\”Nature\” child TV: nanogels optical switch, in the microscale selectively store and display information

Micro-bar code technology is a versatile technology that provides high-throughput and multiplex information is stored as micro- and nano-scale application of biology, medicine and materials science. However, the current barcode technology mainly using fluorescence spectroscopy and fluorescence intensity multiplexing (FI) coding, they are generally susceptible to spectral coding elements overlap. Further, since the concentration of the sample and the external microenvironment variability, it is difficult to obtain quantitative readings. Using a fluorescence lifetime (fluorescence lifetime), may be reproduced to provide an output in the micro-bar code region. With the latest imaging microscopy, fluorescence lifetime may be used as a simple technique to minimize the limitations of conventional fluorescent bar code, over time and provides repeatable quantitative readings. However, the response of the material to find the flexibility to adjust the fluorescence lifetime remains a continuing challenge.

findings

Based on the above issue, Professor Rachel K. O\’Reilly research group from the UK College of Chemistry, University of Birmingham, designed a light generated by the two components can be switched (photo-switchable) nano gel, the gel exhibited variable fluorescence lifetime by energy transfer induced photoisomerisation caused by light irradiation process. Fluorescence lifetime imaging microscopy may be used (FLIM) visually rendering the fluorescence lifetime characteristics of such manipulation remote, which can selectively store and display information on a microscale. And, in the organelles of imaging, fluorescence lifetime imaging may be live cells and subcellular organelles minimize the influence of background. Related outcomes to \”Manipulating the fluorescence lifetime at the sub-cellular via scale photo-switchable barcoding\” in the title, published in \”Nature communciation\”. 《Nature》子刊: 光开关纳米凝胶, 在微尺度上选择性地存储和显示信息

Analytical graphic

1. Design and fabrication of the optical switch Nanogel

Preparation of 《Nature》子刊: 光开关纳米凝胶, 在微尺度上选择性地存储和显示信息
FIG. 1 optical switch polymer Nanogel [ 123]
as a non-destructive sexual stimulation, an important tool for the light to external contact without external manipulation, making it ideal for information flag, the realTracking mark and selectivity. In this study, the researchers designed based on the life of optical bar code system can be switched, the two-component system based gel comprises a nano-substituted maleimide (DTM) and the spiropyran (SP) of the switch . Light by inducing reversible isomerization process may be by Förster resonance energy transfer between the fluorophore and DTM ring-opened form of the photochromic agent SP (FRET) to achieve a similar structure but having multiple lifetime nanogel having to to achieve a controlled, non-coding controllable dynamic fluorescence lifetime.

2. Evaluation of energy transfer in the process of photoisomerization and photophysical behavior

《Nature》子刊: 光开关纳米凝胶, 在微尺度上选择性地存储和显示信息 Figure 2 relates to energy transfer photophysical behavior Nanogel
In the DTM crosslinked polymer environmental protected, emits green light in the 450-600 nm range and having a high fluorescence quantum yield. In contrast, between 500 and 600 nm, in the form of a closed loop SP absorption is negligible, and the open form of the photochromic dye absorption in this wavelength range, over the scheme (FIG. 2a) for the efficient FRET . When varying density functional theory (TD-DFT) calculations show that only when the SP ring-opened isomer as the FRET occurs (Figure 2b). When excited at about 25nm, fluorescence emission DTM holding a green emission (450-600 nm) in a gel nano environment. In contrast, when the irradiation DTM and SP contains nano gel (N2-4), DTM channels gradually reduced green emission, and the emergence of a new peak in the vicinity of 610 nm, indicated that FRET donor / DTM and open loop the formation of a receptor (Fig. 2c) between the SP. As expected, the DTM can be fully restored by irradiation with visible light emission, in the meantime, by the SP switch back to a closed loop to prevent FRET process.

3. The optical stimulus reversible fluorescence lifetime

《Nature》子刊: 光开关纳米凝胶, 在微尺度上选择性地存储和显示信息 FIG. 3 optical switch nano-gel fluorescence lifetime quantification and visualization of
time-correlated single photon counting (TCSPC ) fluorescence lifetime imaging microscopy is used as a decoding technique for visualizing local fluorescence lifetime. First, to compare the decay time by the two-dimensional life at different emission wavelengths monitored resolved Fluorescence Spectroscopy. The observed decrease in fluorescence lifetime of the solution N4 DTM channels, since the light stimulus FRET, ring opening new SP fluorescence decay occurs between 600 and 650 nm. Figure 3a, shown in B, after UV irradiation, may mean life N1-4 ns adjusted from 15 to 28 ns, and the ratio of the linear correlation of the two monomers. Further, by adjusting the ultraviolet irradiation time before reaching the steady stage, the plurality of life can be obtained (FIG. 3C,) in the same gel nanometers. N4 attenuation throughout the life of the UV and visible light in four cycles are completely reversible, there is no measurable change (FIG. 3d). By fluorescence lifetime imaging microscopy (FLIM) further visualized this reversible behavior, which can be observed different lifetimes.

4. Reversible living cells fluorescence lifetime bar code

《Nature》子刊: 光开关纳米凝胶, 在微尺度上选择性地存储和显示信息 FIG. 4 of the optical switch function Nanogel, for lifetime imaging
in living cells [123 ] Finally, researchers have attempted to study the intracellular encoding and decoding processes in the cell structure. For targeting to the mitochondria, after passing through the azide functional group nanogels, alkyne cycloaddition via the azide with TPP nanogels coupling (FIG. 4a). Analysis by confocal fluorescence was observed between the TPP-N6 DTM channel and the red channel colocalization commercial Mito Tracker, TPP-N6 Pearson correlation coefficient (PCC) was 0.85, and no modification TPP (0.57) nano gel, indicating successful location (FIG. 4b) in the mitochondria of these materials. Moreover, tracking agents incorporated group does not affect the reversibility nanogel systems. Compared with the conventional material having the barcode autofluorescence lifetime effect, the nano-gel reversible fluorescence lifetime system provides a strategy to noise ratio can be enlarged by reducing the signal strength.
summed

Herein, researchers have developed a remotely controlling polymer gel nano FRET fluorescence lifetime by one kind of process-induced photoisomerisation. These nanogels system is further adapted to use bar code scanning FLIM fluorescence lifetime, so as to achieve a controllable, reversible and non-invasive method of object tracking and polymorphic fluorescence visualized selectively. As a reading system capable of selectively nanogel quantitative results decoded by the simultaneous extraction of life, thereby allowingXu microscale store information. As a proof of concept, by clicking on the mitochondrial tracer introduced into the reaction nanogels, thereby obtaining a living cell lifespan barcode subcellular size, improved imaging sensitivity. Article link:

https://doi.org/10.1038/s41467-020-16297-3

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