scholarly journals A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy

2015 ◽  
Vol 309 (11) ◽  
pp. C724-C735 ◽  
Author(s):  
Wen Tao ◽  
Michael Rubart ◽  
Jennifer Ryan ◽  
Xiao Xiao ◽  
Chunping Qiao ◽  
...  
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Practical Method ◽  
Two Photon Microscopy ◽  
Cellular Processes ◽  
Two Photon ◽  
Photon Excitation ◽  
Wide Range

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.

Comparison of self-assembled and micelle encapsulated QD chemosensor constructs for biological sensing

2015 ◽  
Vol 185 ◽  
pp. 249-266 ◽  
Author(s):  
Christopher M. Lemon ◽  
Daniel G. Nocera
Keyword(s):  
Self Assembly ◽  
Metabolic Profiling ◽  
Photophysical Properties ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Core Shell ◽  
Biological Sensing ◽  
Two Photon ◽  
Photon Excitation ◽  
Fret Efficiency

Whereas a variety of covalent conjugation strategies have been utilized to prepare quantum dot (QD)-based nanosensors, supramolecular approaches of self-assembly have been underexplored. A major advantage of self-assembly is the ability to circumvent laborious synthetic efforts attendant to covalent conjugation of a chemosensor to functionalized QDs. Here, we combine a CdSe/ZnS core–shell QD with gold(iii) corroles using both self-assembly and micelle encapsulation to form QD nanosensors. Appreciable spectral overlap between QD emission and corrole absorption results in efficient Förster resonance energy transfer (FRET), which may be initiated by one- or two-photon excitation. The triplet state of the gold(iii) corroles is quenched by molecular oxygen, enabling these constructs to function as optical O2 sensors, which is useful for the metabolic profiling of tumours. The photophysical properties, including QD and corrole lifetimes, FRET efficiency, and O2 sensitivity, have been determined for each construct. The relative merits of each conjugation strategy are assessed with regard to their implementation as sensors.

scholarly journals Design of metalloporphyrin-based dendritic nanoprobes for two-photon microscopy of oxygen

2008 ◽  
Vol 12 (12) ◽  
pp. 1261-1269 ◽  
Author(s):  
Artem Y. Lebedev ◽  
Thomas Troxler ◽  
Sergei A. Vinogradov
Keyword(s):  
Electron Transfer ◽  
Energy Transfer ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Triplet States ◽  
Redox Potentials ◽  
Oxygen Quenching ◽  
Two Photon Microscopy ◽  
Two Photon

Metalloporphyrin-based phosphorescent nanoprobes are being developed for two-photon microscopy of oxygen. In these molecular constructs, the generation of porphyrin triplet states following two-photon excitation is induced by the intramolecular Förster-type resonance energy transfer from a covalently attached 2P antenna. In the earlier developed prototypes, electron transfer between the antenna and the metalloporphyrin strongly interferred with the phosphorescence, reducing the sensitivity and the dynamic range of the sensors. By tuning the distances between the antenna and the core, and adjusting their redox potentials, the unwanted electron transfer could be prevented. An array of phosphorescent Pt porphyrins (energy transfer acceptors) and 2P dyes (energy transfer donors) was screened using dynamic quenching of phosphorescence, and the FRET-pair with the minimal ET rate was identified. This pair, consisting of Coumarin-343 and Pt meso-tetra-(4-alkoxyphenyl)porphyrin, was used to construct a probe in which the antenna fragments were linked to the termini of G3 poly(arylglycine) (AG) dendrimer with PtP core. The folded dendrimer formed an insulating layer between the porphyrin and the antenna, simultaneously controlling the rate of oxygen quenching (Stern-Volmer oxygen quenching constant). Modification of the dendrimer periphery with oligoethyleneglycol residues made the probe's signal insensitive to the presence of proteins and other macromolecular solutes.

Sign in / Sign up

Export Citation Format

Share Document