Simultaneous single molecule atomic force and fluorescence lifetime imaging

2010 ◽  
Author(s):  
Olaf Schulz ◽  
Felix Koberling ◽  
Deron Walters ◽  
Marcelle Koenig ◽  
Jacob Viani ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Atomic Force

Fluorescence lifetime imaging system with nm-resolution and single-molecule sensitivity

2002 ◽  
Author(s):  
Michael Wahl ◽  
Hans-Juergen Rahn ◽  
Uwe Ortmann ◽  
Rainer Erdmann ◽  
Martin Boehmer ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Imaging System ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

scholarly journals Targetable Conformationally Restricted Cyanines Enable Photon-Count Limited Applications

2021 ◽  
Author(s):  
Patrick Eiring ◽  
Ryan McLaughlin ◽  
Siddharth Matikonda ◽  
HAN ZHONGYING ◽  
Lennart Grabenhorst ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ring System ◽  
Fluorescence Lifetime Imaging ◽  
Conformationally Restricted ◽  
Lifetime Imaging ◽  
Tertiary Amide ◽  
Imaging Performance

Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to parent species in a range of contexts including their use on a DNA-nano-antenna, in single-molecule Förster resonance energy transfer (FRET) applications, far-red fluorescence lifetime imaging microscopy (FLIM), and single-molecule localization microscopy. These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.

scholarly journals In situ real-time monitoring the mechanism of self-assembly of short peptide supramolecular polymers

2021 ◽  
Author(s):  
Mari C. Mañas-Torres ◽  
Cristina Gila-Vilchez ◽  
Juan Antonio Gonzalez Vera ◽  
Francisco Conejero-Lara ◽  
Victor Blanco ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Self Assembly ◽  
Correlation Spectroscopy ◽  
Supramolecular Polymers ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Single Molecule Fluorescence ◽  
Lifetime Imaging

Making use of the combination of multiparametric Fluorescence Lifetime Imaging Microscopy (FLIM) and single-molecule Fluorescence Lifetime Correlation Spectroscopy (FLCS), we have been able to study early stages of Fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF)...

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 Synchronous, Crosstalk-free Correlative AFM and Confocal Microscopies/Spectroscopies

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Thales F. D. Fernandes ◽  
Oscar Saavedra-Villanueva ◽  
Emmanuel Margeat ◽  
Pierre-Emmanuel Milhiet ◽  
Luca Costa
Keyword(s):  
Fluorescence Lifetime ◽  
Interaction Force ◽  
Metallic Surface ◽  
Fluorescence Lifetime Imaging ◽  
Time Resolved ◽  
Force Microscopy ◽  
Lifetime Imaging ◽  
Atomic Force ◽  
Dimensional Mapping ◽  
Time Acquisition

Abstract Microscopies have become pillars of our characterization tools to observe biological systems and assemblies. Correlative and synchronous use of different microscopies relies on the fundamental assumption of non-interference during images acquisitions. In this work, by exploring the correlative use of Atomic Force Microscopy and confocal-Fluorescence-Lifetime Imaging Microscopy (AFM-FLIM), we quantify cross-talk effects occurring during synchronous acquisition. We characterize and minimize optomechanical forces on different AFM cantilevers interfering with normal AFM operation as well as spurious luminescence from the tip and cantilever affecting time-resolved fluorescence detection. By defining non-interfering experimental imaging parameters, we show accurate real-time acquisition and two-dimensional mapping of interaction force, fluorescence lifetime and intensity characterizing morphology (AFM) and local viscosity (FLIM) of gel and fluid phases separation of supported lipid model membranes. Finally, as proof of principle by means of synchronous force and fluorescence spectroscopies, we precisely tune the lifetime of a fluorescent nanodiamond positioned on the AFM tip by controlling its distance from a metallic surface. This opens up a novel pathway of quench sensing to image soft biological samples such as membranes since it does not require tip-sample mechanical contact in contrast with conventional AFM in liquid.

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