TY - CONF A1 - Schäferling, Michael A1 - Hyppänen, I. A1 - Perälä, N. A1 - Arppe, R. A1 - Soukka, T. T1 - Effects of solvent and excitation power on ratiometric upconversion luminescence based temperature sensing using NaYF4:Yb3+,Er3+ N2 - Thermal sensing using the luminescence intensity ratio of the green Er3+ emissions is affected by the solvent and requires steady-state conditions during the excitation. It is important to keep the excitation power at a moderate level or short exposure times to avoid local heating of aqueous samples. The solvent also determines whether the red emission of Er3+ is excited via a two- or three-photon process. T2 - 2nd conference on properties, design and applications of upconversion nanomaterials CY - Valencia, Spain DA - 04.04.2018 KW - Upconversion luminescence KW - Temperature sensing KW - Excitation path PY - 2018 AN - OPUS4-44819 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Näreoja, T A1 - Deguchi, T A1 - Christ, S. A1 - Peltomaa, R A1 - Prabhakar, N A1 - Fazeli, E A1 - Perälä, N A1 - Rosenholm, J A1 - Arppe, Riika A1 - Soukka, T A1 - Schäferling, Michael T1 - Ratiometric Sensing and Imaging of Intracellular pH Using Polyethylenimine-Coated Photon Upconversion Nanoprobes JF - Analytical Chemistry N2 - Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process, and also acidification of vesicles destined for exocytosis. To monitor these events, the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from the biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low-energy 980 nm laser excitation, which facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake, and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and in in vivo applications, e.g., for the bioimaging of small animal models. KW - PH sensing KW - Upconversion KW - Nanoparticles KW - Fluorescecne imaging PY - 2017 UR - http://pubs.acs.org/doi/abs/10.1021/acs.analchem.6b03223 DO - https://doi.org/10.1021/acs.analchem.6b03223 SN - 0003-2700 SN - 1520-6882 VL - 89 IS - 3 SP - 1501 EP - 1508 PB - American Chemical Society CY - Washington AN - OPUS4-39083 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schäferling, Michael A1 - Näreoja, T. A1 - Deguchi, T. A1 - Fazeli, E. A1 - Peltomaa, R. A1 - Arppe, Riika A1 - Soukka, T. T1 - Sensing and Imaging of Intracellular pH Using Photon Upconversion Based Nanoprobes N2 - Sensing of intracellular pH is of particular interest in biomedical research since structure and function of biomolecules strongly depend on the concentration of protons in their environment. We have functionalized photon upconversion nano-particles (UCNPs) with pH responsive dyes to achieve nanoprobes for intracellular pH determination. The sensing mechanism is based on a resonance energy transfer (UC-RET) from the 550 nm emission of hexagonal nanocrystals of NaYF4: Yb3+,Er3+ to the pH-sensitive fluorophore pHrodoTM Red. The nanocrystals were coated with thin shells of aminosilane or highly branched polyethylenimine (PEI) with several nanometer layer thickness for coupling of the pH indicator. The nanoprobes are calibrated by ratiometric dual wavelength readout at 550 nm (reference signal) and 590 nm (sensor signal) and visualized using a scanning confocal fluorescence microscope with 980 nm excitation wavelength. It was found that PEI coating enables a higher coupling of indicator molecules on the particle surface, better signal to reference ratios in ratiometric readout and an improved cellular uptake compared to the aminosilane coated particles due to a more positive zeta potential. We studied the cellular uptake efficacy of the nanoprobes and determined to which type of compartment, lysosomes, endosomes or cytosol, the probes are targeted to by measuring the pH of their microenvironment. An in situ control was performed in live cells by a treatment with nigericin, whereby the pH of all intracellular compartments is set at extracellular level. Finally, we will show new strategies for the preparation of UCNP-dye conjugates with improved UC-RET efficiency to achieve higher acceptor (sensor) emission. T2 - 1st European / 10th German Biosensor Symposium CY - Potsdam, Germany DA - 20.03.2017 KW - Photon Upconversion KW - Nanoprobes KW - Chemical Sensing KW - PH KW - Live cell imaging PY - 2017 AN - OPUS4-40032 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hyppänen, I. A1 - Perälä, N. A1 - Arppe, Riika A1 - Schäferling, Michael A1 - Soukka, T. T1 - Environmental and excitation power effects on the ratiometric upconversion luminescence based temperature sensing using nanocrystalline NaYF4:Yb3+,Er3+ JF - ChemPhysChem N2 - The luminescence intensity ratio (LIR) of the green emissions of the