A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

2017

Methods

Intravital microscopy (IVM) is an imaging tool that is capable of detecting subcellular signaling or metabolic events as they occur in tissues in the living animal. Imaging in highly scattering biological tissues, however, is challenging because of the attenuation of signal in images acquired at increasing depths. Depth-dependent signal attenuation is the major impediment to IVM, limiting the depth from which significant data can be obtained. Therefore, making quantitative measurements by IVM requires methods that use internal calibration, or alternatively, a completely different way of evaluating the signals. Here, we describe how ratiometric imaging of genetically encoded biosensor probes can be used to make quantitative measurements of changes in the activity of cell signaling pathways. Then, we describe how fluorescence lifetime imaging can be used for label-free measurements of the metabolic states of cells within the living animal.

“…Together, these measurements determine the SBT corrections that are required to quantify the FRET efficiency (E FRET ) of the standards (Fig. 2A) as described earlier [12,13]. …”

Section: Resultsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Theorymentioning

confidence: 99%

See 2 more Smart Citations

Intravital microscopy of biosensor activities and intrinsic metabolic states

Winfree

Hato

2017

Methods

“…The FRET standards were produced using either monomeric (m)Turquoise2 (17) or mNeonGreen (18) as the donor for mScarlet (19). These donor fluorophores were coupled directly to mScarlet through a 10 amino acid (aa) linker as described earlier (20). The FRET standards were used to verify the PIE gating settings for the biosensor measurements.…”

Section: Fret Standards and Biosensorsmentioning

confidence: 99%

PIE-FLIM measurements of two different FRET-based biosensor activities in the same living cells

Reissaus

et al. 2020

Preprint

We report the use of pulsed interleaved excitation-fluorescence lifetime imaging microscopy (PIE-FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells.Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using fluorescence lifetime imaging microscopy (FLIM). The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.

Coreless Fiber‐Based Whispering‐Gallery‐Mode Assisted Lasing from Colloidal Quantum Well Solids

Sak

Taghipour

Delikanlı

et al. 2019

Adv Funct Materials

Whispering gallery mode (WGM) resonators are shown to hold great promise to achieve high-performance lasing using colloidal semiconductor nanocrystals (NCs) in solution phase. However, the low packing density of such colloidal gain media in the solution phase results in increased lasing thresholds and poor lasing stability in these WGM lasers. To address these issues, here optical gain in colloidal quantum wells (CQWs) is proposed and shown in the form of high-density close-packed solid films constructed around a coreless fiber incorporating the resulting whispering gallery modes to induce gain and waveguiding modes of the fiber to funnel and collect light. In this work, a practical method is presented to produce the first CQW-WGM laser using an optical fiber as the WGM cavity platform operating at low thresholds of ≈188 µJ cm −2 and ≈1.39 mJ cm −2 under one-and two-photon absorption pumped, respectively, accompanied with a record low waveguide loss coefficient of ≈7 cm −1 and a high net modal gain coefficient of ≈485 cm −1 . The spectral characteristics of the proposed CQW-WGM resonator are supported with a numerical model of full electromagnetic solution. This unique CQW-WGM cavity architecture offers new opportunities to achieve simple high-performance optical resonators for colloidal lasers.