High-throughput, multi-parametric, and correlative fluorescence lifetime imaging

Poudel, Chetan; Mela, Ioanna; Kaminski, Clemens F.

doi:10.1088/2050-6120/ab7364

Cited by 39 publications

(34 citation statements)

References 147 publications

(223 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In line with previous reports [47], we observed age-dependent coalescence of a-synuclein-YFP into distinct cytoplasmic inclusions ( Figure 1A). Previous fluorescence recovery after photobleaching (FRAP) and FLIM studies have suggested that asynuclein in inclusions exists in a mobile, non-amyloid state [44][45][46][47]. Here, we performed high resolution FLIM to be able to differentiate the protein localised at inclusions from the diffuse (non-aggregated) pool (Figure 1A).…”

Section: A-synuclein Forms Non-amyloid Inclusions In C Elegansmentioning

confidence: 92%

“…To understand the initial events in the a-synuclein aggregation process, we assessed its selfassociation using confocal fluorescence lifetime imaging (FLIM) in the nematode worm Caenorhabditis elegans. The fluorescence lifetime of a fluorophore has been previously shown to be an accurate measure of protein self-assembly and amyloid formation, independent of protein concentration or fluorescence intensity [44][45][46]. Given the optical transparency and well-established genetics of C. elegans, we sought to compare the fluorescence lifetime of a human a-synuclein-YFP fusion protein expressed in body wall muscle cells (OW40 worm model, see Methods).…”

Section: A-synuclein Forms Non-amyloid Inclusions In C Elegansmentioning

confidence: 99%

See 1 more Smart Citation

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

Hardenberg

Sinnige

Casford

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Misfolded a-synuclein is a major component of Lewy bodies, which are a hallmark of Parkinson's disease. A large body of evidence shows that a-synuclein can self-assemble into amyloid fibrils, but the relationship between amyloid formation and Lewy body formation still remains unclear. Here we show, both in vitro and in a C. elegans model of Parkinson's disease, that a-synuclein undergoes liquid-liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies is linked to the arrested maturation of a-synuclein condensates in the presence of lipids and other cellular components.Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting over 2% of the world's population over 65 years of age [1,2]. The molecular origins of this disease are not fully understood, although they have been closely associated with dysregulation of the behaviour of a-synuclein [1, 3, 4], a disordered protein whose function appears to involve the regulation of synaptic vesicle trafficking [5][6][7][8]. This gap in our knowledge of the disease aetiology is no doubt partly responsible for the lack of disease modifying therapies for PD [9][10][11][12].The pathological hallmark of PD is the presence of Lewy bodies within dopaminergic neurons in the brains of affected patients [13]. Although misfolded a-synuclein is a major constituent of Lewy bodies [14,15], ultrastructural and proteome studies have indicated that these aberrant deposits contain a plethora of cellular components, including lipid membranes and organelle fragments [16][17][18][19][20]. These findings have led to the suggestion that the processes associated with the formation of Lewy bodies could be major drivers of neurotoxicity in PD [16,18,21].Since it has also been shown that a-synuclein can aggregate into ordered amyloid fibrils in vitro [22][23][24][25][26] and in vivo [27][28][29][30][31][32], we asked how such a-synuclein aggregation can be reconciled with the formation of highly complex and heterogeneous assemblies such as Lewy bodies. We hypothesised that a-synuclein may be capable of irreversibly capturing cellular components through liquid-liquid phase separation, as this mechanism has been shown to drive the self-assembly of various disease-associated proteins on-pathway to the formation of solid aggregates [33][34][35][36]. Under healthy conditions, the condensation of proteins into a dense liquid droplet state through liquid-liquid phase separation is normally reversible, and exploited in a variety of ways to carry out cellular functions, including RNA metabolism, ribosome biogenesis, DNA damage response and signal transduction [33,34,37]. Upon dysregulation, however, liquid droplets can mature into gel-like deposits, which irreversibly sequester important cellular components and lead to pathological processes [38...

show abstract

Section: A-synuclein Forms Non-amyloid Inclusions In C Elegansmentioning

confidence: 92%

Section: A-synuclein Forms Non-amyloid Inclusions In C Elegansmentioning

confidence: 99%

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

Hardenberg

Sinnige

Casford

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…In the context of TCSPC, this only provides one channel for photon timing with limited throughput [41]. One solution to this issue is parallelization, which theoretically can be applied to any detector/timing electronic setup, given there is enough funding, space, and power available.…”

Section: Tcspc In Ht-flimmentioning

confidence: 99%

“…Traditionally, photomultiplier tubes (PMTs) are used, operating in single-photon counting mode with separate electronics for signal processing and analysis. In the context of TCSPC, this only provides one channel for photon timing with limited throughput [ 41 ]. One solution to this issue is parallelization, which theoretically can be applied to any detector/timing electronic setup, given there is enough funding, space, and power available.…”

Section: High-throughput Flim (Ht-flim)mentioning

confidence: 99%

A Review of New High-Throughput Methods Designed for Fluorescence Lifetime Sensing From Cells and Tissues

Bitton¹,

Sambrano²,

Valentino³

et al. 2021

Front. Phys.

View full text Add to dashboard Cite

Though much of the interest in fluorescence in the past has been on measuring spectral qualities such as wavelength and intensity, there are two other highly useful intrinsic properties of fluorescence: lifetime (or decay) and anisotropy (or polarization). Each has its own set of unique advantages, limitations, and challenges in detection when it comes to use in biological studies. This review will focus on the property of fluorescence lifetime, providing a brief background on instrumentation and theory, and examine the recent advancements and applications of measuring lifetime in the fields of high-throughput fluorescence lifetime imaging microscopy (HT-FLIM) and time-resolved flow cytometry (TRFC). In addition, the crossover of these two methods and their outlooks will be discussed.

show abstract

“…1A). FLI methodologies can be integrated into traditional fluorescence imaging approaches, including widefield, confocal, multiphoton, super-resolution and light-sheet microscopy, as well as macroimaging, fluorescence molecular tomography (FMT) and other optical imaging-based approaches (Ishikawa-Ankerhold et al, 2012;Meyer-Almes, 2017;Sarder et al, 2015;Denicke et al, 2007;Becker et al, 2017;Le Marois and Suhling, 2017;Datta et al, 2020;Poudel et al, 2020;Wang et al, 1992). FLI can also be combined with more specialized approaches such as Förster resonance energy transfer (FRET), which enables sensing of nanoscale interactions.…”

Section: Introductionmentioning

confidence: 99%

Luminescence lifetime imaging of three-dimensional biological objects

Dmitriev

Intes

Barroso

2021

Journal of Cell Science

View full text Add to dashboard Cite

A major focus of current biological studies is to fill the knowledge gaps between cell, tissue and organism scales. To this end, a wide array of contemporary optical analytical tools enable multiparameter quantitative imaging of live and fixed cells, three-dimensional (3D) systems, tissues, organs and organisms in the context of their complex spatiotemporal biological and molecular features. In particular, the modalities of luminescence lifetime imaging, comprising fluorescence lifetime imaging (FLI) and phosphorescence lifetime imaging microscopy (PLIM), in synergy with Förster resonance energy transfer (FRET) assays, provide a wealth of information. On the application side, the luminescence lifetime of endogenous molecules inside cells and tissues, overexpressed fluorescent protein fusion biosensor constructs or probes delivered externally provide molecular insights at multiple scales into protein–protein interaction networks, cellular metabolism, dynamics of molecular oxygen and hypoxia, physiologically important ions, and other physical and physiological parameters. Luminescence lifetime imaging offers a unique window into the physiological and structural environment of cells and tissues, enabling a new level of functional and molecular analysis in addition to providing 3D spatially resolved and longitudinal measurements that can range from microscopic to macroscopic scale. We provide an overview of luminescence lifetime imaging and summarize key biological applications from cells and tissues to organisms.

show abstract

High-throughput, multi-parametric, and correlative fluorescence lifetime imaging

Cited by 39 publications

References 147 publications

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

A Review of New High-Throughput Methods Designed for Fluorescence Lifetime Sensing From Cells and Tissues

Luminescence lifetime imaging of three-dimensional biological objects

Contact Info

Product

Resources

About