near-infrared excited NaYF4:Yb3+,Er3+ nanocrystals is a promising method for temperature sensing. Here, the influence of excitation power density, excitation pulse length, excitation wavelength, silica shell, and solvent on the LIR and its temperature response is reported. The primary objective is to study the LIR mechanism and the impact of measurement and environmental parameters on the calibration and precision of the LIR. The LIR value is demonstrated to be unaffected by the excitation intensity in the studied range. This result is essential, considering the application feasibility of the LIR method as temperature sensor, where the effective excitation power density depends on the sample matrix and the distance excitation light travels in the sample. The pulsed excitation, however, results in an increase in the LIR value upon short pulse width. Silanization of bare nanocrystals has no effect on the LIR values, but the local warming of H2O samples under laser exposure results in slightly increased LIR values compared to other solvents; D2O, oleic acid, and dimethyl sulfoxide. The thermal quenching of luminescence lifetimes of Er3+ emission is proved to be too weak for sensing applications. KW - Photon upconversion KW - Temperature sensing KW - Luminescence intensity ratio KW - Excitation intensity PY - 2017 UR - http://onlinelibrary.wiley.com/doi/10.1002/cphc.201601355/epdf DO - https://doi.org/10.1002/cphc.201601355 SN - 1439-4235 SN - 1439-7641 VL - 18 IS - 6 SP - 692 EP - 701 PB - Wiley-VCH CY - Weinheim AN - OPUS4-39899 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hyppänen, Iko A1 - Höysniemi, Niina A1 - Arppe, Riika A1 - Schäferling, Michael A1 - Soukka, Tero T1 - Environmental impact on the excitation path of the red upconversion emission of nanocrystalline NaYF4:Yb3+,Er3+ JF - Journal of Physical Chemistry C N2 - The mechanism for red upconversion luminescence of Yb−Er codoped materials is not generally agreed on in the literature. Both two-photon and three-photon processes have been suggested as the main path for red upconversion emission. We have studied β-NaYF4:Yb3+,Er3+ nanoparticles in H2O and D2O, and we propose that the nanoparticle environment is a major factor in the selection of the preferred red upconversion excitation pathway. In H2O, efficient multiphonon relaxation (MPR) promotes the two-photon mechanism through green emitting states, while, in D2O, MPR is less effective and the three-photon path involving back energy transfer to Yb3+ is the dominant mechanism. For the green upconversion emission, our results suggest the common two-photon path through the 4F9/2 energy state in both H2O and D2O. KW - Photon upconversion KW - Three photon excitation KW - Multiphonon relaxation KW - Excitation intensity PY - 2017 UR - http://pubs.acs.org/doi/pdf/10.1021/acs.jpcc.7b01019 DO - https://doi.org/10.1021/acs.jpcc.7b01019 SN - 1932-7447 SN - 1932-7455 VL - 121 IS - 12 SP - 6924 EP - 6929 PB - ACS Publications AN - OPUS4-39900 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schäferling, Michael A1 - Deguchi, T. A1 - Christ, S. A1 - Peltomaa, R. A1 - Prabhakar, N. A1 - Rosenholm, J. A1 - Arppe, Riika A1 - Soukka, T. A1 - Näreoja, T. T1 - Nanoprobes for sensing and imaging of intracellular pH based on polyethyleneimide-coated photon upconversion nanoparticles conjugated to a pH sensitive rhodamine dye N2 - Lanthanide-doped photon upconversion nanoparticles (UCNPs) exhibit many advantages compared to Stokes-shifted luminescent probes (organic dyes, quantum dots). Due to the upconversion process, the limitations of photobleaching, autofluorescence and low penetration depths in tissue shown by classical fluorescent probes are avoided. This makes UCNPs particularly useful for applications in complex biological samples. Sensing of intracellular pH is of particular interest in biomedical research since structure and function of biomolecules strongly depend on the concentration of protons in their environment. T2 - UPCON 2016 CY - Wroclaw, Poland DA - 23.05.2016 KW - upconversion KW - nanoprobes KW - chemical sensing PY - 2016 AN - OPUS4-37282 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schäferling, Michael A1 - Deguchi, T. A1 - Christ, S. A1 - Peltomaa, R. A1 - Prabhakar, N. A1 - Rosenholm, J. A1 - Arppe, Riika A1 - Soukka, T. A1 - Näreoja, T. T1 - Sensing and Imaging of Intracellular pH Using Polyethyleneimine-coated Photon Upconversion Based Nanoprobes N2 - Lanthanide-doped photon upconversion nanoparticles (UCNPs) exhibit many advantages compared to conventional Stokes-shifted luminescent probes such as organic dyes and quantum dots. Due to the upconversion (UC) process, which describes the