scholarly journals Back to the Future: Genetically Encoded Fluorescent Proteins as Inert Tracers of the Intracellular Environment

2020 ◽  
Vol 21 (11) ◽  
pp. 4164 ◽  
Author(s):  
Francesco Cardarelli
Keyword(s):  
Fluorescent Proteins ◽  
Ion Concentration ◽  
Fluorescence Labeling ◽  
Living Matter ◽  
Time Resolved ◽  
Intracellular Environment ◽  
Use Of Time ◽  
Cellular Environment ◽  
Molecular Components ◽  
Microscopy Techniques

Over the past decades, the discovery and development of genetically encoded fluorescent proteins (FPs) has brought a revolution into our ability to study biologic phenomena directly within living matter. First, FPs enabled fluorescence-labeling of a variety of molecules of interest to study their localization, interactions and dynamic behavior at various scales—from cells to whole organisms/animals. Then, rationally engineered FP-based sensors facilitated the measurement of physicochemical parameters of living matter—especially at the intracellular level, such as ion concentration, temperature, viscosity, pressure, etc. In addition, FPs were exploited as inert tracers of the intracellular environment in which they are expressed. This oft-neglected role is made possible by two distinctive features of FPs: (i) the quite null, unspecific interactions of their characteristic β-barrel structure with the molecular components of the cellular environment; and (ii) their compatibility with the use of time-resolved fluorescence-based optical microscopy techniques. This review seeks to highlight the potential of such unique combinations of properties and report on the most significative and original applications (and related advancements of knowledge) produced to date. It is envisioned that the use of FPs as inert tracers of living matter structural organization holds a potential for several lines of further development in the next future, discussed in the last section of the review, which in turn can lead to new breakthroughs in bioimaging.

scholarly journals The biochemical resolving power of fluorescence lifetime imaging

2021 ◽  
Author(s):  
Andrew L. Trinh ◽  
Alessandro Esposito
Keyword(s):  
Fluorescence Lifetime ◽  
Resolving Power ◽  
Fluorescence Lifetime Imaging ◽  
Time Resolved ◽  
Instrument Response Function ◽  
Lifetime Imaging ◽  
Use Of Time ◽  
Single Living Cells ◽  
Microscopy Techniques

AbstractA deeper understanding of spatial resolution in microscopy fostered a technological revolution that is now permitting us to investigate the structure of the cell with nanometer resolution. Although fluorescence microscopy techniques enable scientists to investigate both the structure and biochemistry of the cell, the biochemical resolving power of a microscope is a physical quantity that is not well-defined or studied. To overcome this limitation, we carried out a theoretical investigation of the biochemical resolving power in fluorescence lifetime imaging microscopy, one of the most effective tools to investigate biochemistry in single living cells. With the theoretical analysis of information theory and Monte Carlo simulations, we describe how the ‘biochemical resolving power’ in time-resolved sensing depends on instrument specifications. We unravel common misunderstandings on the role of the instrument response function and provide theoretical insights that have significant practical implications in the design and use of time-resolved instrumentation.

scholarly journals FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity

2020 ◽  
Vol 21 (8) ◽  
pp. 3012 ◽  
Author(s):  
Natalia V. Barykina ◽  
Vladimir P. Sotskov ◽  
Anna M. Gruzdeva ◽  
You Kure Wu ◽  
Ruben Portugues ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Dynamic Range ◽  
Fluorescent Proteins ◽  
Confocal Imaging ◽  
Ion Concentration ◽  
Neuronal Cultures ◽  
Two Phase ◽  
Intracellular Environment

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.

Ion Dynamics in Low Frequency RF Plasmas

1983 ◽  
Vol 29 ◽  
Author(s):  
Richard A. Gottscho ◽  
Daniel L. Flamm ◽  
Randolph H. Burton ◽  
Vincent M. Donnelly
Keyword(s):  
Plasma Etching ◽  
Low Frequency ◽  
Ion Concentration ◽  
Ion Dynamics ◽  
Applied Potential ◽  
Time Resolved ◽  
Rf Plasmas ◽  
Use Of Time ◽  
Concentration Time ◽  
State Ionic

ABSTRACTWe describe the use of time-resolved laser-induced fluorescence (TRLIF) and plasma-induced emission (PIE) spectroscopy in studying the dynamics of ion transport, formation, and loss in low frequency RF plasmas, used in plasma etching and deposition. N2+ and Cl2+ ions formed in N2, Cl2, and N2/Cl2 discharges were monitored as a function of both position between the electrodes and magnitude of the applied rf potential. In the discharge center, TRLIF was used to measure ground state ionic lifetimes. In N2/Cl2 mixtures, N2+ was found to charge exchange rapidly with Cl2 and Cl to form Cl2+ and Cl+. In the electrode sheaths, the ion response to the applied potential was evident from periodic depletion of the ion concentration as a result of acceleration by the field. From the spatial variation in the ion concentration time dependence, we deduce that the sheaths expand and contract with the same period as the applied potential.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

scholarly journals Review of time-resolved non-contact electrostatic force microscopy techniques with applications to ionic transport measurements

