This article gives an overview of the various kinds of nanoparticles (NPs) that are widely used for purposes of fluorescent imaging, mainly of cells and tissues. Following an introduction and a discussion of merits of fluorescent NPs compared to molecular fluorophores, labels and probes, the article assesses the kinds and specific features of nanomaterials often used in bioimaging. These include fluorescently doped silicas and sol–gels, hydrophilic polymers (hydrogels), hydrophobic organic polymers, semiconducting polymer dots, quantum dots, carbon dots, other carbonaceous nanomaterials, upconversion NPs, noble metal NPs (mainly gold and silver), various other nanomaterials, and dendrimers. Another section covers coatings and methods for surface modification of NPs. Specific examples on the use of nanoparticles in (a) plain fluorescence imaging of cells, (b) targeted imaging, (c) imaging of chemical species, and (d) imaging of temperature are given next. A final section covers aspects of multimodal imaging (such as fluorescence/nmr), imaging combined with drug and gene delivery, or imaging combined with therapy or diagnosis. The electronic supplementary information (ESI) gives specific examples for materials and methods used in imaging, sensing, multimodal imaging and theranostics such as imaging combined with drug delivery or photodynamic therapy. The article contains 273 references in the main part, and 157 references in the ESI.

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An overview of nanoparticles commonly used in fluorescent bioimaging

Luminescence techniques are among the most widely used detection methods in the life and material sciences. At the core of these methods is an ever-increasing variety of fluorescent reporters (i.e., simple dyes, fluorescent labels, probes, sensors and switches) from different fluorophore classes ranging from small organic dyes and metal ion complexes, quantum dots and upconversion nanocrystals to differently sized fluorophore-doped or fluorophore-labeled polymeric particles. A key parameter for fluorophore comparison is the fluorescence quantum yield (Φf), which is the direct measure for the efficiency of the conversion of absorbed light into emitted light. In this protocol, we describe procedures for relative and absolute determinations of Φf values of fluorophores in transparent solution using optical methods, and we address typical sources of uncertainty and fluorophore class-specific challenges. For relative determinations of Φf, the sample is analyzed using a conventional fluorescence spectrometer. For absolute determinations of Φf, a calibrated stand-alone integrating sphere setup is used. To reduce standard-related uncertainties for relative measurements, we introduce a series of eight candidate quantum yield standards for the wavelength region of ∼350-950 nm, which we have assessed with commercial and custom-designed instrumentation. With these protocols and standards, uncertainties of 5-10% can be achieved within 2 h.

Relative and absolute determination of fluorescence quantum yields of transparent samples

Photon-upconverting nanoparticles (UCNPs) are lanthanide-doped nanocrystals that emit visible light under near-infrared excitation (anti-Stokes emission). This unique optical property precludes background fluorescence and light scattering from biological materials. The emission of multiple and narrow emission lines is an additional hallmark of UCNPs that opens up new avenues for optical encoding. Distinct emission signatures can be obtained if the multiple emission of UCNPs is tuned by their dopant composition or by surface modification with dyes. Tuning the intensity of only one of the multiple emission lines and using another one as a constant reference signal enables the design of ratiometric codes that are resistant to fluctuations in absolute signal intensities. Combining several UCNPs each displaying a distinct set of emission lines expands the coding capacity exponentially and lays the foundation for highly multiplexed analyte detection. This Review highlights the potential of UCNPs for labeling and encoding biomolecules, microspheres, and even whole cells.

Photon-upconverting nanoparticles for optical encoding and multiplexing of cells, biomolecules, and microspheres.

We present a systematic study on the effect of surface ligands on the luminescence properties and colloidal stability of β-NaYF4:Yb3+,Er3+ upconversion nanoparticles (UCNPs), comparing nine different surface coatings to render these UCNPs water-dispersible and bioconjugatable. A prerequisite for this study was a large-scale synthetic method that yields ∼2 g per batch of monodisperse oleate-capped UCNPs providing identical core particles. These ∼23 nm sized UCNPs display an upconversion quantum yield of ∼0.35% when dispersed in cyclohexane and excited with a power density of 150 W cm−2, underlining their high quality. A comparison of the colloidal stability and luminescence properties of these UCNPs, subsequently surface modified with ligand exchange or encapsulation protocols, revealed that the ratio of the green (545 nm) and red (658 nm) emission bands determined at a constant excitation power density clearly depends on the surface chemistry. Modifications relying on the deposition of additional (amphiphilic) layer coatings, where the initial oleate coating is retained, show reduced non-radiative quenching by water as compared to UCNPs that are rendered water-dispersible via ligand exchange. Moreover, we could demonstrate that the brightness of the upconversion luminescence of the UCNPs is strongly affected by the type of surface modification, i.e., ligand exchange or encapsulation, yet hardly by the chemical nature of the ligand.

https://pubs.rsc.org/en/Content/ArticlePDF/2015/NR/C4NR05954A

Water dispersible upconverting nanoparticles: effects of surface modification on their luminescence and colloidal stability

The photoluminescence quantum yield (Φf) that presents a direct measure for the efficiency of the conversion of absorbed photons into emitted photons is one of the spectroscopic key parameters of functional fluorophores. It determines the suitability of such materials for applications in, for example, (bio)analysis, biosensing, and fluorescence imaging as well as as active components in optical devices. The reborn interest in accurate Φf measurements in conjunction with the controversial reliability of reported Φf values of many common organic dyes encouraged us to compare two relative and one absolute fluorometric method for the determination of the fluorescence quantum yields of quinine sulfate dihydrate, coumarin 153, fluorescein, rhodamine 6G, and rhodamine 101. The relative methods include the use of a chain of Φf transfer standards consisting of several “standard dye” versus “reference dye” pairs linked to a golden Φf standard that covers the ultraviolet and visible spectral region, and the use of d...

