Chemie und Prozesstechnik
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Organisationseinheit der BAM
- 1.2 Biophotonik (16) (entfernen)
Objective: The utilization of fluorescein-guided biopsies and resection has been recently discussed as a suitable strategy to improve and expedite operative techniques for the resection of central nervous system (CNS) tumors. However, little is known about the optical properties of sodium fluorescein (NaFl) in human tumor tissue and their potential impact on ex vivo analyses involving fluorescence-based methods.
Methods: Tumor tissue was obtained from a study cohort of an observational study on the utilization of fluorescein-guided biopsy and resection (n=5). The optical properties of fluorescein-stained tissue were compared to the optical features of the dye in vitro and in control samples consisting of tumor tissue of high-grade glioma patients (n=3) without intravenous (i.v.) application of NaFl. The dye-exposed tumor tissues were used for optical measurements to confirm the detectability of NaFl emission ex vivo. The tissue samples were fixed in 4%PFA, immersed in 30% sucrose, embedded in Tissue-Tek OCT compound, and cut to 10 mm cryosections. Spatially resolved emission spectra from tumor samples were recorded on representative slides with a Confocal Laser Scanning Microscope FV1000 (Olympus GmbH, Hamburg, Germany) upon excitation with lexc = 488 nm.
Results: Optical measurements of fluorescein in 0.9% sodium chloride (NaCl) under in vitro conditions showed an absorption maximum of lmax abs = 479 nm as detected with spectrophotometer Specord 200 and an emission peak at lmax em = 538 nm recorded with the emCCD detection system of a custom-made microscope-based single particle setup using a 500 nm long-pass filter. Further measurements revealed pH- and concentration-dependent emission spectra of NaFl. Under ex vivo conditions, confocal laser scanning microscopy of fluorescein tumor samples revealed a slight bathochromic shift and a broadening of the emission band.
Conclusion: Tumor uptake of NaFl leads to changes in the optical properties – a bathochromic shift and broadening of the emission band – possibly caused by the dye’s high pH sensitivity and concentration-dependent reabsorption acting as an innerfilter of the dye’s emission, particularly in the short wavelength region of the Emission spectrum where absorption and fluorescence overlap. Understanding the ex vivo optical properties of fluorescein is crucial for testing and validating its further applicability as an optical probe for intravital microscopy, immunofluorescence localization studies, and flow cytometry analysis.
The absorption and emission properties of organic dyes are generally tuned by altering the substitution pattern. However, tuning the fluorescence lifetimes over a range of several 10 ns while barely affecting the spectral features and maintaining a moderate fluorescence quantum yield is challenging. Such properties are required for lifetime multiplexing and barcoding applications. Here, we show how this can be achieved for the class of fluoranthene dyes, which have substitution-dependent lifetimes between 6 and 33 ns for single wavelength excitation and emission. We explore the substitution-dependent emissive properties in the crystalline solid state that would prevent applications.
Furthermore, by analyzing dye mixtures and embedding the dyes in carboxyfunctionalized 8 μm-sized polystyrene particles, the unprecedented potential of these dyes as labels and encoding fluorophores for time-resolved fluorescence detection techniques is demonstrated.
Redox switches are applied in various fields of research, including molecular lifts, electronic devices and sensors. Switching the absorbance between UV and Vis/NIR by redox processes is of interest for applications in light harvesting or biomedicine.
Here, we present a series of push-pull benzothiadiazole derivatives with high fluorescence quantum yields in solution and in the crystalline solid state. Spectroelectrochemical analysis reveals the switching of UV-absorption in the neutral state to Vis/NIR absorption in the reduced state. We identify the partial irreversibility of the switching process, which appears to be reversible on the cyclic voltammetry timescale.
We present here the design and characterization of a set of spectral calibration beads. These calibration beads are intended for the determination and regular control of the spectral characteristics of fluorescence microscopes and other fluorescence measuring devices for the readout of bead-based assays. This set consists of micrometer-sized polymer beads loaded with dyes from the liquid Calibration Kit Spectral Fluorescence Standards developed and certified by BAM for the wavelength-dependent Determination of the spectral responsivity of fluorescencemeasuring devices like spectrofluorometers. To cover the wavelength Region from 400 to 800 nm, two new near-infrared emissive dyes were included, which were spectroscopically characterized in solution and encapsulated in the beads. The resulting set of beads presents the first step towards a new platform of spectral calibration beads for the determination of the spectral characteristics of fluorescence instruments like fluorescence microscopes, FCM setups, and microtiter plate readers, thereby meeting the increasing demand for reliable and comparable fluorescence data especially in strongly regulated areas, e.g., medical diagnostics. This will eventually provide the basis for standardized calibration procedures for imaging systems as an alternative to microchannel slides containing dye solutions previously reported by us.
Tempo-spectral multiplexing in flow cytometry with lifetime detection using QD-encoded polymer beads
(2020)
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.
In all fluorescence-based techniques, the measured signals contain not only sample-related but also instrument-specific contributions, which limit the direct comparison of fluorescence data obtained e.g. on different devices or at different times and often hamper quantification. To rule out instrumentation as major source of variability of emission data, accepted fluorescence standards and procedures for the control of instrument specifications and long-term performance are required. For flow cytometry (FCM), a broad variety of fluorophore-stained polymer beads differing in emission wavelength and intensity is available for the testing of the alignment, sensitivity, and other parameters of FCM. These calibration tools are intended to facilitate the assessment of instrument performance to ensure reliable measurements and to improve the comparability of FCM experiments.
