Abstract: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 feature… Show more
“…The fluorescence of PSNP-dyad and PSNP-dyad-AMF can be excited at 320 and 560 nm as well as at 480 nm, thereby exciting the dyad and 6-AMF. Excitation at 480 nm requires high dyad loading concentrations of the PSP, as the absorption of the rhodamine unit of the dyad is relatively low at this wavelength (Figure a and Supporting Information, Section S3 on Optical Spectroscopy and Table S3), which can favor self-quenching . Comparative spectroscopic studies of the dyad and PSNP-dyad at different excitation wavelengths confirm the minimum influence of the particle matrix on the dyad’s optical properties, response time, reversibility, and selectivity.…”
A first tricolor fluorescent pH nanosensor is presented, which was rationally designed from biocompatible carboxylated polystyrene nanoparticles and two analyte-responsive molecular fluorophores. Its fabrication involved particle staining with a blue-red-emissive dyad, consisting of a rhodamine moiety responsive to acidic pH values and a pH-inert quinoline fluorophore, followed by the covalent attachment of a fluorescein dye to the particle surface that signals neutral and basic pH values with a green fluorescence. These sensor particles change their fluorescence from blue to red and green, depending on the pH and excitation wavelength, and enable ratiometric pH measurements in the pH range of 3.0−9.0. The localization of the different sensor dyes in the particle core and at the particle surface was confirmed with fluorescence microscopy utilizing analogously prepared polystyrene microparticles. To show the application potential of these polystyrene-based multicolor sensor particles, fluorescence microscopy studies with a human A549 cell line were performed, which revealed the cellular uptake of the pH nanosensor and the differently colored emissions in different cell organelles, that is, compartments of the endosomal-lysosomal pathway. Our results demonstrate the underexplored potential of biocompatible polystyrene particles for multicolor and multianalyte sensing and bioimaging utilizing hydrophobic and/or hydrophilic stimuli-responsive luminophores.
“…The fluorescence of PSNP-dyad and PSNP-dyad-AMF can be excited at 320 and 560 nm as well as at 480 nm, thereby exciting the dyad and 6-AMF. Excitation at 480 nm requires high dyad loading concentrations of the PSP, as the absorption of the rhodamine unit of the dyad is relatively low at this wavelength (Figure a and Supporting Information, Section S3 on Optical Spectroscopy and Table S3), which can favor self-quenching . Comparative spectroscopic studies of the dyad and PSNP-dyad at different excitation wavelengths confirm the minimum influence of the particle matrix on the dyad’s optical properties, response time, reversibility, and selectivity.…”
A first tricolor fluorescent pH nanosensor is presented, which was rationally designed from biocompatible carboxylated polystyrene nanoparticles and two analyte-responsive molecular fluorophores. Its fabrication involved particle staining with a blue-red-emissive dyad, consisting of a rhodamine moiety responsive to acidic pH values and a pH-inert quinoline fluorophore, followed by the covalent attachment of a fluorescein dye to the particle surface that signals neutral and basic pH values with a green fluorescence. These sensor particles change their fluorescence from blue to red and green, depending on the pH and excitation wavelength, and enable ratiometric pH measurements in the pH range of 3.0−9.0. The localization of the different sensor dyes in the particle core and at the particle surface was confirmed with fluorescence microscopy utilizing analogously prepared polystyrene microparticles. To show the application potential of these polystyrene-based multicolor sensor particles, fluorescence microscopy studies with a human A549 cell line were performed, which revealed the cellular uptake of the pH nanosensor and the differently colored emissions in different cell organelles, that is, compartments of the endosomal-lysosomal pathway. Our results demonstrate the underexplored potential of biocompatible polystyrene particles for multicolor and multianalyte sensing and bioimaging utilizing hydrophobic and/or hydrophilic stimuli-responsive luminophores.
“…Thus, there is no need to restrict luminophores and samples for lifetime encoding to materials with mono-exponential decay. This is an important prerequisite of bead-based lifetime barcodes where heterogeneous microenvironments of fluorophores often lead to multi-exponential decays 39 . Figure 5 Impact of signal and background intensity.…”
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.
“…These beads are stained with a single organic dye, here either with PolyAn Red5 45 or with the widely used photostable dye rhodamine 6G (Rh6G) [46][47][48] . The spectroscopic properties of dye-stained PMMA beads have been extensively examined and previously published in a separate study 41 . The encoding dyes were chosen to be excitable at 488 nm and have emission maxima at 550 nm and 690 nm, respectively.…”
Semiconductor quantum dots (QDs) embedded into polymer microbeads are known to be very attractive emitters for spectral multiplexing and colour encoding. Their luminescence lifetimes or decay kinetics have been, however, rarely exploited as encoding parameter, although they cover time ranges which are not easily accessible with other luminophores. We demonstrate here the potential of QDs made from II/VI semiconductors with luminescence lifetimes of several 10 ns to expand the lifetime range of organic encoding luminophores in multiplexing applications using time-resolved flow cytometry (LT-FCM). For this purpose, two different types of QD-loaded beads were prepared and characterized by photoluminescence measurements on the ensemble level and by single-particle confocal laser scanning microscopy. Subsequently, these lifetime-encoded microbeads were combined with dye-encoded microparticles in systematic studies to demonstrate the potential of these QDs to increase the number of lifetime codes for lifetime multiplexing and combined multiplexing in the time and colour domain (tempo-spectral multiplexing). These studies were done with a recently developed novel luminescence lifetime flow cytometer (LT-FCM setup) operating in the time-domain, that presents an alternative to reports on phase-sensitive lifetime detection in flow cytometry.Flow cytometry (FCM), where either single cells or particles are optically detected in a flow, is an established and widespread multiparametric fluorescence technique used for many routine and research applications in biology, medical diagnostics, and food analysis 1-5 . FCM typically relies on organic luminophores for optical encoding and multiplexing as well as analyte quantification 6-10 . State-of-the-art FCM instruments are designed to detect spectral and/or intensity codes and can distinguish a large number of different colour and intensity codes, e.g. in the case of bead-based assays 5,11-13 . Commonly applied spectral multiplexing approaches utilizing organic luminophores are, however, limited by the spectral overlap of the emission bands of the different encoding dyes which limits the number of distinguishable codes and can require signal compensation and cross talk correction 6,14,15 . The exploitation of the luminophore-characteristic luminescence lifetime (LT) as additional encoding parameter could in principle increase the number of accessible codes in flow cytometry analysis, thereby increasing the degree of multiplexing 6,7,14,[16][17][18][19][20][21] . Time-resolved fluorescence measurements in flow cytometry and lifetime encoding have been discussed for decades 8,[22][23][24][25][26][27][28] . The concept of lifetime multiplexing in a flow, however, has not been adopted in routine applications up to now due to the limited measurement time per object resulting in reduced photon count numbers 27,29 . Moreover, the vast majority of reports on lifetime detection in flow cytometry relies on frequency-domain techniques (phase fluorometry), which used to be less expensive as f...
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