación , Microscopía Fluorescente/métodos , Imagenología Tridimensional/métodos , Citoesqueleto , LisosomasRESUMEN
Adaptive optics reflectance-based retinal imaging has proved a valuable tool for the noninvasive visualization of cells in the living human retina. Many subcellular features that remain at or below the resolution limit of current in vivo techniques may be more easily visualized with the same modalities in an ex vivo setting. While most microscopy techniques provide significantly higher resolution, enabling the visualization of fine cellular detail in ex vivo retinal samples, they do not replicate the reflectance-based imaging modalities of in vivo retinal imaging. Here, we introduce a strategy for imaging ex vivo samples using the same imaging modalities as those used for in vivo retinal imaging, but with increased resolution. We also demonstrate the ability of this approach to perform protein-specific fluorescence imaging and reflectance imaging simultaneously, enabling the visualization of nearly transparent layers of the retina and the classification of cone photoreceptor types.
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Adaptive optics scanning light ophthalmoscopy (AOSLO) allows non-invasive visualization of the living human eye at the microscopic scale; but even with correction of the ocular wavefront aberrations over a large pupil, the smallest cells in the photoreceptor mosaic cannot always be resolved. Here, we synergistically combine annular pupil illumination with sub-Airy disk confocal detection to demonstrate a 33% improvement in transverse resolution (from 2.36 to 1.58 µm) and a 13% axial resolution enhancement (from 37 to 32 µm), an important step towards the study of the complete photoreceptor mosaic in heath and disease. Interestingly, annular pupil illumination also enhanced the visualization of the photoreceptor mosaic in non-confocal detection schemes such as split detection AOSLO, providing a strategy for enhanced multimodal imaging of the cone and rod photoreceptor mosaic.
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In vivo imaging of human retinal pigment epithelial (RPE) cells has been demonstrated through multiple adaptive optics (AO)-based modalities. However, whether consistent and complete information regarding the cellular structure of the RPE mosaic is obtained across these modalities remains uncertain due to limited comparisons performed in the same eye. Here, an imaging platform combining multimodal AO-scanning light ophthalmoscopy (AO-SLO) with AO-optical coherence tomography (AO-OCT) is developed to make a side-by-side comparison of the same RPE cells imaged across four modalities: AO-darkfield, AO-enhanced indocyanine green (AO-ICG), AO-infrared autofluorescence (AO-IRAF), and AO-OCT. Co-registered images were acquired in five subjects, including one patient with choroideremia. Multimodal imaging provided multiple perspectives of the RPE mosaic that were used to explore variations in RPE cell contrast in a subject-, location-, and even cell-dependent manner. Estimated cell-to-cell spacing and density were found to be consistent both across modalities and with normative data. Multimodal images from a patient with choroideremia illustrate the benefit of using multiple modalities to infer the cellular structure of the RPE mosaic in an affected eye, in which disruptions to the RPE mosaic may locally alter the signal strength, visibility of individual RPE cells, or even source of contrast in unpredictable ways.
Asunto(s)
Ojo , Verde de Indocianina , Humanos , Colorantes , Angiografía , Células Epiteliales , Pigmentos Retinianos , Angiografía con Fluoresceína , Coroides , Fondo de OjoRESUMEN
We describe a method to speed up microelectromechanical system (MEMS) mirror scanning by > 20x, while also improving scan accuracy. We use Landweber deconvolution to determine an input voltage which would produce a desired output, based on the measured MEMS impulse response. Since the MEMS is weakly nonlinear, the observed behavior deviates from expectations, and we iteratively improve our input to minimize this deviation. This allows customizable MEMS angle vs. time with <1% deviation from the desired scan pattern. We demonstrate our technique by optimizing a point scanning microscope's raster patterns to image mammal submandibular gland and pollen at ~10 frames/s.