ArticleNature protocols2016
A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy.
Article in Nature protocols, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 29 citations in OpenAlex.
- Molecular Spies in Action: Genetically Encoded Fluorescent Biosensors Light up Cellular Signals.Chemical reviews · 2024Review
- Longitudinal intravital microscopy of the mouse kidney: inflammatory responses to abdominal imaging windows.American journal of physiology. Renal physiology · 2024Article
- Opioid-Modulated Receptor Localization and Erk1/2 Phosphorylation in Cells Coexpressing μ-Opioid and Nociceptin Receptors.International journal of molecular sciences · 2023Article
- Multiscale Photoacoustic Tomography of a Genetically Encoded Near-Infrared FRET Biosensor.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2021Article
- The Indiana O'Brien Center for Advanced Renal Microscopic Analysis.American journal of physiology. Renal physiology · 2021Review
- Direct visualization by FRET-FLIM of a putative mechanosome complex involving Src, Pyk2 and MBD2 in living MLO-Y4 cells.PloS one · 2021Article
- Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors.Scientific reports · 2020Article
- PIE-FLIM Measurements of Two Different FRET-Based Biosensor Activities in the Same Living Cells.Biophysical journal · 2020Article
- Biosensors-Based In Vivo Quantification of 2-Oxoglutarate in Cyanobacteria and Proteobacteria.Life (Basel, Switzerland) · 2018Article
- Fit-free analysis of fluorescence lifetime imaging data using the phasor approach.Nature protocols · 2018Article
- Intravital microscopy of biosensor activities and intrinsic metabolic states.Methods (San Diego, Calif.) · 2017Article
Corrections and comments
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Authors and funding
3 authors at 3 institutions in 1 country.
Funding
Abstract
Genetically encoded fluorescent protein (FP)-based biosensor probes are useful tools for monitoring cellular events in living cells and tissues. Because these probes were developed for one-photon excitation approaches, their broad two-photon excitation (2PE) and poorly understood photobleaching characteristics have made their implementation in studies using two-photon laser-scanning microscopy (TPLSM) challenging. Here we describe a protocol that simplifies the use of Förster resonance energy transfer (FRET)-based biosensors in TPLSM. First, the TPLSM system is evaluated and optimized using FRET standards expressed in living cells, which enables the determination of spectral bleed-through (SBT) and the confirmation of FRET measurements from the known standards. Next, we describe how to apply the approach experimentally using a modified version of the A kinase activity reporter (AKAR) protein kinase A (PKA) biosensor as an example-first in cells in culture and then in hepatocytes in the liver of living mice. The microscopic imaging can be accomplished in a day in laboratories that routinely use TPLSM.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.