Multiplexed stimulated emission depletion nanoscopy(mSTED)for 5-color live-cell long-term imaging of organelle interactome

Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation.Here,we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity.By separating live-cell fluorescent probes with similar spectral properties using phasor analysis,our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures.The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.

Rational design of far red to near-infrared rhodamine analogues with huge Stokes shifts for single-laser excitation multicolor imaging

Rhodamine dyes have been widely employed in biological imaging and sensing. However, it is always a challenge to design rhodamine derivatives with huge Stokes shift to address the draconian requirements of single-excitation multicolor imaging. In this work, we described a generally strategy to enhance the Stokes shift of rhodamine dyes by completely breaking their electronic symmetry. As a result, the Stokes shift of novel rhodamine dye DQF-RB-Cl is up to 205 nm in PBS, which is the largest in all the reported rhodamine derivatives. In addition, we successfully realized the single excitation trichromatic imaging of mitochondria, lysosomes and cell membranes by combining DQF-RB-Cl with commercial lysosomal targeting probe Lyso-Tracker Green and membrane targeting dye Dil. This is the organic synthetic dyes for SLE-trichromatic imaging in cells for the first time. These results demonstrate the potential of our design as a useful strategy to develop huge Stokes shift fluorophore for bioimaging.

Multimodal imaging of experimental choroidal neovascularization

AIM:To compare choroidal neovascularization(CNV)lesion measurements obtained by in vivo imaging modalities,with whole mount histological preparations stained with isolectin GS-IB4,using a murine laser-induced CNV model.METHODS:B6 N.Cg-Tg(Csf1 r-EGFP)1 Hume/J heterozygous adult mice were subjected to laser-induced CNV and were monitored by fluorescein angiography(FA),multicolor(MC)fundus imaging and optical coherence tomography angiography(OCTA)at day 14 after CNV induction.Choroidalretinal pigment epithelium(RPE)whole mounts were prepared at the end of the experiment and were stained with isolectin GS-IB4.CNV areas were measured in all different imaging modalities at day 14 after CNV from three independent raters and were compared to choroidal-RPE whole mounts.Intraclass correlation coefficient(ICC)type 2(2-way random model)and its 95%confidence intervals(CI)were calculated to measure the correlation between different raters’measurements.Spearman’s rank correlation coefficient(Spearman’s r)was calculated for the comparison between FA,MC and OCTA data and histology data.RESULTS:FA(early and late)and MC correlates well with the CNV measurements ex vivo with FA having slightly better correlation than MC(FA early Spearman’s r=0.7642,FA late Spearman’s r=0.7097,and MC Spearman’s r=0.7418),while the interobser ver reliability was good for both techniques(FA early ICC=0.976,FA late ICC=0.964,and MC ICC=0.846).In contrast,OCTA showed a poor correlation with ex vivo measurements(Spearman’s r=0.05716)and high variability between different raters(ICC=0.603).CONCLUSION:This study suggests that FA and MC imaging could be used for the evaluation of CNV areas in vivo while caution must be taken and comparison studies should be performed when OCTA is employed as a CNV monitoring tool in small rodents.

Highly-Sensitive Multiplexed in vivo Imaging Using PEGylated Upconversion Nanoparticles

Lanthanide-based upconversion nanoparticles(UCNPs)have been widely explored in various fields,including optical imaging,in recent years.Although earlier work has shown that UCNPs with different lanthanide(Ln3+)dopants exhibit various colors,multicolor-especially in vivo multiplexed biomedical imaging-using UCNPs has rarely been reported.In this work,we synthesize a series of UCNPs with different emission colors and functionalize them with an amphiphilic polymer to confer water solubility.Multicolor in vivo upconversion luminescence(UCL)imaging is demonstrated by imaging subcutaneously injected UCNPs and applied in multiplexed in vivo lymph node mapping.We also use UCNPs for multicolor cancer cell labeling and realize in vivo cell tracking by UCL imaging.Moreover,for the first time we compare the in vivo imaging sensitivity of quantum dot(QD)-based fluorescence imaging and UCNP-based UCL imaging side by side,and find the in vivo detection limit of UCNPs to be at least one order of magnitude lower than that of QDs in our current non-optimized imaging system.Our data suggest that,by virtue of their unique optical properties,UCNPs have great potential for use in highly-sensitive multiplexed biomedical imaging.

Upconversion NaGdF4 nanoparticles for monitoring heat treatment and acid corrosion processes of hair

Lanthanide-based upconversion core-shell NaGdF4 nanocrystals with strong upconversion luminescence and biocompatibility were synthesized by the solvothermal method.The multicolor upconversion emission of these NaGdF4 nanoparticles could be easily obtained by controlling the core-shell compositions.These multicolor core-shell NaGdF4 upconversion nanocrystals could be employed as fluorescent probes for imaging the mouse hair,by which the porous and scalelike structure of the mouse hair were presented clearly.Meanwhile,it was directly shown by fluorescent signals that the mouse hair could resist the corrosion of the strong acid even when the concentration of hydrochloric acid was increased to 36.5%,but could not avoid the carbonization at high temperature of 400 oC.This procedure based on upconversion fluorescent nanoprobes opens a novel route for investigating the basic physical structure and chemical properties of biological tissue and organism.