Time-domain Dual Lifetime Referencing Research Articles

A Transcutaneous Carbon Dioxide Monitor Based on Time-Domain Dual Lifetime Referencing.

The partial pressure of arterial carbon dioxide plays a critical role in assessing the acid-base and respiratory status of the human body. Typically, this measurement is invasive and can only be taken momentarily when an arterial blood sample is drawn. Transcutaneous monitoring is a noninvasive surrogate method that provides a continuous measure of arterial carbon dioxide. Unfortunately, current technology is limited to bedside instruments mainly used in intensive care units. We developed a first-of-its-kind miniaturized transcutaneous carbon dioxide monitor that utilizes a luminescence sensing film and a time-domain dual lifetime referencing method. Gas cell experiments confirmed the monitor's ability to accurately identify changes in the partial pressure of carbon dioxide within the clinically significant range. Compared to the luminescence intensity-based technique, the time-domain dual lifetime referencing method is less prone to measurement errors caused by changes in excitation strength, reducing the maximum error from ∼40% to ∼3% and resulting in more reliable readings. Additionally, we analyzed the sensing film by investigating its behavior under various confounding factors and its susceptibility to measurement drift. Finally, a human subject test demonstrated the effectiveness of the applied method in detecting even slight changes in transcutaneous carbon dioxide, as small as ∼0.7%, during hyperventilation. The prototype, which consumes 30.1 mW of power, is a wearable wristband with compact dimensions of 37 mm× 32 mm.

Extracellular pH Monitoring of Live Single Cells in Microdroplets Using Dual-Labelled Fluorinated Silica Nanoparticles and Time-Domain Dual Lifetime Referencing

Fluorinated silica nanoparticles doped with Ruthenium-tris-1,10-phenanthroline dichloride on the inside and covalently conjugated with perfluorooctyltriethoxysilane and fluorescein isothiocyanate on the outside were developed and served several functions; the fluorination of the particles served to stabilize droplets in a microfluidic system at their interface to the continuous phase for single-cell experiments, and the two dyes provided for intrinsically referenced pH readout according to the time-domain dual lifetime referencing scheme. Apart from eliminating the droplet-to-droplet transport, these nanoparticles at the interface of the droplets generated rigid substrates that were suitable for the proliferation of adherent cells in the droplets without additional matrices. Cancer and non-cancer cell lines with culture media were allowed to proliferate in the droplets and the extracellular pH was monitored. These nanoparticles used in a microdroplet system could measure the pH of the extracellular microenvironment of single cells and provide support for the growth of cells in droplets of around 50 µm diameter. The pHe showed 6.84 ± 0.04 and 6.81 ± 0.04 for cancer cells (MCF-7 and A549, respectively) and 7.36 ± 0.03 for healthy cells (HUVEC), after a 10-h incubation, which can be potentially applied in distinguishing tumor from non-tumor cells. Capable of assisting cell culture and pH sensing in droplet microfluidic systems, the dye-conjugated fluorinated nanoparticles described in this work offer possibilities in a variety of biochemical or environmental analytical applications.

Long-Term Quantitatively Imaging Intracellular Chloride Concentration Using a Core-/Shell-Structured Nanosensor and Time-Domain Dual-Lifetime Referencing Method.

Luminescence lifetime-based nanosensors for chloride ions were designed by incorporating a luminescent ruthenium dye [Ru(1,10-phenanthroline)3] inside silica nanoparticles and chemically labelling their outer surface with chloride ion-sensitive fluorescent dyes (N,N'-bis(carboxypropyl)-9,9'-biacridine). The nanosensor surface was further functionalized with positively charged amino groups to facilitate intracellular uptake via endocytosis and target lysosomes. The nanosensors have an average diameter of 52 nm and are monodispersed in aqueous solutions. Because of the long lifetime of the reference ruthenium dye, the sensor response can be analyzed using the time-domain dual-lifetime referencing (td-DLR) approach. The use of pulsed excitation in td-DLR rather than intense continuous illumination in ratiometric measurements greatly prevents the dye from photobleaching which significantly improves its measurement stability and reproducibility for long-term monitoring. At optimum conditions, the sensor can measure chloride concentration in the range of 0-200 mM with a large ratiometric signal change from 140.9 to 40.2. Combined with our custom-built microscopic td-DLR system, variations of intracellular chloride concentration in lysosomes were imaged quantitatively with a high spatial resolution and accuracy.

