Temperature-sensitive Fluorescent Dye Research Articles

Preparation and characterization of electrospun fluorescent fiber mats as temperature sensors using various polymers

In this study six electrospun fluorescent fiber mats were fabricated using a temperature-sensitive fluorescent dye, a ruthenium complex (tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride, (Ru(dpp)3)), each with one of six different polymers of polyvinyl alcohol (PVA), polyurethane-based hydrogel HydroMed D4 (D4), polyacrylonitrile (PAN), poly(methyl methacrylate) (PMMA), poly(styrene-co-acrylonitrile) (PSAN), and poly(acrylonitrile-co-methyl methacrylate) (PANMM). The electrospun fluorescent fiber mats were characterized as fluorescence temperature sensors in aqueous solution and gaseous environment. The electrospun fluorescent fiber mat fabricated from PAN showed good stability over 100 days, short response time (i.e. 4-5 min), and good reversibility in aqueous solution from 5 to 60 °C, whereas the electrospun fiber mat from PMMA showed relatively high sensitivity and good selectivity in air. These two electrospun fluorescent fiber mats were tested as non-invasive sensors for measuring temperature in biological and environmental processes.

Fluorescence lifetime-based intracellular thermometry for photothermal therapy

Photo-thermal therapy (PTT) is a rapidly developing approach for cancer treatment, that has greatly benefited from the tremendous advances in the synthesis of plasmonic nanoparticles (NPs), which can be used as light sensitive agents. This approach is based on the local induction of hyperthermia via light irradiation of plasmonic NPs in order to thermally kill cancerous cells. Such an approach ensures a high precision of treatment and the mechanism of action makes it as a valid alternative for treatment of malignant neoplasms with a multi-drug resistance. However, in order to minimize the adverse effects of PTT it is necessary to precisely measure and control the achieved temperatures at the tumor foci during light irradiation. Herein we report a novel technique to monitor intracellular temperatures during PTT based on temperature sensitive fluorescent dye, Rhodamine B. These findings may improve the quality of treatment and reduce unwanted adverse effects.

Laser-induced fluorescence thermometry of supercritical CO2 flows inside a micro-channel

This work demonstrates a thermometric technique using laser-induced fluorescence (LIF) in supercritical carbon dioxide flows in a micro-channel. Rhodamine 6G was used as a temperature-sensitive fluorescent dye. The flow conditions were at a pressure of 7.9 MPa and temperature in the range of 23°-90°C. 2D spatial distributions and time-resolved temperature profiles were obtained at this high pressure. Measured LIF signals showed close relations to the temperatures obtained from resistance temperature detectors.

Single-cell temperature mapping with fluorescent thermometer nanosheets.

Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1°C. However, the reliability of intracellular thermometry has been challenged both experimentally and theoretically because it is, in principle, exceedingly difficult to exclude the effects of nonthermal factors on the thermometers. To accurately measure cellular temperatures from outside of cells, we developed novel thermometry with fluorescent thermometer nanosheets, allowing for noninvasive global temperature mapping of cultured single cells. Various types of cells, i.e., HeLa/HEK293 cells, brown adipocytes, cardiomyocytes, and neurons, were cultured on nanosheets containing the temperature-sensitive fluorescent dye europium (III) thenoyltrifluoroacetonate trihydrate. First, we found that the difference in temperature on the nanosheet between nonexcitable HeLa/HEK293 cells and the culture medium was less than 0.2°C. The expression of mutated type 1 ryanodine receptors (R164C or Y523S) in HEK293 cells that cause Ca2+ leak from the endoplasmic reticulum did not change the cellular temperature greater than 0.1°C. Yet intracellular thermometry detected an increase in temperature of greater than ∼2°C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within ±0.2°C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within ±0.1°C. In cardiomyocytes, the temperature was stable within ±0.01°C during contractions when electrically stimulated at 2 Hz. Similarly, when rat hippocampal neurons were electrically stimulated at 0.25 Hz, the temperature was stable within ±0.03°C. The present findings with nonexcitable and excitable cells demonstrate that heat produced upon activation in single cells does not uniformly increase cellular temperature on a global basis, but merely forms a local temperature gradient on the order of ∼1°C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.

In situ temperature monitoring in single-molecule FRET experiments.

Thermodynamic properties of single molecules including enthalpic and entropic contributions are often determined from experiments by a direct control and precise measurement of the local temperature. However, common temperature monitoring techniques using, for example, ultrafine temperature probes can lead to uncertainties as the probe cannot be placed in the vicinity of the molecule of interest. Here, we devised an approach to measure the local temperature in freely diffusing confocal single-molecule Förster Resonance Energy Transfer (smFRET) experiments in situ by directly adding the temperature-sensitive fluorescent dye Rhodamine B, whose fluorescence lifetime serves as a probe of the local temperature in the confocal volume. We demonstrate that the temperature and FRET efficiencies of static and dynamic molecules can be extracted within one measurement simultaneously, without the need of a reference chamber. We anticipate this technique to be particularly useful in the physicochemical analyses of temperature-dependent biomolecular processes from single-molecule measurements.

