scholarly journals Luminescence lifetime imaging of three-dimensional biological objects

2021 ◽  
Vol 134 (9) ◽  
pp. 1-17
Author(s):  
Ruslan I. Dmitriev ◽  
Xavier Intes ◽  
Margarida M. Barroso
Keyword(s):  
Fluorescent Protein ◽  
Multiple Scales ◽  
Three Dimensional ◽  
Luminescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Protein Fusion ◽  
Phosphorescence Lifetime ◽  
Lifetime Imaging ◽  
Molecular Features ◽  
Biological Studies

ABSTRACT 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.

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.

scholarly journals Structure and Location Studies on Key Enzymes in Saponins Biosynthesis of Panax notoginseng

2019 ◽  
Vol 20 (24) ◽  
pp. 6121
Author(s):  
Pengguo Xia ◽  
Yujie Zheng ◽  
Zongsuo Liang
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Three Dimensional ◽  
Panax Notoginseng ◽  
Squalene Epoxidase ◽  
Protein Fusion ◽  
Active Components ◽  
Key Enzymes ◽  
Saponin Biosynthesis

Panax notoginseng is one of the most widely used traditional herbs for the treatment of various diseases, in which saponins were the main active components. At present, the research of P. notoginseng mainly focused on the discovery of new compounds and pharmacology. However, there were few studies on the molecular mechanism of the synthesis of secondary metabolites of P. notoginseng. In our study, four coding sequences (CDS) encoding the key enzymes involved in saponin biosynthesis were cloned, namely farnesyl diphosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), and dammarenediol-II synthase (DS), which contained open reading frame (ORF) of 1029 bp, 1248 bp, 1614 bp, and 2310 bp, and coded 342, 415, 537, and 769 amino acids, respectively. At the same time, their domains, secondary structures, three-dimensional structures, and phylogenetics trees were analyzed by kinds of bioinformatics tools. Their phylogenetics relationships were also analyzed. In addition, GFP (Green fluorescent protein) fusion genes were constructed by the plasmid transformation system to determine the subcellular localization. The results of subcellular localization showed that FPS, SE, and DS were mainly located in cytomembrane and its surrounding, while SS was located both in cytoplasm and cytomembrane. Our findings provided data demonstrating the expression patterns of genes involved in saponin biosynthesis and would facilitate efforts to further elucidate the biosynthesis of the bioactive components in P. notoginseng.

scholarly journals Three-dimensional molecular mapping in a microfluidic mixing device using fluorescence lifetime imaging

2008 ◽  
Vol 33 (16) ◽  
pp. 1887 ◽  
Author(s):  
Tom Robinson ◽  
Prashant Valluri ◽  
Hugh B. Manning ◽  
Dylan M. Owen ◽  
Ian Munro ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Molecular Mapping ◽  
Three Dimensional ◽  
Fluorescence Lifetime Imaging ◽  
Microfluidic Mixing ◽  
Lifetime Imaging

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

scholarly journals Unique actions of GABA arising from cytoplasmic chloride microdomains

2020 ◽  
Author(s):  
Negah Rahmati ◽  
Kieran P. Normoyle ◽  
Joseph Glykys ◽  
Volodymyr I. Dzhala ◽  
Kyle P. Lillis ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Reversal Potential ◽  
Receptor Activation ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Electrophysiological Measurements ◽  
Highly Correlated ◽  
Cation Chloride Cotransporters ◽  
The Impact

AbstractDevelopmental, cellular, and subcellular variations in the direction of neuronal Cl− currents elicited by GABAA receptor activation have been frequently reported, and we found a corresponding variance in the reversal potential (EGABA) for individual interneurons synapsing on a single pyramidal cell. These findings suggest a corresponding variance in the cytoplasmic concentration of Cl− ([Cl−i]). We determined [Cl−]i by: 1) two-photon imaging of the Cl− sensitive, ratiometric fluorescent protein SuperClomeleon (sCLM); 2) Fluorescence Lifetime IMaging (FLIM) of the Cl− sensitive fluorophore MEQ; and 3) electrophysiological measurements of EGABA. These methods collectively demonstrated stable [Cl−]i microdomains in individual neurons in vivo. Fluorometric and electrophysiological estimates of local [Cl−]i were highly correlated. [Cl−]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters (CCCs) but steadily decreased after inhibiting the polymerization of the anionic macromolecule actin. These studies highlight the existence of functionally significant neuronal Cl− microdomains that modify the impact of GABAergic inputs.

scholarly journals Optical refocusing three-dimensional wide-field fluorescence lifetime imaging microscopy

2012 ◽  
Vol 20 (2) ◽  
pp. 960 ◽  
Author(s):  
Qiang Wu ◽  
Shangyu Guo ◽  
Yinxing Ma ◽  
Feng Gao ◽  
Chengliang Yang ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Three Dimensional ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

Singlet Oxygen Phosphorescence Lifetime Imaging Based on a Fluorescence Lifetime Imaging Microscope

2015 ◽  
Vol 119 (14) ◽  
pp. 3393-3399 ◽  
Author(s):  
Wenming Tian ◽  
Liezheng Deng ◽  
Shengye Jin ◽  
Heping Yang ◽  
Rongrong Cui ◽  
...  
Keyword(s):  
Singlet Oxygen ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Phosphorescence Lifetime ◽  
Lifetime Imaging ◽  
Phosphorescence Lifetime Imaging
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