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pH and oxygen are amongst the most important and frequently measured analytes in the life and material sciences, indicating, e.g., diseases and corrosion processes. This includes the optical monitoring of pH in living cells for studying cellular internalization pathways, such as phagocytosis, endocytosis, and receptor ligand internalization with the aid of molecular and nanoscale fluorescent sensors. Nanoparticle (NP)-based sensors, that are labeled or stained with a multitude of sensor dyes, have several advantages as compare to conventional molecular probes like enhanced brightness, i.e., amplified signals, ease of designing ratiometric systems by combining analyte sensitive and inert reference dyes, and increased photostability. Moreover, this can enable the use of hydrophobic dyes in aqueous environments. Versatile templates and carriers for the fabrication of nanosensors by the staining and/or labelling with different fluorophores and sensor molecules or surface functionalized NP like silica (SiO2-NP) and polystyrene (PS-NP) particles provide. Here we present the design of a versatile platform of color emissive nanosensors and stimuli-responsive microparticles for the measurement of pH, oxygen, and other targets utilizing both types of matrices and sets of spectrally distinguishable sensor and reference dyes and their characterization and demonstrate the applicability of representative sensor particle for cellular studies.
Dye-biomolecule conjugates and NIR-fluorescent particles for targeting of disease-related biomarkers
(2011)
Indispensable for fluorescence imaging are highly specific and sensitive molecular probes that absorb and emit in the near infrared (NIR) spectral region and respond to or target molecular species or processes. Here, we present approaches to targeted fluorescent probes for in vivo imaging in the intensity and lifetime domain exploiting NIR dyes. Screening schemes for the fast identification of suitable fluorophores are derived and design criteria for highly emissive optical probes. In addition, as a signal amplification strategy that enables also the use of hydrophobic NIR fluorophores as fluorescent reporters, first steps towards versatile strategies for the preparation of NIR-fluorescent polymeric particles are presented that can be utilized also for the design of targeted and analyte-responsive probes.
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.
Polystyrene nanoparticles (PS-NPs) were doped with an oxygen-sensitive near-infrared (NIR)-emissive palladium meso-tetraphenylporphyrin and an inert reference dye which are both excitable at 635 nm. The nanosensors were characterized with special emphasis on fundamental parameters such as absolute photoluminescence quantum yield and fluorescence lifetime. The PS-NPs were employed for ratiometric dual-wavelength and lifetime-based photoluminescent oxygen sensing. They were efficiently taken up by cultured murine alveolar macrophages, yielding a characteristic and reversible change in ratiometric response with decreasing oxygen concentration. This correlated with the cellular hypoxic status verified by analysis of hypoxia inducible factor-1α (HIF-1α) accumulation. In addition, the surface of PS-NPs was functionalized with polyethylene glycol (PEG) and the monoclonal antibody herceptin, and their binding to HER2/neu-overexpressing tumor cells was confirmed in vitro. First experiments with tumor-bearing mouse revealed a distinctive ratiometric response within the tumor upon hypoxic condition induced by animal sacrifice. These results demonstrate the potential of these referenced NIR nanosensors for in vitro and in vivo imaging that present a new generation of optical probes for oncology.
Water-Soluble Aza-BODIPYs: Biocompatible Organic Dyes for High Contrast In Vivo NIR-II Imaging
(2020)
A simple NIR-II emitting water-soluble system has been developed and
applied in vitro and in vivo. In vitro, the fluorophore quickly accumulated in 2D and 3D cell cultures and rapidly reached the tumor in rodents, showing high NIR-II contrast for up to 1 week. This very efficient probe possesses all the qualities necessary for translation to the clinic as well as for the development of NIR-II emitting materials.