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Bioanalytical, diagnostic, and security applications require the fast and sensitive determination of a steadily increasing number of analytes or events in parallel in a broad variety of detection formats.[1,2] Ideal candidates for spectral encoding and multiplexing schemes are luminescent nanocrystals like semiconductor quantum dots (QDs), particularly Cd-containing II/VI QDs with their narrow and symmetric emission bands. With the availability of relatively simple and inexpensive instrumentation for time-resolved fluorescence measurements, similar strategies utilizing the compound-specific parameter fluorescence lifetime or fluorescence decay kinetics become increasingly attractive.[3-5] The potential of different types of QDs like II/VI, III/V and Cd-free ternary QDs such as AgInS (AIS) QDs for lifetime-based encoding and multiplexing has been, however, barely utilized, although the lifetimes of these nanocrystals cover a time windows which is barely accessible with other fluorophores. Here we present a brief insight into the photophysics of AIS QDs and show the potential of dye- and QD-encoded beads for lifetime-based encoding and detection schemes in conjunction with flow cytometry and fluorescence lifetime imaging microscopy
Research of division Biophotonics at the Federal Institute for Materials Research and Testing (BAM) covers several topics including photophysics of molecular and nanocrystalline emitters, the development of signal enhancement, multiplexing, and barcoding strategies, surface group quantification, the rational design of different types of stimuli-responsive optical probes, and concepts and reference materials for the validation of optical-spectroscopic measurements. In the following representative examples for each of these topics are given. Also, current developments like single particle spectroscopy and flow cytometry with lifetime detection and newly certified fluorescence quantum yield standards are presented.
Photoluminescence applications in the life and material sciences require bright molecular and nanocrystalline emitters, stimuli-responsive optical probes, signal enhancement, multiplexing, and barcoding strategies and traceable methods to quantify the signal-relevant optical properties of luminescent materials at the ensemble and single molecule/particle level. In this context, current research at Division Biophotonics of BAM is presented ranging from dye and nanocrystal photophysics, absolute measurements of photoluminescence quantum yields in the UV/vis/NIR/SWIR, lifetime multiplexing, and the development of different types of fluorescence standards for validating optical-spectroscopic measurements.
Aiming at the development of a non-destructive optical measurement methods for the characterization of nano-sized airborne particulate pollutants we performed fluorescence spectroscopic investigations of their relevant emission properties. Here, we present first results from studies of soot particles from synthetic aerosols deposited on PTFE membranes.
Applications of luminescent nanomaterials like semiconductor nanocrystals (QDs) and lanthanide-based upconversion nanocrystals (UCNPs) in the life sciences such as bioimaging studies or their use as reporter in assays call for a correlation of the photoluminescence (PL) properties of these nanomaterials on ensemble and single particle levels. This is particularly relevant within the context of continuously decreasing detection limits. Aiming at optimum nanomaterials for spectroscopic and microscopic applications, we examine the optical properties of QDs like II/VI QDs and cadmium-free AgInS2/ZnS QDs (AIS/ZnS) and UCNPs of different chemical composition, size, and particle architecture for ensembles and single particles. This includes PL spectra, PL quantum yields (ΦF), brightness values, blinking behavior, and PL decay kinetics. For UCNPs with their nonlinear spectrally converted PL excited by sequential multiphoton absorption, these measurements were also done as a function of excitation power density (P). Special emphasis is dedicated to the performance parameters ΦF and brightness, that determine signal size and provide a measure for nanocrystal quality.[1-5]
Systematic studies of the excitation energy dependence (EED) [6] of the PL properties of II/VI and ternary AgInS2/ZnS QDs reveal the potential of this relatively simple method for providing insights into the electronic energy structure of QDs. The intrinsic nature of the inhomogeneous broadening of the PL bands of AIS/ZnS QDs was confirmed by single particle spectroscopy.[5] By combining P-dependent integration spectroscopy and single particle measurements of UCNPs, using a new custom-made setup, consisting of different lasers, an inverted microscope, different detectors, and an AFM, we could study the P-dependent optical properties of these nonlinear emitters from ~10 W/cm2 up to ~105 W/cm2. These results provide optimum dopant ion concentrations for bioanalytical, spectroscopic, and microscopic applications of UCNP.
Acknowledgement. Financial support by grants RE1203/12-3 and RE1203/20-1 (support of F. Weigert, L. Dhamo, and F. Frenzel) from German Research Council (DFG) is acknowledged.
The rational synthesis and use of nanomaterials require the characterization of many different properties, ranging from particle size and size distribution over surface chemistry to more applicationrelevant features like optical, electrochemical, and magnetic properties. In the following, several methods for the characterization of functional groups on nanomaterials, like polymer and silica nanoparticles, semiconductor quantum dots, and lanthanide-based upconversion nanocrystals are presented. Additionally, procedures for the measurement of the key spectroscopic performance parameters of nanomaterials with linear and nonlinear photoluminescence, such as the photoluminescence quantum yield, are presented for the UV/vis/NIR/SWIR.
The comparison of different emitter classes and the rational design of the next generation of molecular and nanoscale reporters require accurate and quantitative photo-luminescence measurements. This is of special importance for all photoluminescence applications in the life and material sciences and nanobiophotonics. In the following, procedures for the determination of the spectroscopic key parameter photoluminescence quantum yield, i.e., the number of emitted per absorbed photons, in the UV/vis/NIR/SWIR are presented including pitfalls and achievable uncertainties and material-specific effects related to certain emitter classes are addressed.
A brief introduction to fluorescence spectroscopy will be provided, ranging from typically measured fluorescence quantities over instrument-specific contributions to measured fluorescence signals to selected applications. In this context, an overview of the photoluminescence properties of molecular and nanoscale luminescence reporters will be given including a brief insight into their photophysics and fluorescence standards designed by division Biophotonics for the calibration and instrument performance validation of fluorescence measuring devices will be presented.
A brief perspective of BAM on advanced materials is presented including examples for nanomaterials and other systems presenting advanced materials with special emphasis on characterization methods used in different division of BAM for the determination of functional or safety parameters of such materials. In this respect, also ongoing activities of the capacity building project nanoplatform of BAM aiming at the development of nanometer-sized reference materials are briefly summarized.
Photoluminescence techniques are amongst the most widely used tools in the life sciences, with new and exciting applications in medical diagnostics and molecular imaging continuously emerging. Advantages include their comparative ease of use, unique sensitivity, non-invasive character, and potential for multiplexing, remote sensing, and miniaturization. General drawbacks are, however, signals, that contain unwanted wavelength- and polarization contributions from instrument-dependent effects, which are also time-dependent due to aging of instrument-components, and difficulties to measure absolute fluorescence intensities. Moreover, scattering systems require special measurement geometries and the interest in new optical reporters with emission > 1000 nm strategies for reliable measurements in the second diagnostic for the comparison of material performance and the rational design of new fluorophores with improved properties.
Here, we present strategies to versatile method-adaptable liquid and solid fluorescence standards for different fluorescence parameters. Examples are multi-emitter glasses, spectral fluorescence standards, and quantum yield standards for the UV/vis/NIR.