Comparison of Methods and Achievable Uncertainties for the Relative and Absolute Measurement of Photoluminescence Quantum Yields

There is an increasing interest in chromophores absorbing and emitting in the near-infrared (NIR) spectral region, e.g., for applications as fluorescent reporters for optical imaging techniques and hence, in reliable methods for the characterization of their signal-relevant properties like the fluorescence quantum yield (Φf) and brightness. The lack of well established Φf standards for the NIR region in conjunction with the need for accurate Φf measurements in transparent and scattering media encouraged us to built up an integrating sphere setup for spectrally resolved measurements of absolute fluorescence traceable to radiometric scales. Here, we present the design of this setup and its characterization and validation including an uncertainty budget for the determination of absolute Φf in the visible and NIR. To provide the basis for better measurements of Φf in the spectral window from ca. 600 to 1000 nm used, e.g., for optical imaging, the absolute Φf of a set of NIR chromophores covering this spectral...

Integrating Sphere Setup for the Traceable Measurement of Absolute Photoluminescence Quantum Yields in the Near Infrared

The commercial availability of stand-alone setups for the determination of absolute photoluminescence quantum yields (pf) in conjunction with the increasing use of integrating sphere accessories for spectrofluorometers is expected to have a considerable influence not only on the characterization of chromophore systems for use in optical and opto-electronic devices, but also on the determination of this key parameter for (bio)analytically relevant dyes and functional luminophores. Despite the huge potential of systems measuring absolute pf values and the renewed interest in dependable data, evaluated protocols for even the most elementary case, the determination of the fluorescence quantum yield of transparent dilute solutions of small organic dyes with integrating sphere methods, are still missing. This encouraged us to evaluate the performance and sources of uncertainty of a simple commercial integrating sphere setup with dilute solutions of two of the best characterized fluorescence quantum yield standards, quinine sulfate dihydrate and rhodamine 101, strongly differing in spectral overlap between absorption and emission. Special attention is dedicated to illustrate common pitfalls of this approach, thereby deriving simple procedures to minimize measurement uncertainties and improve the comparability of data for the broad community of users of fluorescence techniques.

Evaluation of a commercial integrating sphere setup for the determination of absolute photoluminescence quantum yields of dilute dye solutions.

Die vorliegende Erfindung betrifft eine Vorrichtung und eine Verfahren zur Bestimmung der Photolumineszenzquantenausbeute und weiterer optischer Eigenschaften einer Probe. Es ist Aufgabe der vorliegenden Erfindung, eine Vorrichtung und ein Verfahren anzugeben, mit denen neben der Bestimmung der Quantenausbeute auch eine Bestimmung von Reflexion und Transmission einer Probe schnell, kostengunstig und zuverlassig realisiert werden konnen. Dazu werden ein erster Spiegel (S1), ein zweiter Spiegel (S2) und ein dritter Spiegel (S3) vorgesehen, wobei der erste Spiegel (S1) und der zweite Spiegel (S2) in Bezug auf die Ulbricht-Kugel (26) fixiert sind und der dritte Spiegel (S3) drehbar gelagert ist, und wobei erster Spiegel (S1), zweiter Spiegel (S2) und dritter Spiegel (S3) derart angeordnet sind, dass das von der Messlichtquelle (10) emittierte Licht in einer ersten Winkelstellung (48) des dritten Spiegels (S3) vom dritten Spiegel (3) zum ersten Spiegel (S1) und nachfolgend vom ersten Spiegel (S1) durch die erste Offnung (1) in die Ulbricht-Kugel (26) reflektiert und in einer zweiten Winkelstellung (46) des dritten Spiegels (S3) vom dritten Spiegel (3) zum zweiten Spiegel (S2) und nachfolgend vom zweiten Spiegel (S2) durch die zweite Offnung (5) in die Ulbricht-Kugel (26) reflektiert wird.

Vorrichtung und Verfahren zur Bestimmung der Photolumineszenzquantenausbeute und weiterer optischer Eigenschaften einer Probe

The device has an integrating sphere (26), and a sample holder for holding a sample (28) in a middle point of the sphere. First and second mirrors are fixed with respect to the sphere, and a third mirror is rotatably supported. The mirrors are arranged such that light emitted by a lamp (10) is reflected from the third mirror to the first mirror and from the first mirror to the sphere in an angular position of the third mirror, and reflected from the third mirror to the second mirror and from the second mirror to the sphere in another angular position of the third mirror. An independent claim is also included for a method for determining luminescence quantum efficiency and optical characteristics of a sample.

Cornelia Lochmann

Papers

Integrating Sphere Setup for the Traceable Measurement of Absolute Photoluminescence Quantum Yields in the Near Infrared

Evaluation of a commercial integrating sphere setup for the determination of absolute photoluminescence quantum yields of dilute dye solutions.

Vorrichtung und Verfahren zur Bestimmung der Photolumineszenzquantenausbeute und weiterer optischer Eigenschaften einer Probe

Device and method for determining the photoluminescence quantum yield and other optical characteristics of a sample