As a step towards an improved comparability of fluorescence data, with special emphasis on spectroscopic methods measuring nano- and micrometer-sized fluorescent objects, we are currently developing a set of fluorescent polystyrene (PS) beads loaded with luminophores from the certified BAM-Kit “Spectral fluorescent standards”, initially developed for the calibration of fluorescence spectrometers. Here, we present first results from studies of these fluorophore-loaded polymer beads. Moreover, new beads are made to supplement this kit by encapsulating near-infrared (NIR)-emissive luminophores in PS beads to cover the UV/VIS, and NIR wavelength range.
These beads are designed for calibration of flow cytometers and other fluorescence imaging systems to meet the increasing demand for reliable and comparable fluorescence data especially in strongly regulated areas like e.g. medical diagnostics.
A new fluorescent pentaphene derivative is presented that differs
from hexabenzocoronene (HBC) by one carbon atom in the basal
plane skeleton. A 500% increased fluorescence quantum yield
is measured compared to the HBC derivative. The pentaphene
compound, obtained by a modified Scholl oxidation, is also emissive
in the solid-state, due to the packing motif in the crystal.
Time-resolved flow cytometry
(2019)
The fast identification of a large number of analytes or events is increasingly required in bioanalytical, diagnostic, and security applications. The versatility and straightforward use make multiparametric fluorescence techniques particularly interesting as detection techniques. An established method for high-throughput single-cell and single-particle measurements is flow cytometry (FCM). Using only spectral encoding without further intensity information, state-of-the-art instruments equipped with several light sources and detectors can resolve almost 20 different color codes. However, this is not sufficient to answer complex research questions, e.g. in cell biology and immunology. In contrast, routine applications demand low-cost and sometimes even portable instruments and thus a minimum number of instrument components. Thus, there are currently two main research directions in FCM: the development of methods that can either address increasingly complex analytical challenges or provide low-cost and robust approaches for routine multiplex analyses. Common spectral multiplexing approaches face limitations in both directions. On the one hand, spectral overlap of labels restricts the number of codes and makes elaborate correction schemes necessary. On the other hand, even for lower degrees of multiplexing often a sophisticated optical setup is needed. An alternative to spectral multiplexing and intensity encoding is to exploit the luminescence lifetime (LT) as an encoding parameter. This can allow for extending the parameter space in combination with spectral encoding or result in more simple and compact devices due to fewer optical components. The availability of fast electronics enables miniaturized and portable lifetime measurement setups at relatively low cost. LT-FCM requires to master LT determination with a limited number of detected photons due to the short interaction time of the encoded objects with the laser spot. In this study, we address this issue for time-domain cytometry and present a novel lifetime flow cytometry (LT-FCM) platform based on a compact setup and straightforward time-domain measurements utilizing LT-encoded luminescent beads. Moreover, we present the realization of a first bioanalytical assay with LT-encoded beads.
There is an increasing interest in bridging the gap between the photoluminescence (PL) properties of nanomaterials like semiconductor nanocrystals (QDs) commonly assessed in ensemble studies and the PL features of single QDs for life sciences applications such as bioimaging studies or use in microfluidic assays. The fluorescence quantum yield (ΦF) is a key performance parameter for all molecular and nanoscale emitters, increasingly employed in nanoscience, nanotechnology, and medical diagnostics.
ΦF determines not only the signal size together with the reporter´s molar extinction coefficient, yet it is particularly relevant for nanocrystals like QDs with coordinatively bound surface ligands and size- and surface chemistry-dependent PL characteristics.
The importance of ΦF measurements at ultralow concentration encouraged us to explore the potential of fluorescence correlation spectroscopy (FCS) for the relative determination of ΦF of ligand-stabilized CdTe nanocrystals in comparison to molecular dyes with closely matching spectral properties and known ΦF.
We describe a FCS-based method for the relative determination of ΦF of dispersed QDs at ultralow concentrations, and procedures to overcome QD-inherent challenges like complex and power-dependent blinking behavior as well as ligand- and QD-specific aggregation. We could demonstrate the potential of this approach by comparison with steady state ensemble measurements.
The fluorescence lifetime is a key property of fluorophores that can be utilized for microenvironment probing, analyte sensing, and multiplexing as well as barcoding applications. For the rational design of lifetime probes and barcodes, theoretical methods have been developed to enable the ab initio prediction of this parameter, which depends strongly on interactions with solvent molecules and other chemical species in the emitters' immediate environment. In this work, we investigate how a conductor-like screening model (COSMO) can account for variations in fluorescence lifetimes that are caused by such fluorophore−solvent interactions. Therefore, we calculate vibrationally broadened fluorescence spectra using the nuclear ensemble method to obtain distorted molecular geometries to sample the electronic transitions with time-dependent density functional theory (TDDFT). The influence of the solvent on fluorescence lifetimes is accounted for with COSMO. For example, for 4-hydroxythiazole fluorophore containing different heteroatoms and acidic and basic moieties in aprotic and protic solvents of varying polarity, this approach was compared to experimentally determined lifetimes in the same solvents. Our results demonstrate a good correlation between theoretically predicted and experimentally measured fluorescence lifetimes except for the polar solvents Ethanol and acetonitrile that can specifically interact with the heteroatoms and the carboxylic acid of the thiazole derivative.