Michael Schäferling

The Art of Fluorescence Imaging with Chemical Sensors

Fluorescence imaging techniques involving chemical sensors are essential tools in many fields of science and technology because they enable the visualization of parameters which exhibit no intrinsic color or fluorescence, for example, oxygen, pH value, CO(2), H(2)O(2), Ca(2+), or temperature, to name just a few. This Review aims to highlight the state of the art of fluorescence sensing and imaging, starting from a comprehensive overview of the basic functional principles of fluorescent probes (or indicators) and the design of sensor materials. The focus is directed towards the progress made in the development of multiple sensors and methods for their signal read out. Imaging methods involving optical sensors are applied in quite diverse scientific areas, such as medical research, aerodynamics, and marine research.

Dual Fluorescence Sensor for Trace Oxygen and Temperature with Unmatched Range and Sensitivity

An optical dual sensor for oxygen and temperature is presented that is highly oxygen sensitive and covers a broad temperature range. Dual sensing is based on luminescence lifetime measurements. The novel sensor contains two luminescent compounds incorporated into polymer films. The temperature-sensitive dye (ruthenium tris-1,10-phenanthroline) has a highly temperature-dependent luminescence and is incorporated in poly(acrylonitrile) to avoid cross-sensitivity to oxygen. Fullerene C70 was used as the oxygen-sensitive probe owing to its strong thermally activated delayed fluorescence at elevated temperatures that is extremely oxygen sensitive. The cross-sensitivity of C70 to temperature is accounted for by means of the temperature sensor. C70 is incorporated into a highly oxygen-permeable polymer, either ethyl cellulose or organosilica. The two luminescent probes have different emission spectra and decay times, and their emissions can be discriminated using both parameters. Spatially resolved sensing is achieved by means of fluorescence lifetime imaging. The response times of the sensor to oxygen are short. The dual sensor exhibits a temperature operation range between at least 0 and 120 degrees C, and detection limits for oxygen in the ppbv range, operating for oxygen concentrations up to at least 50 ppmv. These ranges outperform all dual oxygen and temperature sensors reported so far. The dual sensor presented in this study is especially appropriate for measurements under extreme conditions such as high temperatures and ultralow oxygen levels. This dual sensor is a key step forward in a number of scientifically or commercially important applications including food packaging, for monitoring of hyperthermophilic microorganisms, in space technology, and safety and security applications in terms of detection of oxygen leaks.

Targeted luminescent near-infrared polymer-nanoprobes for in vivo imaging of tumor hypoxia.

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.

Red- and Green-Emitting Iridium(III) Complexes for a Dual Barometric and Temperature-Sensitive Paint

A new dual luminescent sensitive paint for barometric pressure and temperature (T) is presented. The green-emitting iridium(III) complex [Ir(ppy)(2)(carbac)] (ppy=2-phenylpyridine; carbac=1-(9H-carbazol-9-yl)-5,5-dimethylhexane-2,4-dione) was applied as a novel probe for T along with the red-emitting complex [Ir(btpy)(3)], (btpy=2-(benzo[b]thiophene-2-yl)pyridine) which functions as a barometric (in fact oxygen-sensitive) probe. Both iridium complexes were dissolved in different polymer materials to achieve optimal responses. The probe [Ir(ppy)(2)(carbac)] was dispersed in gas-blocking poly(acrylonitrile) microparticles in order to suppress any quenching of its luminescence by oxygen. The barometric probe [Ir(btpy)(3)], in turn, was incorporated in a cellulose acetate butyrate film which exhibits good permeability for oxygen. The effects of temperature on the response of the oxygen probe can be corrected by simultaneous optical determination of T, as the poly(acrylonitrile) microparticles containing the temperature indicator are incorporated into the film. The phosphorescent signals of the probes for T and barometric pressure, respectively, can be separated by optical filters due to the approximately 75 nm difference in their emission maxima. The dual sensor is applicable to luminescence lifetime imaging of T and barometric pressure. It is the first luminescent dual sensor material for barometric pressure/T based exclusively on the use of Ir(III) complexes in combination with luminescence lifetime imaging.

Fluorescence Quenching of the Europium Tetracycline Hydrogen Peroxide Complex by Copper (II) and Other Metal Ions

The europium–tetracycline complex [Eu(Tc)] is known to show only weak fluorescence with an emission maximum at 615 nm. On addition of hydrogen peroxide (HP), the strongly fluorescent [Eu(Tc)(HP)] complex is formed, which displays a 15-fold stronger luminescence intensity. This study describes the decrease in luminescence intensity of the [Eu(Tc)(HP)] complex in aqueous solution in the presence of Cu2+, Fe3+, Ag+, Al3+, Zn2+, Co2+, Ni2+, Mn2+, Ca2+, and Mg2+. Static and dynamic quenching can be induced by Cu2+, and these processes were quantified by means of their quenching constants. Stern–Volmer plots were also derived from lifetime imaging measurements accomplished by the rapid lifetime determination (RLD) technique based on microwell plate assays, and also by the time-correlated single photon counting (TCSPC) technique. According to those data, a time-resolved fluorescent method for copper determination can be proposed that is based on dynamic quenching of the [Eu(Tc)(HP)] complex by Cu2+ ions. The response to copper concentrations is linear up to 1.6 μmol L−1, providing a detection limit of 0.2 μmol L−1.