Manipulation and Immobilization of a Single Fluorescence Nanosensor for Selective Injection into Cells.

Manipulation and injection of single nanosensors with high cell viability is an emerging field in cell analysis. We propose a new method using fluorescence nanosensors with a glass nanoprobe and optical control of the zeta potential. The nanosensor is fabricated by encapsulating a fluorescence polystyrene nanobead into a lipid layer with 1,3,3-trimethylindolino-6′-nitrobenzopyrylospiran (SP), which is a photochromic material. The nanobead contains iron oxide nanoparticles and a temperature-sensitive fluorescent dye, Rhodamine B. The zeta potential of the nanosensor switches between negative and positive by photo-isomerization of SP with ultraviolet irradiation. The positively-charged nanosensor easily adheres to a negatively-charged glass nanoprobe, is transported to a target cell, and then adheres to the negatively-charged cell membrane. The nanosensor is then injected into the cytoplasm by heating with a near-infrared (NIR) laser. As a demonstration, a single 750 nm nanosensor was picked-up using a glass nanoprobe with optical control of the zeta potential. Then, the nanosensor was transported and immobilized onto a target cell membrane. Finally, it was injected into the cytoplasm using a NIR laser. The success rates of pick-up and cell immobilization of the nanosensor were 75% and 64%, respectively. Cell injection and cell survival rates were 80% and 100%, respectively.

A novel heating area design of temperature-jump microfluidic chip for synchrotron radiation solution X-ray scattering

In this paper, we present a novel design scheme of temperature-jump (T-jump) area for microfluidic device. Numerical simulation and experimental research of thermal characteristics of the solution in microchannels is completed. Numerical simulation of the temperature-jump microchannel is analyzed to study the heat transfer characteristics by comparing performance of three proposed configurations. Calculation of the power requirement is discussed in the dimensional design of microheater. Temperature-sensitive fluorescent dye is applied to investigate the temperature field of microchannel heated by a designed microheater. It is found that the T-jump microfluidic device can provide rapid heating for solutions with strong convection heat transfer ability.

Characterization of the evaporation of interacting droplets using combined optical techniques

A monodisperse droplet stream is injected into a high-temperature enclosure supplied with air heated up to 540 °C. The two-color laser-induced fluorescence (2cLIF) is used for measuring the droplet temperature. The liquid fuel is seeded by pyrromethene 597-C8, which is a temperature-sensitive fluorescent dye. Calibration tests are performed for different types of fuels including ethanol and several alkanes and some of their mixtures. Morphology-dependent resonances (MDRs) are identified as a possible adverse effect for temperature measurements. Due to MDRs, lasing of pyrromethene 597-C8 may occur within fluorescent droplets and affect drastically the fluorescence signal upon which temperature measurement relies. The determination of the droplet size and velocity is achieved by means of quantitative shadow imaging. A double cavity PIV laser is focused on a piece of PMMA doped with a fluorescent dye to produce the background illumination of the droplets. A PIV camera is used to capture the drop motion between the pulses of the laser cavities. A large range of initial distance parameters (the ratio between the inter-droplet distance and the droplet diameter) is explored for different liquid fuels (ethanol, isohexane, n-heptane, n-decane, n-dodecane) and their mixtures. To put forward the effects of the interactions between the droplets, size and temperature measurements are compared to the isolated droplet whose evolution can be predicted with the use of classical models. Comparisons reveal that the inter-droplet spacing and also the fuel volatility play an important role in the reduction of the heat and mass transfers for these interacting droplets. Finally, the ability of the 2cLIF techniques to address the case of multicomponent droplet is also demonstrated.

Dielectrophoretic immobilisation of antibodies on microelectrode arrays

A silicon based chip device with a regular array of more than 100,000 cylindrical sub-microelectrodes has been developed for the dielectrophoretic (DEP) manipulation of nanoparticles and molecules in solution. It was fabricated by a standard CMOS (complementary metal oxide semiconductor) compatible process. The distribution of the electrical field gradient was calculated to predict the applicability of the setup. Heating due to field application was determined microscopically using a temperature sensitive fluorescent dye. Depending on voltage and frequency, temperature increase was found to be compatible with protein function. Successful field controlled immobilisation of biomolecules from solution was demonstrated with the autofluorescent protein R-phycoerythrin (RPE) and with fluorescently labelled IgG antibodies. Biological activity after DEP application was proven by immobilisation of an anti-RPE antibody and subsequent binding of RPE. These results demonstrate that the developed chip system allows the directed immobilisation of proteins onto microelectrodes by dielectrophoresis without the need for any chemical modification and that protein function is preserved. Being based on standard lithographical methods, further miniaturisation and on-chip integration of electronics towards a multiparameter single cell analysis system appear near at hand.

