Analytische Chemie
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FLiMFlow – Recent achievements in flow cytometry with lifetime detection and lifetime-encoded beads
(2016)
Flow cytometry is a widespread technique in biological research and clinical applications. Two different directions are currently of importance in development of new methods in this field. Whereas analyses in research become increasingly complex and require a growing number of simultaneously detectable codes and fluorescent labels, also low-cost methods and portable devices are needed in routine application.
Lifetime encoding could present an alternative to common spectral multiplexing. On the one hand, it addresses the need for additional codes by combining spectral and lifetime multiplexing. On the other hand, using only lifetime encoding could help to reduce instrument costs by keeping the number of excitation sources and detectors low.
Here, we report on our recent progress in employing dye-stained lifetime-encoded polymer microparticles as a model system for lifetime encoding in flow cytometry. The discrimination of two lifetime codes was achieved with two bead sets. Moreover, the simultaneous detection of a spectrally different ligand fluorescence signal could be demonstrated.
Flow cytometry is a common tool in biological research and clinical analyses. In current developments, there are two different tendencies of interest. Firstly, we face the need for analysis methods that are capable of addressing more and more involved analysis tasks, i.e., an increasing number of fluorescent codes and markers is required. Secondly, low-cost diagnostic tests, e.g. in disease recognition, are needed in routine application.
Lifetime encoding could be an attractive alternative to commonly applied color (spectral) encoding. By combining spectral and lifetime multiplexing, the number of simultaneously detectable codes might be increased by adding lifetime codes to the parameter space. Otherwise, instrumentation costs could be lowered using only lifetime encoding and thus avoiding costly excitation light sources and detectors.
Here, we report on our recent progress in time-resolved flow cytometry using dye-stained lifetime-encoded polymer microparticles as a model system. We could show that the discrimination of two lifetime codes is feasible. Moreover, the simultaneous detection of a spectrally different ligand fluorescence signal excited at the same wavelength as the lifetime code fluorescence could be demonstrated.
For the application of time-resolved detection in the framework of flow cytometry, suitable luminescence lifetime code carriers are required. Here we report on our achievements concerning strategies to increase the accessible range of lifetime values and to realize continuously tunable lifetimes.
To that end, we investigated polymer (PMMA) microbeads stained with mixtures of dyes exhibiting different fluorescence decay kinetics. At the expense of spectrally varying decay kinetics, it is possible to modify the lifetime by changing the dye concentration ratio.
Moreover, semiconductor quantum dots incorporated into polymer beads were studied as alternative luminophores outperforming organic dyes with respect to long luminescence lifetimes, flexible choice of excitation wavelength and narrow spectral emission width.
Our experiments demonstrate that lifetime adaption with dye mixing is basically feasible and semiconductor quantum dots represent promising candidates for long-lifetime codes.
Multiparametric analyses involving optical techniques like flow cytometry are at the core of studying complex systems in biological research and diagnostic applications. However, for fluorescence-based techniques, the number of reporters distinguishable in spectral multiplexing is limited by spectral overlap and requires a multitude of excitation light sources and detection Systems. Intensity encoding often used for bead assays suffers from problems regarding dye concentration control and excitation light intensity fluctuations. An alternative is luminescence lifetime encoding, particularly to minimize instrument costs.
Here, we report on dye-stained polymer microparticles for lifetime encoding in flow cytometry with different organic dyes. This ranges from studies of the impact of parameters like dye loading concentration and particle diameter on fluorescence decay behavior to the demonstration of lifetime code reading and simultaneous ligand fluorescence signal detection with single-wavelength excitation in a flow.
Flow cytometry is a widely used method in biological research and medical diagnostics. Depending on the respective application, two opposing directions of development are currently of interest. On the one hand, there is a need for analyses of growing complexity employing more and more fluorescent labels and codes. On the other hand, cost-effective methods and portable, miniaturized, and robust instruments are desired.
Commonly performed spectral multiplexing utilizing a color code suffers from several problems, such as the sensitivity of fluorescence intensity measurements to fluctuations in excitation light intensity and dye concentration and hence, photobleaching, dye leaking for certain encoding procedures, and spectral crosstalk, limiting the achievable number of detection channels. Moreover, it typically requires several costly excitation light sources. An innovative alternative can be lifetime multiplexing and the discrimination between different encoding fluorophores and carrier beads based on their fluorescence decay kinetics. In order to examine the potential of this approach, dye encoded beads (lifetime encoded surface chemistry) were prepared using several fluorophores from different dye classes and their suitability for lifetime discrimination in a flow was tested in conjunction with a custom designed flow cytometer equipped with a pulsed light source and a fast detector.
In a first step, the spectroscopic properties of micrometer-sized dye-stained PMMA beads were studied by means of steady state and time-resolved photoluminescence measurements. For the performance of studies on the practical use of these microbeads in flow cytometry applications, a custom-built demonstrator model for a flow system was employed.
Our results demonstrated that lifetime discrimination and simultaneous readout of a ligand fluorescence signal for analyte quantification with a set of dye-stained polymer microbeads at single wavelength excitation is feasible. These studies are expected to pave the road for new applications of fluorescence lifetime multiplexing within the framework of time-domain flow cytometry and bead-based assays.
Dye-stained lifetime-encoded polymer microbeads for application in time-resolved flow cytometry
(2017)
Flow cytometry is a standard analytical tool for biological research and in medical applications. There are different requirements triggering recent device and method development depending on the desired field of application. One trend is governed by the need for an increasing number of simultaneously detectable codes, i.e., fluorescent labels. The other one focuses on cost-effective methods and development of miniaturized, portable devices.
