Fluorescence Lifetime of Fluorescent Proteins

Jung, Gregor; Brockhinke, Andreas; Gensch, Thomas; Hötzer, Benjamin; Schwedler, Stefanie; Veettil, Seena Koyadan

doi:10.1007/4243_2011_14

Cited by 15 publications

(25 citation statements)

References 101 publications

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“…2A). Signals could be observed in the cytoplasm and the endosomal membranes as previously shown [33]. The emission of the cellular fluorescence (Fig.…”

Section: Resultssupporting

confidence: 74%

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Visualization of Cu²⁺ uptake and release in plant cells by fluorescence lifetime imaging microscopy

et al. 2011

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A principal objective in life sciences is the visualization of biochemical processes. Fluorescence-based techniques are widely used to demonstrate transport of relevant substances across cellular membranes. In this paper we report a novel noninvasive, real-time fluorescence lifetime imaging microscopy method for visualizing uptake and release of divalent copper ions (Cu 2+ ) in vivo. For this purpose, we employed a green fluorescent protein (GFP) form able to change its fluorescence lifetime upon Cu 2+ binding. We demonstrate that this technique is selective for Cu 2+ . We show the reversible decrease of the fluorescence lifetime of GFP from 2.2 to 1.6 ns in Escherichia coli and from 1.8 to 1.3 ns in root cells of Arabidopsis after the addition of Cu 2+ . Cu 2+ uptake of epidermal tobacco cells leads to a drop of the GFP lifetime from 2.5 to 2.2 ns. In summary, the spatially resolved visualization of Cu 2+ distribution in vivo is demonstrated in prokaryote and eukaryote cells.

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“…2A). Signals could be observed in the cytoplasm and the endosomal membranes as previously shown [33]. The emission of the cellular fluorescence (Fig.…”

Section: Resultssupporting

confidence: 74%

“…This is necessary also no direct quenching of the lifetime upon binding of other metal ions was observed, in order to exclude a blocking of the binding sites. Applying lifetime‐based measurements to GFP mutants with longer lifetimes could extend the sensor’s dynamic range [33].…”

Section: Discussionmentioning

confidence: 99%

Visualization of Cu²⁺ uptake and release in plant cells by fluorescence lifetime imaging microscopy

et al. 2011

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“…Recording the fluorescence lifetime has the advantage of being independent on the fluorophore concentration and the excitation intensity, for a large variety of fluorescent proteins. 10 Sequences of FLIM images allow for visualizing of the local changes of copper availability in cellular systems and plant cells.…”

Section: Do Not Distributementioning

confidence: 99%

“…This parameter can be measured and visualized under fluorescent microscopes using the FLIM technique. 10 Figure 2. Proposed principle for visualization of intracellular Cu 2+ homeostasis.…”

Section: +mentioning

confidence: 99%

Investigation of copper homeostasis in plant cells by fluorescence lifetime imaging microscopy

Hötzer

Ivanov

Bauer

et al. 2012

Plant Signaling & Behavior

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“…However, pixel‐wise fitting models usually require prior knowledge of the photophysics of the probe in the conditions of interest, which is not always available or easy to obtain. For example, a probe in itself may have a multi‐exponential decay, as is the case for CFP . In some cases, the nature of the emitting molecule is also not known, as in when probing endogenous fluorescence .…”

Section: Introductionmentioning

confidence: 99%

Noise‐Corrected Principal Component Analysis of fluorescence lifetime imaging data

Marois

Labouesse

Suhling

et al. 2016

Journal of Biophotonics

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Fluorescence Lifetime Imaging (FLIM) is an attractive microscopy method in the life sciences, yielding information on the sample otherwise unavailable through intensity-based techniques. A novel Noise-Corrected Principal Component Analysis (NC-PCA) method for time-domain FLIM data is presented here. The presence and distribution of distinct microenvironments are identified at lower photon counts than previously reported, without requiring prior knowledge of their number or of the dye's decay kinetics. A noise correction based on the Poisson statistics inherent to Time-Correlated Single Photon Counting is incorporated. The approach is validated using simulated data, and further applied to experimental FLIM data of HeLa cells stained with membrane dye di-4-ANEPPDHQ. Two distinct lipid phases were resolved in the cell membranes, and the modification of the order parameters of the plasma membrane during cholesterol depletion was also detected. Noise-corrected Principal Component Analysis of FLIM data resolves distinct microenvironments in cell membranes of live HeLa cells.

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Fluorescence Lifetime of Fluorescent Proteins

Cited by 15 publications

References 101 publications

Visualization of Cu²⁺ uptake and release in plant cells by fluorescence lifetime imaging microscopy

Visualization of Cu²⁺ uptake and release in plant cells by fluorescence lifetime imaging microscopy

Investigation of copper homeostasis in plant cells by fluorescence lifetime imaging microscopy

Noise‐Corrected Principal Component Analysis of fluorescence lifetime imaging data

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Fluorescence Lifetime of Fluorescent Proteins

Cited by 15 publications

References 101 publications

Visualization of Cu2+ uptake and release in plant cells by fluorescence lifetime imaging microscopy

Visualization of Cu2+ uptake and release in plant cells by fluorescence lifetime imaging microscopy

Investigation of copper homeostasis in plant cells by fluorescence lifetime imaging microscopy

Noise‐Corrected Principal Component Analysis of fluorescence lifetime imaging data

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Visualization of Cu²⁺ uptake and release in plant cells by fluorescence lifetime imaging microscopy

Visualization of Cu²⁺ uptake and release in plant cells by fluorescence lifetime imaging microscopy