2019 ◽  
Vol 10 ◽  
pp. 617-633 ◽  
Author(s):  
Aaron Mascaro ◽  
Yoichi Miyahara ◽  
Tyler Enright ◽  
Omur E Dagdeviren ◽  
Peter Grütter
Keyword(s):  
Atomic Force Microscopy ◽  
Electrostatic Force ◽  
Oscillation Period ◽  
Electrostatic Force Microscopy ◽  
Time Varying ◽  
Time Resolved ◽  
Battery Materials ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Techniques

Recently, there have been a number of variations of electrostatic force microscopy (EFM) that allow for the measurement of time-varying forces arising from phenomena such as ion transport in battery materials or charge separation in photovoltaic systems. These forces reveal information about dynamic processes happening over nanometer length scales due to the nanometer-sized probe tips used in atomic force microscopy. Here, we review in detail several time-resolved EFM techniques based on non-contact atomic force microscopy, elaborating on their specific limitations and challenges. We also introduce a new experimental technique that can resolve time-varying signals well below the oscillation period of the cantilever and compare and contrast it with those previously established.

Determination of Carbon Content in Steel Using Laser-Induced Breakdown Spectroscopy

1992 ◽  
Vol 46 (9) ◽  
pp. 1382-1387 ◽  
Author(s):  
J. A. Aguilera ◽  
C. Aragón ◽  
J. Campos
Keyword(s):  
Carbon Content ◽  
Matrix Effects ◽  
Sample Surface ◽  
Nitrogen Atmosphere ◽  
Laser Induced Breakdown Spectroscopy ◽  
Time Resolved ◽  
Breakdown Spectroscopy ◽  
Use Of Time ◽  
Laser Induced Breakdown

Laser-induced breakdown spectroscopy has been used to determine carbon content in steel. The plasma was formed by focusing a Nd:YAG laser on the sample surface. With the use of time-resolved spectroscopy and generation of the plasma in nitrogen atmosphere, a precision of 1.6% and a detection limit of 65 ppm have been obtained. These values are similar to those of other accurate conventional techniques. Matrix effects for the studied steels are reduced to a small slope difference between the calibration curves for stainless and nonstainless steels.

Steady-state and time-resolved two-photon fluorescence microscopy: a versatile tool for probing cellular environment and function

2007 ◽  
Vol 76 (3) ◽  
pp. C115-C121 ◽  
Author(s):  
Stefan Denicke ◽  
Jan-Eric Ehlers ◽  
Raluca Niesner ◽  
Stefan Quentmeier ◽  
Karl-Heinz Gericke
Keyword(s):  
Steady State ◽  
Fluorescence Microscopy ◽  
Time Resolved ◽  
Two Photon ◽  
Cellular Environment ◽  
Versatile Tool ◽  
And Function ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence

Luminescence Intermittency and Quantum Efficiency of Individual Porous Si Nanoparticles.

1999 ◽  
Vol 560 ◽  
Author(s):  
Michael D. Mason ◽  
Grace M. Credo ◽  
Paul J. Carson ◽  
Steven K. Buratto
Keyword(s):  
Quantum Efficiency ◽  
Colloidal Suspension ◽  
Porous Si ◽  
Time Resolved ◽  
High Quantum Efficiency ◽  
Si Nanoparticles ◽  
Shear Force Microscopy ◽  
Spectrally Resolved ◽  
Single Particle Spectroscopy ◽  
Microscopy Techniques

ABSTRACTWe have recently observed spectrally resolved vibronic structure and luminescence intermittency from nanometer-size porous silicon nanocrystals. In this study we examine the quantum efficiency of a single nanoparticle and show that emitting nanoparticles do so with near unity quantum efficiency. This result suggests that the emission from porous Si nanoparticles, and thus bulk porous Si, results from a small number of high quantum efficiency emitters. In our previous work we have shown that our nanoparticles contain more than one coupled chromophore. In order to examine these effects more closely we employ several spectroscopy and microscopy techniques including: 1) single-particle spectroscopy, 2) shear-force microscopy, and 3) time-resolved spectroscopy, on a colloidal suspension of size-selected, surface-oxidized nanoparticles. In addition we apply statistical techniques to provide a more complete picture of the coupling between chromophores in a given nanoparticle.

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