Fluorophore encoding is usually based on spectral encoding. However, this approach is hampered by, e.g., spectral crosstalk. Additionally, the sensitivity of fluorescence intensity measurements to fluctuations in excitation light intensity and dye concentration limits the achievable number of detection channels. Moreover, spectral multiplexing typically requires several costly excitation light sources. Lifetime multiplexing and the discrimination between different encoding fluorophores and carrier beads based on their fluorescence decay kinetics could present an innovative alternative. Encoded beads, i.e., beads with lifetime codes corresponding to the surface chemistry, have been employed to evaluate the feasibility of this approach with a custom designed flow cytometer equipped with a pulsed light source and a fast detector for time-resolved measurements in a flow.
In a first step, we used steady state and time-resolved photoluminescence measurements for the spectroscopic characterization of micrometer-sized dye-stained PMMA beads. Subsequently, the potential use of these microbeads for flow cytometry applications was analyzed with a prototype flow cytometer with lifetime detection.
With our proof-of-concept studies, we could demonstrate that lifetime discrimination and simultaneous readout of a ligand fluorescence signal for analyte quantification is feasible with a set of dye-stained polymer microbeads at single wavelength excitation. These studies are expected to pave the road for new applications of fluorescence lifetime multiplexing in time-domain flow cytometry and bead-based assays in general.
Many applications of nanometer- and micrometer-sized particles include their surface functionalization with linkers, sensor molecules, and analyte recognition moieties like (bio)ligands. This requires knowledge of the chemical nature and number of surface groups accessible for subsequent coupling reactions. Particularly attractive for the quantification of these groups are spectrophotometric and fluorometric assays, which can be read out with simple instrumentation. In this respect, we present here a novel Family of cleavable spectrophotometric and multimodal reporters for conjugatable amino and carboxyl surface groups on nano- and microparticles. This allows determination of particle-bound labels, unbound reporters in the supernatant, and reporters cleaved off from the particle surface, as well as the remaining thiol groups on particle, by spectrophotometry and inductively coupled optical emission spectrometry (32S ICP-OES). Comparison of the performance of these cleavable reporters with conductometry and conventional labels, utilizing changes in intensity or Color of absorption or emission, underlines the analytical potential of this versatile concept which elegantly circumvents Signal distortions by scattering and Encoding dyes and enables straightforward validation by method comparison.
Dye-stained micrometer-sized polymer beads are important tools in the life sciences with applications in biomedical, biochemical, and clinical research. Here, bead-based assays are increasingly used, for example, in DNA sequencing and the detection of autoimmune diseases or pathogenic microorganisms. Moreover, stained beads are employed as calibration tools for fluorescence microscopy and flow cytometry methods with increasing complexity. To address the requirements concerning the relevant fluorescence features, the spectroscopic properties of representative polymer beads with diameters ranging from about 1 to 10 μm stained with varying concentrations of rhodamine 6G were systematically assessed. The observed dependence of the spectral properties, fluorescence decay kinetics, and fluorescence quantum yields on bead size and dye loading concentration is attributed to different fluorescence characteristics of fluorophores located in the particle core and near-surface dye molecules. Supported by the fluorescence anisotropy measurements, the origin of the observed alteration of fluorescence features is ascribed to a combination of excitation energy transfer and polarity-related effects that are especially pronounced at the interface of the bead and the surrounding medium. The results of our studies underline the need to carefully control and optimize all Parameters that can affect the fluorescence properties of the dye-stained beads.
Time-resolved flow cytometry represents an alternative to commonly applied spectral or intensity multiplexing in bioanalytics. At present, the vast majority of the reports on this topic focuses on phase-domain techniques and specific applications. In this report, we present a flow cytometry platform with time-resolved detection based on a compact setup and straightforward time-Domain measurements utilizing lifetime-encoded beads with lifetimes in the nanosecond range. We provide general assessment of time-domain flow cytometry and discuss the concept of this platform to address achievable resolution limits, data analysis, and requirements on suitable encoding dyes. Experimental data are complemented by numerical calculations on photon count numbers and impact of noise and measurement time on the obtained lifetime values.
Aldehyde-functionalized materials have found widespread use in bioconjugation applications. For example, coupling of aldehyde surface groups with proteins, peptides or amine-functionalized oligonucleotides can readily produce biomolecule-decorated chip and bead surfaces for multiplex analyses. Furthermore, aldehyde-modified nanoparticles can possess bioadhesive properties that can prolong their retention time in biological compartments. These emerging novel bioanalytical and biomedical applications call for reliable tools and methods to detect and quantify accessible aldehyde functionalities on material surfaces.
We report on a versatile concept to quantify the accessible aldehyde moieties on particle surfaces through the specific binding and subsequent release of small reporter molecules such as fluorescent dyes and non-fluorescent chromophores utilizing acylhydrazone formation as a reversible covalent labeling strategy. This is representatively demonstrated for a set of polymer microparticles with different aldehyde labeling densities. Excess reporter molecules can be easily removed by washing, eliminating inaccuracies caused by unspecific adsorption to hydrophobic surfaces. Cleavage of hydrazones at acidic pH assisted by a carbonyl trap releases the fluorescent reporters rapidly and quantitatively and allows for their fluorometric detection at low concentration. Importantly, this strategy separates the signal-generating molecule from the bead surface, thereby circumventing common issues associated with light scattering and signal distortions due to binding-induced changes in reporter fluorescence as well as quenching dye-dye interactions on crowded material surfaces. In addition, we demonstrate that the release of a non-fluorescent chromophore via disulfide cleavage and subsequent quantification by absorption spectroscopy gives comparable results, verifying that both assays are capable of rapid and sensitive quantification of aldehydes on microbead surfaces. These strategies enable a quantitative comparison of bead batches with different functionalization densities, and a qualitative prediction of their coupling efficiencies in bioconjugations, as demonstrated in reductive amination reactions with Streptavidin.