Method for simultaneous luminescence sensing of two species using optical probes of different decay time, and its application to an enzymatic reaction at varying temperature

AbstractChemical sensing, imaging and microscopy based on the use of fluorescent probes has so far been limited almost exclusively to the detection of a single parameter at a time. We present a scheme that can overcome this limitation by enabling optical sensing of two parameter simultaneously and even at identical excitation and emission wavelengths of two probes provided (a) their decay times are different enough to enable two time windows to be recorded, and (b) the emission of the shorter-lived probe decays to below the detectable limit while that of the other still can be measured. We refer to this new scheme as the dual lifetime determination (DLD) method and show that it can be widely varied by appropriate choice of probes and experimental settings. DLD is demonstrated to work by sensing oxygen and temperature independently from each other by making use of two probes, one for oxygen (a platinum porphyrin dissolved in polystyrene), and one for temperature [a europium complex dissolved in poly(vinyl methylketone)]. DLD was applied to monitor the consumption of oxygen in the glucose oxidase-catalyzed oxidation of glucose at varying temperatures. The scheme is expected to have further applications in cellular assays and biophysical imaging. FigurePrinciple behind the dual lifetime determination (DLD) method

Time-Resolved Luminescence Imaging of Hydrogen Peroxide Using Sensor Membranes in a Microwell Format:

We demonstrate an optical imaging scheme for hydrogen peroxide in a microwell-based format using the europium(III) tetracycline complex as the fluorescent probe, which is incorporated into a polyacrylonitrile-co-polyacrylamide polymer matrix. The resulting sensor membranes are integrated into a 96-microwell plate. Hydrogen peroxide can be visualized by means of time-resolved luminescence lifetime imaging. The imaging system consists of a fast, gated charge-coupled device (CCD) camera and a pulsed array of 96 light emitting diodes (LEDs). Fluorescence lifetime images are acquired in different modes (rapid lifetime determination, RLD, and phase delay rationing, PDR) and compared with conventional intensity-based methods with respect to sensitivity and the dynamic range of the sensor. The lowest limits of detection can be achieved by the RLD method. The response time of the sensor is comparatively high, typically in the range of 10 to 20 minutes, but the response is reversible. The largest signal changes are observed at pH values between 6.5 and 7.5.

Porphyrin-functionalized oligo- and polythiophenes

The synthesis and properties of a series of bi- and terthiophenes 3–8 substituted with meso-tetraphenylporphyrin (TPP) groups via an isolating oxaalkyl chain is described. Electrooxidative polymerization leads to the corresponding metal complexed porphyrin-functionalized polythiophenes P4–P8. The electrochemical and spectroscopic properties of the polymer films reveal the superimposition of the electronic properties of the individual π-systems. Spectroelectrochemical experiments and conductivity measurements point to a mixed charge transport mechanism. Polaronic and bipolaronic delocalization on the conjugated chains combined with electron hopping processes via the porphyrin redox centers result in high stability of the polymers against overoxidation. Importantly, hybrid materials have been obtained which at the same time exhibit properties of a conducting polymer and a redox polymer that can be used for the detection of polychlorinated phenols in amperometric sensors.

Background-free referenced luminescence sensing and imaging of pH using upconverting phosphors and color camera read-out.

Fluorescence background and problems with proper signal referencing severely disrupt the read-out of luminescence sensors and images. We present a pH sensor film in combination with a simple read-out technique that overcomes issues of background signals and autofluorescence. It consists of micrometer-sized upconversion phosphors (UCPs) and a pH indicator (Neutral Red) that absorbs their green emission. Both are embedded in a proton permeable hydrogel matrix. The UCPs generate green and red luminescence upon excitation with IR light of 980 nm wavelength. Solely the green light of the UCPs is affected by the pH indicator, while the red emission acts as inert reference signal for ratiometric measurements. The emission peaks of the UCPs match the red and green color channels of standard digital cameras. Thereby, the devised sensor film can be used for referenced ratiometric sensing and 2D imaging of pH using a color camera read-out. The sensor setup using common and hand-held devices is cheap and straightforward and allows for point-of-care measurements. Finally, pH measurements in human serum samples show the potential of this sensor for imaging free of interfering background signals.

Referenced dual pressure- and temperature-sensitive paint for digital color camera read out.

The first fluorescent material for the referenced simultaneous RGB (red green blue) imaging of barometric pressure (oxygen partial pressure) and temperature is presented. This sensitive coating consists of two platinum(II) complexes as indicators and a reference dye, each of which is incorporated in appropriate polymer nanoparticles. These particles are dispersed in a polyurethane hydrogel and spread onto a solid support. The emission of the (oxygen) pressure indicator, PtTFPP, matches the red channel of a RGB color camera, whilst the emission of the temperature indicator [Pt(II) (Br-thq)(acac)] matches the green channel. The reference dye, 9,10-diphenylanthracene, emits in the blue channel. In contrast to other dual-sensitive materials, this new coating allows for the simultaneous imaging of both indicator signals, as well as the reference signal, in one RGB color picture without having to separate the signals with additional optical filters. All of these dyes are excitable with a 405 nm light-emitting diode (LED). With this new composite material, barometric pressure can be determined with a resolution of 22 mbar; the temperature can be determined with a resolution of 4.3 °C.