Dataset Information

Bioimaging with Macromolecular Probes Incorporating Multiple BODIPY Fluorophores.

ABSTRACT: Seven macromolecular constructs incorporating multiple borondipyrromethene (BODIPY) fluorophores along a common poly(methacrylate) backbone with decyl and oligo(ethylene glycol) side chains were synthesized. The hydrophilic oligo(ethylene glycol) components impose solubility in aqueous environment on the overall assembly. The hydrophobic decyl chains effectively insulate the fluorophores from each other to prevent detrimental interchromophoric interactions and preserve their photophysical properties. As a result, the brightness of these multicomponent assemblies is approximately three times greater than that of a model BODIPY monomer. Such a high brightness level is maintained even after injection of the macromolecular probes in living nematodes, allowing their visualization with a significant improvement in signal-to-noise ratio, relative to the model monomer, and no cytotoxic or behavioral effects. The covalent scaffold of these macromolecular constructs also permits their subsequent conjugation to secondary antibodies. The covalent attachment of polymer and biomolecule does not hinder the targeting ability of the latter and the resulting bioconjugates can be exploited to stain the tubulin structure of model cells to enable their visualization with optimal signal-to-noise ratios. These results demonstrate that this particular structural design for the incorporation of multiple chromophores within the same covalent construct is a viable one to preserve the photophysical properties of the emissive species and enable the assembly of bioimaging probes with enhanced brightness.

SUBMITTER: Thapaliya ER

PROVIDER: S-EPMC5906127 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Bioimaging with Macromolecular Probes Incorporating Multiple BODIPY Fluorophores.

Thapaliya Ek Raj ER Zhang Yang Y Dhakal Pravat P Brown Adrienne S AS Wilson James N JN Collins Kevin M KM Raymo Françisco M FM

Bioconjugate chemistry 20170501 5

Seven macromolecular constructs incorporating multiple borondipyrromethene (BODIPY) fluorophores along a common poly(methacrylate) backbone with decyl and oligo(ethylene glycol) side chains were synthesized. The hydrophilic oligo(ethylene glycol) components impose solubility in aqueous environment on the overall assembly. The hydrophobic decyl chains effectively insulate the fluorophores from each other to prevent detrimental interchromophoric interactions and preserve their photophysical proper ...[more]

PMID: 28430413

Similar Datasets

Project description:Fluorescence microscopy and detection have become indispensible for understanding organization and dynamics in biological systems. Novel fluorophores with improved brightness, photostability, and biocompatibility continue to fuel further advances but often rely on having minimal background. The visualization of interactions in very high biological background, especially for proteins or bound complexes at very low copy numbers, remains a primary challenge. Instead of focusing on molecular brightness of fluorophores, we have adapted the principles of high-sensitivity absorption spectroscopy to improve the sensitivity and signal discrimination in fluorescence bioimaging. Utilizing very long wavelength transient absorptions of kinetically trapped dark states, we employ molecular modulation schemes that do not simultaneously modulate the background fluorescence. This improves the sensitivity and ease of implementation over high-energy photoswitch-based recovery schemes, as no internal dye reference or nanoparticle-based fluorophores are needed to separate the desired signals from background. In this Account, we describe the selection process for and identification of fluorophores that enable optically modulated fluorescence to decrease obscuring background. Differing from thermally stable photoswitches using higher-energy secondary lasers, coillumination at very low energies depopulates transient dark states, dynamically altering the fluorescence and giving characteristic modulation time scales for each modulatable emitter. This process is termed synchronously amplified fluorescence image recovery (SAFIRe) microscopy. By understanding and optically controlling the dye photophysics, we selectively modulate desired fluorophore signals independent of all autofluorescent background. This shifts the fluorescence of interest to unique detection frequencies with nearly shot-noise-limited detection, as no background signals are collected. Although the fluorescence brightness is improved slightly, SAFIRe yields up to 100-fold improved signal visibility by essentially removing obscuring, unmodulated background (Richards, C. I.; J. Am. Chem. Soc. 2009, 131, 4619). While SAFIRe exhibits a wide, linear dynamic range, we have demonstrated single-molecule signal recovery buried within 200 nM obscuring dye. In addition to enabling signal recovery through background reduction, each dye exhibits a characteristic modulation frequency indicative of its photophysical dynamics. Thus, these characteristic time scales offer opportunities not only to expand the dimensionality of fluorescence imaging by using dark-state lifetimes but also to distinguish the dynamics of subpopulations on the basis of photophysical versus diffusional time scales, even within modulatable populations. The continued development of modulation for signal recovery and observation of biological dynamics holds great promise for studying a range of transient biological phenomena in natural environments. Through the development of a wide range of fluorescent proteins, organic dyes, and inorganic emitters that exhibit significant dark-state populations under steady-state illumination, we can drastically expand the applicability of fluorescence imaging to probe lower-abundance complexes and their dynamics.

Dataset Information

Bioimaging with Macromolecular Probes Incorporating Multiple BODIPY Fluorophores.

Publications

Bioimaging with Macromolecular Probes Incorporating Multiple BODIPY Fluorophores.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets