Combined Selective Plane Illumination Microscopy and FRAP Maps Intranuclear Diffusion of NLS-GFP

Hobson, Chad M.; Falvo, Michael R.; Superfine, Richard

doi:10.1016/j.bpj.2020.07.001

Cited by 11 publications

(7 citation statements)

References 34 publications

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“…Lastly, we expect that light sheet microscopy will expand as a tool ideally suited for in vivo measurements, due to the reduced levels of photobleaching and toxicity compared to those of other fluorescence microscopy techniques. This has already been combined with several F-techniques, − encouraging further advances in the field.…”

Section: Optical Methods Of Analysismentioning

confidence: 99%

Illuminating the Path to Target GPCR Structures and Functions

et al. 2020

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G-Protein-coupled receptors (GPCRs) are ubiquitous within eukaryotes, responsible for a wide array of physiological and pathological processes. Indeed, the fact that they are the most drugged target in the human genome is indicative of their importance. Despite the clear interest in GPCRs, most information regarding their activity has been so far obtained by analyzing the response from a “bulk medium”. As such, this Perspective summarizes some of the common methods for this indirect observation. Nonetheless, by inspecting approaches applying super-resolution imaging, we argue that imaging is perfectly situated to obtain more detailed structural and spatial information, assisting in the development of new GPCR-targeted drugs and clinical strategies. The benefits of direct optical visualization of GPCRs are analyzed in the context of potential future directions in the field.

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Section: Optical Methods Of Analysismentioning

confidence: 99%

Illuminating the Path to Target GPCR Structures and Functions

et al. 2020

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“…Methods for probing material properties, such as viscosity, surface tension and component dynamics (for example, diffusion and mobile fraction), can be applied in vitro and in live cells 40 , 44 , 185 (Box 4 ); this information can be leveraged as a read-out to detect a change in condensate milieu, and/or to gain insights into the mechanisms driving a condensatopathy or a c-mod.…”

Section: Building a C-mod Discovery Platformmentioning

confidence: 99%

Modulating biomolecular condensates: a novel approach to drug discovery

Mitrea¹,

Mittasch²,

Gomes³

et al. 2022

Nat Rev Drug Discov

191

106

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In the past decade, membraneless assemblies known as biomolecular condensates have been reported to play key roles in many cellular functions by compartmentalizing specific proteins and nucleic acids in subcellular environments with distinct properties. Furthermore, growing evidence supports the view that biomolecular condensates often form by phase separation, in which a single-phase system demixes into a two-phase system consisting of a condensed phase and a dilute phase of particular biomolecules. Emerging understanding of condensate function in normal and aberrant cellular states, and of the mechanisms of condensate formation, is providing new insights into human disease and revealing novel therapeutic opportunities. In this Perspective, we propose that such insights could enable a previously unexplored drug discovery approach based on identifying condensate-modifying therapeutics (c-mods), and we discuss the strategies, techniques and challenges involved.

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“…To enable mobility analysis within heterogeneous media, several strategies have been considered for recovery of full spatially-resolved diffusion maps, in which the diffusion coefficient is independently recovered in each pixel or domain in an image. Superfine and co-workers leveraged selective plane illumination to photobleach an entire two-dimensional slice through a sample and generate a fluorescence recovery image of NLS-GFP and 53BP1-mCherry in live cells . The authors used a combination of a cylindrical lens and an annulus to produce a line Bessel sheet for photobleaching the YZ plane.…”

Section: Introductionmentioning

confidence: 99%

“…Superfine and co-workers leveraged selective plane illumination to photobleach an entire two-dimensional slice through a sample and generate a fluorescence recovery image of NLS-GFP and 53BP1-mCherry in live cells. 28 The authors used a combination of a cylindrical lens and an annulus to produce a line Bessel sheet for photobleaching the YZ plane. Horizontal imaging of the fluorescence recovery was achieved using a right-angle reflective prism.…”

Section: ■ Introductionmentioning

confidence: 99%

Periodic Photobleaching with Structured Illumination for Diffusion Imaging

et al. 2023

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The use of periodically structured illumination coupled with spatial Fourier-transform fluorescence recovery after photobleaching (FT-FRAP) was shown to support diffusivity mapping within segmented domains of arbitrary shape. Periodic "comb-bleach" patterning of the excitation beam during photobleaching encoded spatial maps of diffusion onto harmonic peaks in the spatial Fourier transform. Diffusion manifests as a simple exponential decay of a given harmonic, improving the signal to noise ratio and simplifying mathematical analysis. Image segmentation prior to Fourier transformation was shown to support pooling for signal to noise enhancement for regions of arbitrary shape expected to exhibit similar diffusivity within a domain. Following proof-ofconcept analyses based on simulations with known ground-truth maps, diffusion imaging by FT-FRAP was used to map spatiallyresolved diffusion differences within phase-separated domains of model amorphous solid dispersion spin-cast thin films. Notably, multi-harmonic analysis by FT-FRAP was able to definitively discriminate and quantify the roles of internal diffusion and exchange to higher mobility interfacial layers in modeling the recovery kinetics within thin amorphous/amorphous phase-separated domains, with interfacial diffusion playing a critical role in recovery. These results have direct implications for the design of amorphous systems for stable storage and efficacious delivery of therapeutic molecules.

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Combined Selective Plane Illumination Microscopy and FRAP Maps Intranuclear Diffusion of NLS-GFP

Cited by 11 publications

References 34 publications

Illuminating the Path to Target GPCR Structures and Functions

Illuminating the Path to Target GPCR Structures and Functions

Modulating biomolecular condensates: a novel approach to drug discovery

Periodic Photobleaching with Structured Illumination for Diffusion Imaging

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