Abstract
AbstractThe discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live‐cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs.
🔬 Techniques
✨ Fluorophores
🏛️ Research Organizations (ROR)
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📊 Figures
Figure 1
A: Cartoon illustrating FRET between green and red FPs fused to interacting DNA-binding proteins. Energy transfer can only occur when the FPs are positioned close to one another by the interactions of...
Figure 2
Spectral imaging of a cell producing the mTFP1-5aa-Venus fusion protein. A : The cell was illuminated at the donor excitation wavelength and spectral measurements were acquired from the ROI indicated ...
Figure 3
A simplified Perrin-Jablonski energy level diagram for a fluorescent molecule. The arrows represent absorption of excitation photon energy causing the transition from the lowest vibrational levels of ...
Figure 4
The Time Domain (TD) and Frequency-domain (FD) FLIM Methods. A: TD FLIM requires a pulsed excitation source with a femtosecond pulse width. The pulsed laser is coupled to the scanning system of the mi...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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