Luminescent dual sensors reveal extracellular pH-gradients and hypoxia on chronic wounds that disrupt epidermal repair

Wound repair is a quiescent mechanism to restore barriers in multicellular organisms upon injury. In chronic wounds, however, this program prematurely stalls. It is known that patterns of extracellular signals within the wound fluid are crucial to healing. Extracellular pH (pHe) is precisely regulated and potentially important in signaling within wounds due to its diverse cellular effects. Additionally, sufficient oxygenation is a prerequisite for cell proliferation and protein synthesis during tissue repair. It was, however, impossible to study these parameters in vivo due to the lack of imaging tools. Here, we present luminescent biocompatible sensor foils for dual imaging of pHe and oxygenation in vivo. To visualize pHe and oxygen, we used time-domain dual lifetime referencing (tdDLR) and luminescence lifetime imaging (LLI), respectively. With these dual sensors, we discovered centripetally increasing pHe-gradients on human chronic wound surfaces. In a therapeutic approach, we identify pHe-gradients as pivotal governors of cell proliferation and migration, and show that these pHe-gradients disrupt epidermal barrier repair, thus wound closure. Parallel oxygen imaging also revealed marked hypoxia, albeit with no correlating oxygen partial pressure (pO2)-gradient. This highlights the distinct role of pHe-gradients in perturbed healing. We also found that pHe-gradients on chronic wounds of humans are predominantly generated via centrifugally increasing pHe-regulatory Na+/H+-exchanger-1 (NHE1)-expression. We show that the modification of pHe on chronic wound surfaces poses a promising strategy to improve healing. The study has broad implications for cell science where spatial pHe-variations play key roles, e.g. in tumor growth. Furthermore, the novel dual sensors presented herein can be used to visualize pHe and oxygenation in various biomedical fields.

Application of Optical pH Sensors in the Microfluidic Free-flow Isoelectric Focusing of Biomolecules

We present methods for fabrication and application of microfluidic chips for free-flow electrophoresis (FFE) that allow integrated pH sensing. Monitoring of the pH gradient in microfluidic free-flow isoelectric focusing (FFIEF) allows on-line adjustment of the parameters for electrophoresis and evaluation of the isoelectric point (pI) of focused analyte bands almost in real time. The fabrication of these assemblies is demonstrated via multistep photopolymerization or a laser cutting and lamination technique. Integration of pH sensing layers is accomplished via photopolymerization or inkjet printing of hydrogels that contain covalently attached pH probes. These microchips were characterized and employed for the FFIEF of proteins, peptides and plasma. Multispectral microscopic imaging was used to separate the pH sensor and the biomolecule fluorescence. We also show pH sensing in FFE based on time-domain dual lifetime referencing (t-DLR), a microreactor for integrated labelling prior to the FFIEF and the isoelectric focusing of unlabeled compounds.

Luminescent Dual Sensors Reveal Extracellular pH-Gradients and Hypoxia on Chronic Wounds That Disrupt Epidermal Repair

Wound repair is a quiescent mechanism to restore barriers in multicellular organisms upon injury. In chronic wounds, however, this program prematurely stalls. It is known that patterns of extracellular signals within the wound fluid are crucial to healing. Extracellular pH (pHe) is precisely regulated and potentially important in signaling within wounds due to its diverse cellular effects. Additionally, sufficient oxygenation is a prerequisite for cell proliferation and protein synthesis during tissue repair. It was, however, impossible to study these parameters in vivo due to the lack of imaging tools. Here, we present luminescent biocompatible sensor foils for dual imaging of pHe and oxygenation in vivo. To visualize pHe and oxygen, we used time-domain dual lifetime referencing (tdDLR) and luminescence lifetime imaging (LLI), respectively. With these dual sensors, we discovered centripetally increasing pHe-gradients on human chronic wound surfaces. In a therapeutic approach, we identify pHe-gradients as pivotal governors of cell proliferation and migration, and show that these pHe-gradients disrupt epidermal barrier repair, thus wound closure. Parallel oxygen imaging also revealed marked hypoxia, albeit with no correlating oxygen partial pressure (pO2)-gradient. This highlights the distinct role of pHe-gradients in perturbed healing. We also found that pHe-gradients on chronic wounds of humans are predominantly generated via centrifugally increasing pHe-regulatory Na+/H+-exchanger-1 (NHE1)-expression. We show that the modification of pHe on chronic wound surfaces poses a promising strategy to improve healing. The study has broad implications for cell science where spatial pHe-variations play key roles, e.g. in tumor growth. Furthermore, the novel dual sensors presented herein can be used to visualize pHe and oxygenation in various biomedical fields.