conversion of NIR light into shorter wavelength radiation, the limitations of photobleaching, autofluorescence and low penetration depths in tissue shown by classical fluorescent probes absorbing in the UV/vis range are avoided. This makes UCNPs particularly useful for applications in complex samples occurring in bioanalysis, biomedicine and imaging. Sensing of intracellular pH is of particular interest in biomedical research since structure and function of biomolecules strongly depend on the concentration of protons in their environment. We described previously a UCNP nanosensor for pH based on a resonance energy transfer from hexagonal nanocrystals of NaYF4: Yb3+,Er3+ to a pH-sensitive fluorophore (pHrodoTM Red).[1] The nanocrystals were coated with a thin shell of aminosilane with several nanometer layer thickness for coupling of the pH indicator. In this contribution we present a new generation of UC nanoprobes that are coated with a layer of highly branched polyethylenimine (PEI). The PEI coating enables a higher coupling of indicator molecules on the particle surface, better signal to reference ratios in ratiometric readout and an improved cellular uptake compared to the aminosilane coated particles due to a more positive zeta potential. Again, pHrodoTM Red is used as pH indicator, sensitized by the 550 nm emission of the UCNPs. The nanoprobes are calibrated by ratiometric dual wavelength readout at 550 nm (reference signal) and 590 nm (sensor signal) and visualized using a scanning confocal fluorescence microscope with 980 nm excitation wavelength. We studied the cellular uptake efficacy of the nanoprobes and determined to which type of compartment, lysosomes, endosomes or cytosol, the probes are targeted to by measuring the pH of their microenvironment. An in situ control was performed in live cells by a treatment with nigericin, whereby the pH of all intracellular compartments is set at extracellular level. Our results suggest that the PEI coating facilitated endosomal escape of the nanoprobes. T2 - Europtrode XIII CY - Graz, Austria DA - 20.03.2016 KW - upconversion KW - chemical sensing KW - nanoprobes PY - 2016 AN - OPUS4-37283 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schäferling, Michael T1 - Nanoparticle-based luminescent probes for intracellular sensing and imaging of pH JF - WIREs Nanomedicine and Nanobiotechnology N2 - Fluorescence imaging microscopy is an essential tool in biomedical research. Meanwhile, various fluorescent probes are available for the staining of cells, cell membranes, and organelles. Though, to monitor intracellular processes and dysfunctions, probes that respond to ubiquitous chemical parameters determining the cellular function such as pH, pO2, and Ca2+ are required. This review is focused on the progress in the design, fabrication, and application of photoluminescent nanoprobes for sensing and imaging of pH in living cells. The advantages of using nanoprobes carrying fluorescent pHindicators compared to single molecule probes are discussed as well as their limitations due to the mostly lysosomal uptake by cells. Particular attention is paid to ratiometric dual wavelength nanosensors that enable intrinsic referenced measurements. Referencing and proper calibration procedures are basic prerequisites to carry out reliable quantitative pH determinations in complex samples such as living cells. A variety of examples will be presented that highlight the diverseness of nanocarrier materials (polymers, micelles, silica, quantum dots, carbon dots, gold, photon upconversion nanocrystals, or bacteriophages), fluorescent pH indicators for the weak acidic range, and referenced sensing mechanisms, that have been applied intracellularly up to now. KW - pH sensing KW - Luminescent probes KW - Imaging PY - 2016 DO - https://doi.org/10.1002/wnan.1366 SN - 1939-0041 SN - 1939-5116 VL - 8 IS - 3 SP - 378 EP - 413 PB - Wiley-Blackwell CY - Hoboken, New Jersey, USA AN - OPUS4-36367 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CHAP A1 - Schäferling, Michael ED - Meyers, R.A. T1 - Fluorescence-based biosensors T2 - Encyclopedia of Analytical Chemistry N2 - Biosensors, as defined by Pure and Applied Chemistry, are ‘chemical sensors in which the recognition System utilizes a biochemical mechanism. The biological recognition system translates information from the biochemical domain, usually an analyte concentration, into a chemical or physical output signal with a defined sensitivity’.