scholarly journals Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging

2012 ◽  
Vol 48 (42) ◽  
pp. 5127 ◽  
Author(s):  
Antonella Battisti ◽  
Michelle A. Digman ◽  
Enrico Gratton ◽  
Barbara Storti ◽  
Fabio Beltram ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Fluorescence Lifetime Imaging ◽  
Ph Measurements ◽  
Lifetime Imaging

Functionalized CdSe/ZnS Quantum Dots for Intracellular pH Measurements by Fluorescence Lifetime Imaging Microscopy

ACS Sensors ◽  
2020 ◽  
Vol 5 (7) ◽  
pp. 2106-2117 ◽  
Author(s):  
Pedro J. Pacheco-Liñán ◽  
Iván Bravo ◽  
María L. Nueda ◽  
José Albaladejo ◽  
Andrés Garzón-Ruiz
Keyword(s):  
Quantum Dots ◽  
Intracellular Ph ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Ph Measurements ◽  
Lifetime Imaging

scholarly journals Fluorescence lifetime imaging of pH along the secretory pathway

2021 ◽  
Author(s):  
Peter Linders ◽  
Martin ter Beest ◽  
Geert van den Bogaart
Keyword(s):  
Temporal Resolution ◽  
Fluorescence Lifetime ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Excitation Wavelength ◽  
Fluorescence Lifetime Imaging ◽  
Ph Homeostasis ◽  
Ph Changes ◽  
Lifetime Imaging ◽  
Wide Range

Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein (GFP), pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution unsuitable to follow the rapid transit of cargo between organelles. We therefore applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway. Then, we analyze the dynamic pH changes within cells treated with Brefeldin A, a COPI coat inhibitor. Finally, we followed the pH changes of newly-synthesized molecules of the inflammatory cytokine tumor necrosis factor (TNF)-α while it was in transit from the endoplasmic reticulum via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution, and can be used to assess organellar pH in disease models.

The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging

2013 ◽  
Vol 405 (12) ◽  
pp. 3983-3987 ◽  
Author(s):  
Sandrine Poëa-Guyon ◽  
Hélène Pasquier ◽  
Fabienne Mérola ◽  
Nicolas Morel ◽  
Marie Erard
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Protein A ◽  
Ph Sensor ◽  
Cyan Fluorescent Protein ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Enhanced Cyan Fluorescent Protein

pH-sensitive perylene bisimide probes for live cell fluorescence lifetime imaging

2014 ◽  
Vol 2 (39) ◽  
pp. 6792-6801 ◽  
Author(s):  
D. Aigner ◽  
R. I. Dmitriev ◽  
S. M. Borisov ◽  
D. B. Papkovsky ◽  
I. Klimant
Keyword(s):  
Intracellular Ph ◽  
Fluorescence Lifetime ◽  
Ph Sensitive ◽  
Live Cell ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Perylene Bisimide ◽  
Lifetime Imaging

Several new perylene bisimide (PBI) probes comprising oligo-guanidine conjugates and cationic hydrogel nanoparticle structures were designed for sensing intracellular pH in live cell fluorescence lifetime imaging microscopy (FLIM).

scholarly journals Carbon Dots for Intracellular pH Sensing with Fluorescence Lifetime Imaging Microscopy

Nanomaterials ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 604 ◽  
Author(s):  
Maojia Huang ◽  
Xinyue Liang ◽  
Zixiao Zhang ◽  
Jing Wang ◽  
Yiyan Fei ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Fluorescence Lifetime ◽  
Fluorescence Intensity ◽  
Carbon Dots ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Gradual Change ◽  
Ph Sensing ◽  
Lifetime Imaging