The Impact of 10% α-Hydroxy Acid Emulsion on Skin pH

Background/Aims: The effects of a 10% α-hydroxy acid (AHA) oil/water (O/W) emulsion on the pH of human skin surface (pH_ss) and stratum corneum (SC; pH_sc) were evaluated in vivo. Methods: The AHA O/W emulsion was applied to an area on the volar forearm of male volunteers (n = 12), and then wiped off after 10 min. Prior to application and over the following 3 h, the pH_ss was measured. We used glass electrode measurements and time domain dual lifetime referencing (tdDLR) with luminescent sensor foils. In another experiment (n = 5), the impact of the AHA O/W emulsion on the pH_sc gradient was measured by tape stripping of the SC of the volar forearm after application of the AHA O/W emulsion. Results: Compared with pH_ss values prior to treatment [5.2 ± 1.7 (tdDLR) or 4.8 ± 0.5 (electrode)], the pH_ss was significantly reduced 10 min after application [4.0 ± 0.3 (tdDLR) or 4.1 ± 0.1 (electrode)] and the pH_ss remained significantly reduced over the measurement period of 3 h [after 3 h: 4.4 ± 0.2 (tdDLR) or 4.5 ± 0.3 (electrode)]. The AHA O/W emulsion significantly reduced the pH_sc even down to deep layers of the SC. Conclusion: After a 10-min application time, the 10% AHA O/W emulsion reduces the pH_ss (for at least 3 h) and pH_sc in deep layers of the SC.

Time-resolved pH imaging in marine sediments with a luminescent planar optode

A laboratory-based optical pH sensor for 2-dimensional pH imaging at benthic interfaces is presented. The sensor consists of a single-layer hydrogel matrix embedding the fluorescent pH indicator 2′, 7′-dihexyl-5(6)-Noctadecyl-carboxamidofluorescein ethyl ester (DHFAE) and the phosphorescent ruthenium(II)-Tris-4,7-diphenyl1,10-phenanthroline incorporated in nanoparticles, serving as an inert reference standard. The measuring principle is based on time domain dual-lifetime referencing (t-DLR). The fluorophore/phosphor couple is simultaneously excited by a green LED (λ max 530 nm) pulsed in the microsecond range, and the sensor emission is recorded by a fast-gateable CCD camera. For each pH image, intensity images in 2 time windows (1 during and 1 after the excitation phase) are taken. The ratio of these 2 images is proportional to the pH of the sample and not affected by the overall signal intensity. The sensor has a dynamic range suitable for marine conditions (pH 7.3 to 9.3) with an apparent pK a of 8.3. The sensor was long-term stable (months) when kept in darkness and had a response time of < 200 s when going from pH 8.3 to 7.6. Light proved to have a negative effect on the sensor performance due to photobleaching of the pH indicator, resulting in a negative drift in the signal ratio at higher pH after prolonged light exposure. The spatial resolution (83 by 83 μm/pixel) of the sensor was capable of resolving smallscale spatial variability in the pH at a heterogeneous sediment-water interface, and time series of calibrated pH images also expressed a marked temporal variability in the pH distribution across this interface. Hotspots with intensified microbial activity were observed, and pH minima along burrow walls of polychaetes indicated elevated diagenetic activity in these zones. Individually extracted profiles from the pH images agreed well with independently measured pH microelectrode profiles, confirming the robustness of the approach.

Fluorescent imaging of pH with optical sensors using time domain dual lifetime referencing.

We present a referenced scheme for fluorescence intensity measurements that is useful for imaging applications. It is based on the conversion of the fluorescence intensity information into a time-dependent parameter. A phosphorescent dye is added in the form of approximately 10-microm particles to the sample containing the pH-sensitive fluorescent indicator. Both the reference dye and the pH probe are excited simultaneously by a blue LED, and an overall luminescence is measured. In the time-resolved imaging method presented here, two images taken at different time gates were recorded using a CCD camera. The first image is recorded during excitation and reflects the luminescence signal of both the fluorophore (pH) and the phosphor (reference). The second image, which is measured after a certain delay (after switching off the light source), is solely caused by the long-lived phosphorescent dye. Because the intensity of the fluorophore contains the information on pH, whereas phosphorescence is pH-independent, the ratio of the images displays a referenced intensity distribution that reflects the pH at each picture element (pixel). The scheme is useful for LED light sources and CCD cameras that can be gated with square pulses in the microsecond range. The fundamentals and potential of this new method, to which we refer as time domain dual lifetime referencing (t-DLR), are demonstrated.