(1) It is also appointed that chemical or biological sensors contain two basic components connected in series: a chemical or biomolecular recognition System (receptor) and a physicochemical transducer. According to this prerequisite, this overlook is confined to sensor devices that combine a biomolecular recognition element with an optical signal transducer. Homogeneous or intracellular assays using fluorescent molecular probes or nanoparticles are not considered, although they are frequently termed as molecular sensors or nanosensors in the literature. Fluorescence-based biosensors are generalized as those devices that derive an analytical signal from a photoluminescent (either fluorescence or phosphorescence) emission process. Chemi- or bioluminescent detection systems are only briefly discussed in this review. Biosensors are used for a wide variety of tasks, including detection of compounds of biomedical, environmental or defense interest; on-line monitoring for process control; quality control of foodstuffs; selective detection of compounds undergoing a chemical separation; and screening of drug compounds. Advantages of such devices include high selectivity, rapid response times, reusability, amenability to remote analysis, and immunity to electrical interferences. The selective nature of complexation between biomolecule and analyte and the small size of sensor devices can be combined with advanced detection techiques such as total internal reflection (TIR) spectroscopy. This results in an ability to measure analytes in complex matrices with unsurpassed sensitivity. Such samples may include highly scattering components such as milk or whole blood,(11) or relatively inaccessible locations such as groundwater wells, or even intracellular environments. The key limitation of such devices mainly centers on the poor stability of biological compounds, which can lead to a substantial drift in instrumental response over time. The so-called Cambridge Definition appoints another characteristic property of sensors. Therein, they are defined as ‘miniaturized devices which can deliver real-time and on-line information on the presence of specific compounds or ions in even complex samples’. Accordingly, a sensor is expected to respond reversibly and continuously. With the exception of some enzymatic sensors, these conditions are not fulfilled in case of most biosensors. Particularly, in devices where immunological reagents or DNA are used as recognition elements, they show a lack of reversibility and operate only as a ‘one-shot’ screen, without the potential for continuous, quantitative analysis. Nevertheless, the designations immunosensors or DNA sensors became accepted for such analytical or diagnostic tools. KW - Biosensor KW - Fluorescence KW - Immunoassay KW - Enzyme sensor KW - DNA sensor PY - 2016 UR - http://onlinelibrary.wiley.com/doi/10.1002/9780470027318.a0206.pub3/abstract SN - 9780470027318 DO - https://doi.org/10.1002/9780470027318.a0206.pub3 SP - 1 EP - 51 PB - John Wiley & Sons AN - OPUS4-38741 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Wang, X.-d. A1 - Meier, R.J. A1 - Schäferling, Michael A1 - Bange, S. A1 - Lupton, J.M. A1 - Sperber, M. A1 - Wegener, J. A1 - Ondrus, V. A1 - Beifuss, U. A1 - Henne, U. A1 - Klein, C. A1 - Wolfbeis, Otto S. T1 - Two-photon excitation temperature nanosensors based on a conjugated fluorescent polymer doped with an Europium probe JF - Advanced Optical Materials N2 - A strongly fluorescent organic semiconducting polymer doped with a highly temperature dependent fluorescent europium(III) complex is converted into a nanosized material that is capable of optically sensing temperature (T) in the range from 0 to 50 °C via two-photon excitation at 720 nm. The nanosensors are prepared from a blue-fluorescent polyfluorene that acts as both a lightharvesting antenna (to capture two-photon energy) and an energy donor in a fluorescence resonance energy transfer (FRET) system. The photonic energy absorbed by the polymer is transferred to the T-sensitive red-luminescent europium complex contained in the nanoparticles. The close spatial proximity of the donor and the acceptor warrants efficient FRET. A poly(ethylene glycol)- co-poly(propylene oxide) block copolymer is also added to render the particles biocompatible. It is shown that T can be calculated from a) the intensity of the luminescence of the europium complex, b) the ratio of the intensities of the red and blue luminescence, or c) the T-dependent luminescence lifetime of the Eu(III) complex. KW - Optical sensor KW - Temperature sensor KW - Nanosensor KW - FRET system PY - 2016 UR - http://onlinelibrary.wiley.com/doi/10.1002/adom.201600601/abstract DO - https://doi.org/10.1002/adom.201600601 SN - 2195-1071 VL - 4 IS - 11 SP - 1854 EP - 1859 PB - Wiley-VCH CY - Weinheim AN - OPUS4-38737 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -