Optimizing Long-Term Live Cell Imaging

Lac, Alex; Lam, Austin Le; Heit, Bryan

doi:10.1007/978-1-0716-2051-9_3

Cited by 5 publications

(4 citation statements)

References 32 publications

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“…We also observed large variability in photostability, which is the resistance of the chromophore to bleaching by bright light. Bleaching is often a limitation in long-term live-imaging studies 49 , whereas it is an advantage in assays such as fluorescence recovery after photobleaching (FRAP), in which fast fluorescence decay enhances signal 50 . We isolated two designs that exhibited higher photostability than GFP (photostable.1 & photostable.2, with seven mutations each from PROSS-eGFP) and many significantly less photostable designs (Fig.…”

Section: Resultsmentioning

confidence: 99%

Designed active-site library reveals thousands of functional GFP variants

et al. 2023

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Mutations in a protein active site can lead to dramatic and useful changes in protein activity. The active site, however, is sensitive to mutations due to a high density of molecular interactions, substantially reducing the likelihood of obtaining functional multipoint mutants. We introduce an atomistic and machine-learning-based approach, called high-throughput Functional Libraries (htFuncLib), that designs a sequence space in which mutations form low-energy combinations that mitigate the risk of incompatible interactions. We apply htFuncLib to the GFP chromophore-binding pocket, and, using fluorescence readout, recover >16,000 unique designs encoding as many as eight active-site mutations. Many designs exhibit substantial and useful diversity in functional thermostability (up to 96 °C), fluorescence lifetime, and quantum yield. By eliminating incompatible active-site mutations, htFuncLib generates a large diversity of functional sequences. We envision that htFuncLib will be used in one-shot optimization of activity in enzymes, binders, and other proteins.

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Section: Resultsmentioning

confidence: 99%

Designed active-site library reveals thousands of functional GFP variants

et al. 2023

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Section: Resultsmentioning

confidence: 99%

Designed active-site library reveals thousands of functional GFP variants

Weinstein

Aldaravi

Lipsh‐Sokolik

et al. 2022

Preprint

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Mutations in a protein active site can lead to dramatic and useful changes in protein activity. The active site, however, is extremely sensitive to mutations due to a high density of molecular interactions, drastically reducing the likelihood of obtaining functional multipoint mutations. We introduce an atomistic and machine-learning-based approach called htFuncLib to design a sequence space in which mutations form low-energy combinations that mitigate the risk from incompatible interactions. We screened 11 million htFuncLib designs that targeted the GFP chromophore-binding pocket and isolated >16,000 unique fluorescent designs encoding as many as eight active-site mutations. Many designs exhibit substantial and useful diversity in stability and activity. By eliminating incompatible active-site mutations, htFuncLib generates a greater diversity of functional sequences than evolutionary or mutational scanning approaches for optimizing enzymes, binders, and other functional proteins.

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“…Thus, COSM is presented as an alternative approach to achieve brightfield optical sectioning [2,16]. This optical sectioning technique, generally, is linked to fluorescence microscopy, where photodamage and phototoxicity issues have been noted [17,18]. Adopting conventional brightfield microscopy for optical sectioning is of great significance because some of the biological specimens are not suitable for fluorescence imaging, it offers label-free imaging modalities, and it is simple, fast, and has a low cost.…”

Section: Introductionmentioning

confidence: 99%

Multiplane Image Restoration Using Multivariate Curve Resolution: An Alternative Approach to Deconvolution in Conventional Brightfield Microscopy

2023

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Three-dimensional reconstruction in brightfield microscopy is challenging since a 2D image includes from in-focus and out-of-focus light which removes the details of the specimen’s structures. To overcome this problem, many techniques exist, but these generally require an appropriate model of Point Spread Function (PSF). Here, we propose a new images restoration method based on the application of Multivariate Curve Resolution (MCR) algorithms to a stack of brightfield microscopy images to achieve 3D reconstruction without the need for PSF. The method is based on a statistical reconstruction approach using a self-modelling mixture analysis. The MCR-ALS (ALS for Alternating Least Square) algorithm under non-negativity constraints, Wiener, Richardson–Lucy, and blind deconvolution algorithms were applied to silica microbeads and red blood cells images. The MCR analysis produces restored images that show informative structures which are not noticeable in the initial images, and this demonstrates its capability for the multiplane reconstruction of the amplitude of 3D objects. In comparison with 3D deconvolution methods based on a set of No Reference Images Quality Metrics (NR-IQMs) that are Standard Deviation, ENTROPY Average Gradient, and Auto Correlation, our method presents better values of these metrics, showing that it can be used as an alternative to 3D deconvolution methods.

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Optimizing Long-Term Live Cell Imaging

Cited by 5 publications

References 32 publications

Designed active-site library reveals thousands of functional GFP variants

Designed active-site library reveals thousands of functional GFP variants

Designed active-site library reveals thousands of functional GFP variants

Multiplane Image Restoration Using Multivariate Curve Resolution: An Alternative Approach to Deconvolution in Conventional Brightfield Microscopy

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