A Labeling Strategy for Living Specimens in Long-Term/Super-Resolution Fluorescence Imaging

Abstract

Despite the urgent need to image living specimens for cutting-edge biological research, most existing fluorescent labeling methods suffer from either poor optical properties or complicated operations required to realize cell-permeability and specificity. In this study, we introduce a method to overcome these limits—taking advantage of the intrinsic affinity of bright and photostable fluorophores, no matter if they are supposed to be live-cell incompatible or not. Incubated with living cells and tissues in particular conditions (concentration and temperature), some Atto and BODIPY dyes show live-cell labeling capability for specific organelles without physical cell-penetration or chemical modifications. Notably, by using Atto 647N as a live-cell mitochondrial marker, we obtain 2.5-time enhancement of brightness and photostability compared with the most commonly used SiR dye in long-term imaging. Our strategy has expanded the scientist's toolbox for understanding the dynamics and interactions of subcellular structures in living specimens.

Three-dimensional multi-color optical nanoscopy at sub-10-nm resolution based on small-molecule organic probes

“…The results showed that MAA-Actin-TMR colocalized well with EGFP-Lifeact (Pearson’s coefficient: 0.94), and MAA-Lyso-Atto 565 colocalized well with LysoTracker Green (Pearson’s coefficient: 0.9), indicating the high specificity of the probes. Notably, not all the fluorescent dyes are suitable for constructing the actin probes, since strongly lipophilic and cationic dyes 9 may cause nonspecific labeling (e.g., Atto 647N). Besides, cyanine derives (e.g., Alexa Fluor 647 and Cy5) should also be avoided because of their weak resistance to the following expansion process.…”

Antibody Functionalization of Ultrasmall Fluorescent Core–Shell Aluminosilicate Nanoparticle Probes for Advanced Intracellular Labeling and Optical Super Resolution Microscopy

“…While such Ab–dye immunoconjugates are successful at specific targeting, for example of particular proteins, their optical performance is limited. Organic fluorophores are prone to both non-radiative decay processes and photobleaching that limit their photon production and photostability, respectively, for example, for applications involving extended study periods. − To overcome these issues, researchers have turned toward nanoparticles such as quantum dots, polymer nanoparticles, or silica nanoparticles. ,− As a result of their favorable biodistribution and pharmacokinetics profiles, increasing attention has recently been focused on ultrasmall optical probes with sizes below the cutoff for renal clearance, that is, diameters below 10 nm. − Furthermore, ultrasmall nanoparticles are ideal probes for studying nanoscale structures as they minimize both steric hindrance of the labeled entity as well as the distance between the fluorescent probe and structure of interest, leading to more accurate size estimations . We demonstrate that efficiently attaching Abs to such ultrasmall nanoparticles and leading to highly specific targeting probes enabling successful imaging of several subcellular structures with low fluorescence background and super-resolution below the diffraction limit of optical light is non-trivial and requires substantial efforts to generate robust and reproducible protocols.…”