scholarly journals Structured illumination microscopy for in-vivo human retinal imaging: a theoretical assessment

2012 ◽  
Vol 20 (23) ◽  
pp. 25700 ◽  
Author(s):  
Sabah Chetty ◽  
Steve Gruppetta
Keyword(s):  
Retinal Imaging ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Theoretical Assessment

scholarly journals Super-resolution ophthalmoscopy: Virtually structured detection for resolution improvement in retinal imaging

2020 ◽  
pp. 153537022097053
Author(s):  
Xincheng Yao ◽  
Rongwen Lu ◽  
Benquan Wang ◽  
Yiming Lu ◽  
Tae-Hoon Kim
Keyword(s):  
Eye Movements ◽  
Human Subjects ◽  
Numerical Aperture ◽  
Retinal Imaging ◽  
Super Resolution ◽  
Practical Implementation ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging

Quantitative retinal imaging is essential for advanced study and clinical management of eye diseases. However, spatial resolution of retinal imaging has been limited due to available numerical aperture and optical aberration of the ocular optics. Structured illumination microscopy has been established to break the diffraction-limit resolution in conventional light microscopy. However, practical implementation of structured illumination microscopy for in vivo ophthalmoscopy of the retina is challenging due to inevitable eye movements that can produce phase artifacts. Recently, we have demonstrated the feasibility of using virtually structured detection as one alternative to structured illumination microscopy for super-resolution imaging. By providing the flexibility of digital compensation of eye movements, the virtually structured detection provides a feasible, phase-artifact-free strategy to achieve super-resolution ophthalmoscopy. In this article, we summarize the technical rationale of virtually structured detection, and its implementations for super-resolution imaging of freshly isolated retinas, intact animals, and awake human subjects.

scholarly journals Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

2015 ◽  
Vol 112 (32) ◽  
pp. E4390-E4399 ◽  
Author(s):  
Mathew Stracy ◽  
Christian Lesterlin ◽  
Federico Garza de Leon ◽  
Stephan Uphoff ◽  
Pawel Zawadzki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Spatial Organization ◽  
Search Time ◽  
Population Level ◽  
Bacterial Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Superresolution Microscopy

Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.

scholarly journals Tension-dependent stretching and folding of ZO-1 controls the localization of its interactors

2017 ◽  
Author(s):  
Domenica Spadaro ◽  
Shimin Le ◽  
Thierry Laroche ◽  
Isabelle Mean ◽  
Lionel Jond ◽  
...  
Keyword(s):  
Tight Junction ◽  
Molecular Mechanisms ◽  
Magnetic Tweezers ◽  
Dependent Manner ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Epithelial Homeostasis ◽  
Junction Protein ◽  
Tensile Forces

Tensile forces regulate epithelial homeostasis, but the molecular mechanisms behind this regulation are poorly understood. Using structured illumination microscopy and proximity ligation assays we show that the tight junction protein ZO-1 undergoes actomyosin tension-dependent stretching and folding in vivo. Magnetic tweezers experiments using purified ZO-1 indicate that pN-scale tensions (~2-4 pN) are sufficient to maintain the stretched conformation of ZO-1, while keeping its structured domains intact. Actomyosin tension and substrate stiffness regulate the localization and expression of the transcription factor DbpA and the tight junction membrane protein occludin in a ZO-1/ZO-2-dependent manner, resulting in modulation of gene expression, cell proliferation, barrier function and cyst morphogenesis. Interactions between the N-terminal (ZPSG) and C-terminal domains of ZO-1 prevent binding of DbpA to the ZPSG, and folding is antagonized by heterodimerization with ZO-2. We propose that tensile forces regulate epithelial homeostasis by activating ZO proteins through stretching, to modulate their protein interactions and downstream signaling.

scholarly journals Dynamic super-resolution structured illumination imaging in the living brain

2019 ◽  
Vol 116 (19) ◽  
pp. 9586-9591 ◽  
Author(s):  
Raphaël Turcotte ◽  
Yajie Liang ◽  
Masashi Tanimoto ◽  
Qinrong Zhang ◽  
Ziwei Li ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Optical Aberrations ◽  
Sample Motion ◽  
Heterogeneous Tissue ◽  
Conventional Microscopy ◽  
Super Resolution Microscopy

Cells in the brain act as components of extended networks. Therefore, to understand neurobiological processes in a physiological context, it is essential to study them in vivo. Super-resolution microscopy has spatial resolution beyond the diffraction limit, thus promising to provide structural and functional insights that are not accessible with conventional microscopy. However, to apply it to in vivo brain imaging, we must address the challenges of 3D imaging in an optically heterogeneous tissue that is constantly in motion. We optimized image acquisition and reconstruction to combat sample motion and applied adaptive optics to correcting sample-induced optical aberrations in super-resolution structured illumination microscopy (SIM) in vivo. We imaged the brains of live zebrafish larvae and mice and observed the dynamics of dendrites and dendritic spines at nanoscale resolution.

scholarly journals UNC-45a promotes myosin folding and stress fiber assembly

2017 ◽  
Vol 216 (12) ◽  
pp. 4053-4072 ◽  
Author(s):  
Jaakko I. Lehtimäki ◽  
Aidan M. Fenix ◽  
Tommi M. Kotila ◽  
Giuseppe Balistreri ◽  
Lassi Paavolainen ◽  
...  
Keyword(s):  
Large Fraction ◽  
Gradient Centrifugation ◽  
Proteasome Inhibition ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Dynamic Component ◽  
Fiber Assembly

Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.

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