The monitoring of intracellular pH is of great importance for understanding intracellular trafficking and functions. It has various limitations for biosensing based on the fluorescence intensity or spectra study. In this research, pH-sensitive carbon dots (CDs) were employed for intracellular pH sensing with fluorescence lifetime imaging microscopy (FLIM) for the first time. FLIM is a highly sensitive method that is used to detect a microenvironment and it can overcome the limitations of biosensing methods based on fluorescence intensity. The different groups on the CDs surfaces changing with pH environments led to different fluorescence lifetime values. The CDs aqueous solution had a gradual change from 1.6 ns to 3.7 ns in the fluorescence lifetime with a pH range of 2.6–8.6. Similar fluorescence lifetime changes were found in pH buffer-treated living cells. The detection of lysosomes, cytoplasm, and nuclei in living cells was achieved by measuring the fluorescence lifetime of CDs. In particular, a phasor FLIM analysis was used to improve the pH imaging. Moreover, the effects of the coenzymes, amino acids, and proteins on the fluorescence lifetime of CDs were examined in order to mimic the complex microenvironment inside the cells.

scholarly journals Unravelling the mechanisms controlling heme supply and demand

2021 ◽  
Vol 118 (22) ◽  
pp. e2104008118
Author(s):  
Galvin C.-H. Leung ◽  
Simon S.-P. Fung ◽  
Andrea E. Gallio ◽  
Robert Blore ◽  
Dominic Alibhai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Supply And Demand ◽  
Fluorescence Lifetime Imaging ◽  
Prosthetic Group ◽  
Lifetime Imaging ◽  
Free Heme ◽  
Green Fluorescent ◽  
Cellular Compartments

In addition to heme’s role as the prosthetic group buried inside many different proteins that are ubiquitous in biology, there is new evidence that heme has substantive roles in cellular signaling and regulation. This means that heme must be available in locations distant from its place of synthesis (mitochondria) in response to transient cellular demands. A longstanding question has been to establish the mechanisms that control the supply and demand for cellular heme. By fusing a monomeric heme-binding peroxidase (ascorbate peroxidase, mAPX) to a monomeric form of green-fluorescent protein (mEGFP), we have developed a heme sensor (mAPXmEGFP) that can respond to heme availability. By means of fluorescence lifetime imaging, this heme sensor can be used to quantify heme concentrations; values of the mean fluorescence lifetime (τMean) for mAPX-mEGFP are shown to be responsive to changes in free (unbound) heme concentration in cells. The results demonstrate that concentrations are typically limited to one molecule or less within cellular compartments. These miniscule amounts of free heme are consistent with a system that sequesters the heme and is able to buffer changes in heme availability while retaining the capability to mobilize heme when and where it is needed. We propose that this exchangeable supply of heme can operate using mechanisms for heme transfer that are analogous to classical ligand-exchange mechanisms. This exquisite control, in which heme is made available for transfer one molecule at a time, protects the cell against the toxic effect of excess heme and offers a simple mechanism for heme-dependent regulation in single-molecule steps.

scholarly journals Fluorescence lifetime imaging of compartmental pH dynamics using red fluorescent protein sensors in live cells

2018 ◽  
Vol 32 (S1) ◽  
Author(s):  
Emily Haynes ◽  
Megha Rajendran ◽  
Angeline Lyon ◽  
Nicholas Noinaj ◽  
Richard Day ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Red Fluorescent Protein ◽  
Lifetime Imaging ◽  
Protein Sensors

scholarly journals Interaction of Poxvirus Intracellular Mature Virion Proteins with the TPR Domain of Kinesin Light Chain in Live Infected Cells Revealed by Two-Photon-Induced Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Microscopy

2010 ◽  
Vol 84 (24) ◽  
pp. 12886-12894 ◽  
Author(s):  
Ananya Jeshtadi ◽  
Pierre Burgos ◽  
Christopher D. Stubbs ◽  
Anthony W. Parker ◽  
Linda A. King ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Surface Protein ◽  
Fluorescence Lifetime Imaging ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Infected Cells ◽  
Tpr Domain

ABSTRACT Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 ± 0.1 ns to 2.1 ± 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.

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