Leica Classroom | FRET Imaging of the LAS X Functional Iceberg
With the development of microscopic imaging technology, researchers have higher and higher requirements for imaging resolution. For this reason, Leica has introduced ultra-high resolution STED and high resolution Hyvolution based on the latest generation of SP8 confocal microscope. On the other hand, simple image acquisition and resolution enhancements are no longer sufficient for many researchers, and they require more advanced and powerful imaging functions and image processing functions.
As a platform for Leica microscopy imaging, LAS X software not only provides perfect image quality, but also integrates many advanced applications with powerful post-processing data analysis functions, including image 2D, 3D measurement and analysis functions, FRAP function, FRET Features, Hyvolution high-resolution capabilities, live data mode, etc., can provide flexible imaging solutions according to different needs of users.
The next small series will introduce FRET as an example to its application in the field of biomedicine.
Figure 1. LAS X software's powerful image post-processing and advanced imaging modules.
Introduction to FRET
FRET (Förster resonance energy transfer) occurs when the distance between the donor fluorescent molecule and the acceptor fluorescent molecule is sufficiently close and the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor (within 10 nm). When a donor molecule absorbs a photon of a certain frequency and is excited to a higher electron energy state, energy is transferred to the adjacent acceptor molecule by resonance before the electron returns to the ground state. After FRET occurs, the fluorescence intensity of the donor is much lower than when it is present alone, while the fluorescence emitted by the acceptor is greatly enhanced, and the fluorescence lifetime of the donor is also shortened by the occurrence of FRET (FLIM-FRET application).
Therefore, FRET can detect the interaction between molecules and the folding and conformational changes of molecules from the two dimensions of fluorescence intensity and fluorescence lifetime.
Figure 2 FRET generation principle.
Application of FRET in the field of biomedicine
1) The article "AFRET biosensor reveals spatiotemporal activation and functions of aurora kinaseA in living cells" published in 2016 on the nature communication has constructed a novel biosensor through FRET technology, which can be realized by the sensor. The study of air conditioning control and function of Aurora kinase A protein in cells.
Firstly, the authors demonstrated that the biosensor GFP-AURKA-mCherry can correctly reflect the activation of Aurora kinase A by FLIM-FRET and immunoblotting in vitro and in vivo, and its FRET condition is its conformational change. Thr288 is caused by phosphorylation. Through positioning and functional recovery experiments, the author further proves that the biosensor can correctly perform the function of Aurora kinaseA.
Figure 3 demonstrates the GFP-AURKA-mCherry biosensor by FLIM-FRET technique in vitro .
Next, through the biosensor, the authors successfully observed the regulation of spatiotemporal activation of Aurora kinaseA in living cells, and proved that it was activated during the G1 phase, and coordinated the regulation of microtubule stability by TPX2 and CEP192 proteins.
Figure 4 TPX2 and CEP192 activate GFP-AURKA-mCherry during the G1 period .
Aurora kinase A is a recognized drug target for the treatment of cancer. Due to its low expression level, it is very difficult to track it in living cells. The biosensor is based on FRET technology, and the Aurora kinase A can be successfully studied under the condition of low expression by FLIM-FRET technology. Therefore, the biosensor has a signal pathway research and cancer drug screening for Aurora kinase A. Significance.
2) Another article published on the oncotarget " HER2-HER3 dimer quantification by FLIM-FRET predicts breast cancer metastatic relapse independently of HER2 IHC status" using FLIM-FRET technology to detect the level of HER2-HER3 dimer in breast cancer patients . Elevated expression of HER2 is an important indicator in the diagnosis of breast cancer, but it cannot be used to detect breast cancer caused by only elevated HER2-HER3 dimers. Therefore, new detection methods are urgently needed. This article is based on FLIM-FRET technology to successfully detect the level of HER2-HER3 dimer, thus becoming a new diagnostic method for detecting breast cancer caused by elevated HER2-HER3, which is important for the occurrence and recovery of breast cancer. significance.
Figure 5 detects HER2-HER3 dimer levels by the FLIM-FRET technique .
summary
The LAS X software provides a dedicated FRET technology operation guide. The samples can be detected by FRET-AB (acceptor photo-bleaching) and FRET-SE (sensitizedemission) respectively. Users only need to follow the operation guide step by step. The FRET data of the sample can be easily obtained.
Figure 6 FRET operation wizard in LAS X software .
For other advanced imaging modes, such as FRAP, Hyvolution, etc., users can also quickly collect data according to the corresponding operation wizard. Combined with LAS X's powerful image post-processing and analysis capabilities, reliable data is easily available.
references:
1. Giulia Bertolin et.al., A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells, Nature Communications, 2016 Sep.
2. Gregory Weitsman et.al., HER2-HER3dimer quantification by FLIM-FRET predicts breast cancer metastatic relapseindependently of HER2 IHC status, oncotarget, 2016 July.
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