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    Home > ACS center. SCI.: fluorescent label with spontaneous scintillation mark designed for super-resolution imaging

    ACS center. SCI.: fluorescent label with spontaneous scintillation mark designed for super-resolution imaging

    • Last Update: 2019-10-17
    • Source: Internet
    • Author: User
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    Small molecule fluorophores are basic tools for biological research Rhodamine has been widely used in biological imaging, such as biomarkers, cell staining, environmental detection, etc., due to its high fluorescence quantum yield, good light stability, structural modifiability and reversible transformation between non fluorescent lactone (L) and fluorescent zwitterions (z; equilibrium constant: kl-z) Among them, by adjusting kl-z to reduce the formation of lipophilic non fluorescent lactones, the permeability of cells can be improved, and "fluorescent" ligands can be produced, which show significant fluorescent signals after binding with biological targets Recently, Dr Luke D Levis of the Jennifer Institute of Howard Hughes Medical Research Institute in the United States has designed and synthesized a new type of fluorescence group janelia fluor (jf526) based on the rhodamine fluorescence group, which forms a self labeling ligand for the staining of internal structures and the spontaneous scintillation labeling of super-resolution epidemic free fluorescence Relevant achievements were published on ACS central science under the title of "rational design of fluorescent and spontaneity blinding labels for super resolution imaging" (DOI: 10.1021 / acscientsci 9b00676) (source: ACS Central Science) tetramethyl Si rhodamine (Sir, 1; figure 1a) is a common fluorescent dye, which shows far-infrared wavelength, maximum absorption wavelength of 643 nm, emission wavelength of 662 nm, fluorescence quantum yield of 0.41 (Table 1), and excellent optical stability The ligand based on Sir has a low kl-z value (0.0034), indicating that it is in the form of non fluorescent lactone in aqueous solution and has high permeability However, such ligands, when combined with molecular targets, will shift the equilibrium to zwitterions and generate fluorescence (Figure 1b) In addition, the author found that the substitution of N-dimethylamino in the fluorophore with the Quaternary azabutane ring can improve the properties of the substance Therefore, the author introduced 3-fluoroazacyclobutane into the structure to obtain jf635 (3, λ ABS / λ EM = 635nm / 652nm, ε w = 1000m-1cm-1, Φ = 0.54), which shows low kl-z value (< 0.0001) and strong fluorescence The fluorescent dye 4 - 7 (Figure 1C, table 1) was also obtained by a similar method Then, the fluorescence groups jf552 (9) and jf526 (10) were obtained with rhodamine as the basic framework In addition, 3,3-difluoroanzobutane was used to replace the azacyclobutane ring in jf608 (4, λ ABS / λ EM = 608nm / 631nm, Φ = 0.67) The compound jf585 (5, λ ABS / λ EM = 585nm / 609nm, Φ = 0.78) with bright fluorescence was obtained (source: ACS Central Science) later, the author studied the properties of Si rhodamine 110 (8) Compound 8 (SIR 110, λ ABS / λ EM = 587nm / 609nm, Φ = 0.53; figure 2a) was obtained from compound 11 and tert Butyl carbamate by PD catalyzed cross coupling reaction and then deprotected by TFA Compared with Sir, the blue shift of Sir 110 is ~ 50 nm, but the quantum yield of fluorescence increases In this paper, we synthesized the Sir 110 halo tag ligand (8 HTL, figure 2b) from 6 and tested its spectral properties It was found that its fluorescence absorption increased by 7 times (Figure 2C) after binding with halo tag protein, and it had a linear relationship with the trend of kl-z (Figure 1E) The dye can be used to label cell imaging (Figure 2D) and has better photostability (Figure 2e) The above results further prove that the low kl-z value is closely related to the strong fluorescence of rhodamine (source: ACS Central Science) later, the author devoted to develop a general strategy for the synthesis of jf552 derivatives, hoping to apply to the substitution of different azacyclobutane functional groups A series of condensation, coupling and deprotection reactions were carried out with 3-bromo-4-fluorophenol (17) as raw materials to obtain the conjugates 6 TMP and 9 TMP (scheme 1c) of jf552, jf552 HaloTag ligands 9 HTL (scheme 1b), jf549 and jf552 Then, we evaluated the fluorescent labeling effect of these substances in yeast cell nucleus Among them, 6 HTL showed poor labeling effect (Figure 3a), while 9 HTL showed strong fluorescence signal (Figure 3B) under the same conditions (source: ACS Central Science) then, the author combined the structural modification methods of compounds 7 and 9 to obtain a new fluorescence group rhodamine hexafluoride 10 (Figure 4a) The author synthesized 6-carboxyl derivative (scheme 1b) by the way similar to jf552 ligand Similarly, the author obtained jf526 HaloTag ligand (10 HTL) and jf526 snap tag ligand (10 STL, figure 4b) through a series of cross coupling and deprotection reactions, and compared the imaging effect with jf525 HaloTag (7 HTL) in living cells The results show that whether HaloTag (Figure 4D, e) or snap tag ligand (Figure 4f, g) is used, jf526 has higher fluorescence intensity than jf525 (source: ACS Central Science) the author further synthesized a variety of jf526 ligands to explore the application of dyes in polychromatic microscopy, Jf526 Hoechst (10 HST) was used for DNA staining, jf526 Taxol (10 TXL) for microtubule staining and jf526 pepsin inhibitor A (10 PEP) for lysosome staining (Figure 4B and 5a) Then the author realized the monochromatic, bichromatic and trichromatic wash free imaging experiments (Figure 5b-d) with these materials At the same time, we use 10pep and jf646 Hoechst (2hst, figure 6a) to realize the two-color 3d-sim imaging in living cells, and 10txl to realize the multi-color super-resolution step imaging (Figure 6b) of microtubules In particular, jf526 Taxol (10 TXL, microtubule), jf646 snap tag ligand (2 STL, endoplasmic reticulum) and jf585 HaloTag ligand (5 HTL, mitochondria) were used for trichromatic sted imaging of living cells (Figure 6c) The double color graticule photomicrography (Figure 6D) was realized with 10 PEP and 2 HST (source: ACS Central Science) finally, the author further used the lactone zwitterion equilibrium of rhodamine dye for single molecule location microimaging (smlm) Hydroxymethyl sir (HM sir, 32) mainly exists in the form of non fluorescent spiro ether, but under the condition of physiological pH, it can protonation to produce transient fluorescence (Figure 7a) In order to evaluate the performance of hm-jf526 in smlm experiment, 37 labeled Goat anti mouse second antibody was used, and then anti β - tubulin first antibody was immunostained in fixed cells Smlm images show the fine structure of microtubules (Figure 7E) Compared with the diffraction limited image, rhodamine dye provides a higher resolution mitochondrial smlm image (Figure 7f-h) (source: ACS Central Science) in a word, the author used various modification methods to change the kl-z value of Rhodamine, and proved the importance of kl-z in the reasonable design of spontaneous flashing rhodamine fluorescence group At the same time, the author explored a universal method to reconstruct the structure of fluorescent clusters, which is conducive to the design and synthesis of more small molecules for functional imaging in living cells and organisms.
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