Nanocrystals and Nanoparticles Versus Molecular Fluorescent Labels as Reporters for Bioanalysis and the Life Sciences: A Critical Comparison

Abstract

At the core of photoluminescence techniques are suitable fluorescent labels and reporters, the spectroscopic properties of which control the limit of detection, the dynamic range, and the potential for multiplexing. Many applications including recent developments in intracellular labeling rely on well established molecular chromophores such as small organic dyes or fluorescent proteins. However, one of the most exciting -but also controversial -advances in reporter technology, the emerging development and application of luminescent nanoparticles with unique optical properties, yet complicated surface chemistry paves new roads for fluorescence imaging and sensing as well as for in vitro and in vivo labeling. Here, we compare and evaluate the differences in physico-chemical properties of common fluorophores, focusing on traditional organic dyes and luminescent nanocrystals with size-dependent features. The ultimate goal is to provide a better understanding of the advantages and limitations of both classes of chromophores, facilitate fluorophore choice for users of fluorescence techniques, and address future challenges in the rational design and manipulation of nanoparticulate labels and probes.

Comparative Analysis of Fluorescence Reporter Signals Based on Intensity, Anisotropy, Time-Resolution, and Wavelength-Ratiometry

“…There are many variants in choosing the dye donor-acceptor pair in which two correspondent bands are well separated on the wavelength scale or produce different lifetimes. Meantime, we observe increasing popularity of lanthanide chelates [37] and Quantum Dots [38,39] as FRET donors, which is mainly because of their increased brightness and longer emission lifetimes [40]. If the acceptor is excited not directly but by the energy transferred from the donor, its lifetime increases to that of the donor [41].…”

PET/Fluorescence Imaging: An Overview of the Chemical Strategies to Build Dual Imaging Tools

“…Indeed, their small size and tunable optical characteristics make them suitable dyes for imaging applications in biological systems. 176,177 Fluorescent NPs used for bimodal PET/FLI applications are most of the time functionalized with a BFCA and a targeting vector, both being grafted by covalent bond. Here again, activated forms of BFCA such as isothiocyanate 136,155−158 or N-hydroxysuccinimide 148,149 are used.…”

“…Alternatively to the grafting of a fluorescent dye into the NPs, some of them display inherent fluorescence properties like Quantum Dots (QDs) that are also widely used for bimodal application. Indeed, their small size and tunable optical characteristics make them suitable dyes for imaging applications in biological systems. , Fluorescent NPs used for bimodal PET/FLI applications are most of the time functionalized with a BFCA and a targeting vector, both being grafted by covalent bond. Here again, activated forms of BFCA such as isothiocyanate ,− or N -hydroxysuccinimide , are used.…”

Interlaboratory Comparison on Absolute Photoluminescence Quantum Yield Measurements of Solid Light Converting Phosphors with Three Commercial Integrating Sphere Setups

“…The latter equals the ratio of the number of emitted and absorbed photons, providing the conversion efficiency of absorbed into emitted photons. , From the material or sample side, the size of the measurable fluorescence signal is determined by the product of the luminophore’s absorption coefficient or absorption cross section at the excitation wavelength and Φ f , termed brightness . Therefore, Φ f is frequently used to select optimum emitters for applications, e.g., in medical diagnostics, solid-state lighting, and converter materials for light-emitting diodes (LEDs) and solar concentrators. ,,− Also, Φ f measurements are an essential part of photophysical and mechanistic studies and provide the basis for the design of next-generation functional luminescent materials. This underlines the importance of reliable and reproducible Φ f measurements for the scientific community, manufacturers, and users of commercial luminescent materials, as well as international standardization organizations like the International Electrotechnical Commission (IEC).…”