Suppression of photobleaching‐induced artifacts in frequency‐domain FLIM by permutation of the recording order

Münster, E.; Gadella, Theodorus

doi:10.1002/cyto.a.20013

Cited by 72 publications

(51 citation statements)

References 25 publications

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“…It should be noted that this comparison is at Poisson limited signal/noise which is superior to GOI performance. Our data also show that frame rates in excess of 2 Hz are readily achievable with the system, without the limitations imposed by GOI based systems [41][42][43][44].…”

Section: Comparing Lifetime Analysis Techniques For Image Acquisitionsupporting

confidence: 51%

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

Poland¹,

Krstajić²,

Monypenny³

et al. 2015

Biomed. Opt. Express

111

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Abstract:We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction. 73-76 (1990). 9. F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2(12), 932-940 (2005). 10. J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23(9), 655-657 (1998) ©2015 Optical Society of America

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Section: Comparing Lifetime Analysis Techniques For Image Acquisitionsupporting

confidence: 51%

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

Poland¹,

Krstajić²,

Monypenny³

et al. 2015

Biomed. Opt. Express

111

View full text Add to dashboard Cite

show abstract

“…The phase and modulation were determined from a series of images taken at 12 phase settings using software provided by the manufacturer and converted to phase lifetimes with correction for photobleaching as described (van Munster and Gadella, 2004). Fluorescein dianion (lifetime 4.1 ns) was used as a reference (Magde et al, 1999).…”

Section: Antibodies and Plasmidsmentioning

confidence: 99%

Recruitment of adenomatous polyposis coli and β-catenin to axin-puncta

et al. 2008

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The adenomatous polyposis coli (APC) tumour suppressor is a multifunctional protein involved in the regulation of Wnt signalling and cytoskeletal dynamics. Little is known about how APC controls these disparate functions. In this study, we have used APC-and axin-fluorescent fusion proteins to examine the interactions between these proteins and show that the functionally distinct populations of APC are also spatially separate. Axin-RFP forms cytoplasmic punctate structures, similar to endogenous axin puncta. Axin-RFP recruits b-catenin destruction complex proteins, including APC, b-catenin, glycogen synthase kinase-3-b (GSK3-b) and casein kinase-1-a (CK1-a). Recruitment into axin-RFP puncta sequesters APC from clusters at cell extensions and this prevents its microtubule-associated functions. The interaction between APC-GFP and axin-RFP within the cytoplasmic puncta is direct and dramatically alters the dynamic properties of APC-GFP. However, recruitment of APC to axin puncta is not absolutely required for b-catenin degradation. Instead, formation of axin puncta, mediated by the DIX domain, is required for b-catenin degradation. An axinDDIX mutant did not form puncta, but still mediated recruitment of destruction complex proteins and phosphorylation of b-catenin. We conclude that there are distinct pools of APC and that the formation of axin puncta, rather than the axin/APC complex, is essential for b-catenin destruction.

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“…Samples were excited by means of a 514 nm Argon laser modulated at 75.1 MHz and a BP 530-560 nm emission filter was used to detect YFP fluorescence. FLIM stacks of 12 phase images permutated in recording order (van Munster and Gadella, 2004b) were acquired with an exposure time of 0.4-1.5 seconds each.…”

Section: Fluorescence Lifetime Imaging Microscopymentioning

confidence: 99%

Plant G protein heterotrimers require dual lipidation motifs of Gα and Gγ and do not dissociate upon activation

Adjobo‐Hermans

Goedhart

Gadella

2006

Journal of Cell Science

111

117

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In plants one bona fide Gα subunit has been identified, as well as a single Gβ and two Gγ subunits. To study the roles of lipidation motifs in the regulation of subcellular location and heterotrimer formation in living plant cells, GFP-tagged versions of the Arabidopsis thaliana heterotrimeric G protein subunits were constructed. Mutational analysis showed that the Arabidopsis Gα subunit, GPα1, contains two lipidation motifs that were essential for plasma membrane localization. The Arabidopsis Gβ subunit, AGβ1, and the Gγ subunit, AGG1, were dependent upon each other for tethering to the plasma membrane. The second Gγ subunit, AGG2, did not require AGβ1 for localization to the plasma membrane. Like AGG1, AGG2 contains two putative lipidation motifs, both of which were necessary for membrane localization. Interaction between the subunits was studied using fluorescence resonance energy transfer (FRET) imaging by means of fluorescence lifetime imaging microscopy (FLIM). The results suggest that AGβ1 and AGG1 or AGβ1 and AGG2 can form heterodimers independent of lipidation. In addition, FLIM-FRET revealed the existence of GPα1-AGβ1-AGG1 heterotrimers at the plasma membrane. Importantly, rendering GPα1 constitutively active did not cause a FRET decrease in the heterotrimer, suggesting no dissociation upon GPα1 activation.

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Suppression of photobleaching‐induced artifacts in frequency‐domain FLIM by permutation of the recording order

Cited by 72 publications

References 25 publications

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

Recruitment of adenomatous polyposis coli and β-catenin to axin-puncta

Plant G protein heterotrimers require dual lipidation motifs of Gα and Gγ and do not dissociate upon activation

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