A Microfluidic Device for High Throughput Measurements of Thermotaxis

E. coli bacteria have the ability to sense thermal gradients and congregate in locations with a preferred temperature. Studies of this phenomenon, called thermotaxis, have been complicated by the lack of tools to enable the generation of well-defined thermal gradients and the collection of data on motile E. coli in these gradients. Here we report a microfluidic experimental system which allows the collection of data on the migration of bacteria in thermal gradients at a high throughput. In our microfluidic device, a bacterial suspension slowly flows through a 10 mm long, 0.5 mm wide microchannel that has a linear temperature gradient across it. It takes ∼100 seconds for cells to reach the end of the microchannel, thus they have sufficient time to sample the temperature gradient and find their preferred position in the gradient. By analyzing the distribution of cells across the microchannel near its end, we collect data on the thermotaxis of >10,000 individual E. coli cells per experiment. The temperature gradient is generated by the continuous circulation of temperature-controlled water through two large-diameter channels flanking the microchannel. The temperature gradient is evaluated using a temperature-sensitive fluorescent dye. The microfluidic chip is made from a single cast of PDMS, making it easy to fabricate and replicate with high fidelity. We have used the experimental system to perform preliminary experiments on wild type E. coli in a variety of linear thermal gradients and have observed the congregation of cells centered about a temperature of 35–37° C.

Characteristics of the turbulent/non-turbulent interface of a non-isothermal jet

The turbulent/non-turbulent interface of a jet is characterized by sharp jumps ('discontinuities') in the conditional flow statistics relative to the interface. Experiments were carried out to measure the conditional flow statistics for a non-isothermal jet, i.e. a cooled jet. These experiments are complementary to previous experiments on an isothermal Re=2000 jet, where, in the present experiments on a non-isothermal jet, the thermal diffusivity is intermediate to the diffusivity of momentum and the diffusivity of mass. The experimental method is a combined laser-induced fluorescence/particle image velocimetry method, where a temperature-sensitive fluorescent dye (rhodamine 6G) is used to measure the instantaneous temperature fluctuations. The results show that the cooled jet can be considered to behave like a self-similar jet without any significant buoyancy effects. The detection of the interface is based on the instantaneous temperature, and provides a reliable means to detect the interface. Conditional flow statistics reveal the superlayer jump in the conditional vorticity and in the temperature.

Functional gel-microbead manipulated by optical tweezers for local environment measurement in microchip

A novel on-chip environment measurement with functional gel-microtool was developed. Environment measurement gel-microtool was fabricated by connecting the gel-microbeads impregnated with indicators in a microchip. In this artcle, Bromothymol blue (BTB) and Bromocresol green (BCG) were employed as pH indicators. BTB and BCG have the different indicator range. Rhodamine B is temperature sensitive fluorescent dye and is used for temperature measurement. Gel-microbead is made by salting-out of hydrophilic photo-crosslinkable resin and is manipulated by optical tweezers. Moreover, gel-microbead is polymerized by UV illumination and connected to other gel-microbead under an electrolyte solution. The connection of gel-microbeads is performed by contact of gel-microbeads under UV illumination. Environment measurement gel-microtool with an arbitrary shape is fabricated by connection of the gel-microtool impregnated with arbitrary indicator. Multiple environments measurement gel-microtool included with several indicators is realized by assembly of the gel-microbeads impregnated with different indicators. Environment measurement is performed by detecting the color and the fluorescence intensity of each gel-microbead. We succeeded in the on-chip fabrication of the environment measurement gel-microtools such as circular pH measurement gel-microtool and wide range pH measurement gel-microtool in a microchip.

複数環境計測のための機能性ゲルツールのオンチップ作製

A novel on-chip environment measurement with functional gel-tool was developed. Environment measurement gel-tool was fabricated by connecting the gel-microbeads impregnated with indicators in a microchip. In this paper, Bromothymol blue (BTB) and Bromocresol green (BCG) were employed as pH indicators. BTB and BCG have the different indicator range. Rhodamine B is temperature sensitive fluorescent dye and is used for temperature measurement. Gel-microbead is made by salting-out of hydrophilic photo-crosslinkable resin and is manipulated by optical tweezers. Moreover, gel-microbead is polymerized by UV illumination. The connection of gel-microbeads is performed by contact of gel-microbeads under UV illumination in an electrolyte solution of a certain concentration. Multiple environments measurement gel-tool included with several indicators is realized by assembly of the gel-microbeads impregnated with different indicators. This technique can be employed for compensation of the dependence of the indicator on environment except for the target environment. Environment measurement is performed by detecting the color and the fluorescence intensity of each gel-microbead. We succeeded in the on-chip fabrication of the environment measurement gel-tools such as wide range pH measurement gel-tool and pH and temperature measurement gel-tool in a microchip.

온도 민감 형광을 이용한 마이크로 스케일 표면온도 측정

A technique for measuring surface temperature field in micro scale is newly proposed, which uses temperature-sensitive fluorescent (TSF) dye coated on the surface and is easily implemented with a fluorescence microscope and a CCD camera. The TSF dye is chosen among mixtures of various chemical compositions including rhodamine B as the fluorescent dye to be most sensitive to temperature change. In order to examine the effectiveness of this temperature measurement technique, numerical analysis and experiment on transient conduction heat transfer for two different substrate materials, i. e., silicon and glass, are performed. In the experiment, to accurately measure the temperature with high resolution temperature calibration curves were obtained with very fine spatial units. The experimental results agree qualitatively well with the numerical data in the silicon and glass substrate cases so that the present temperature measurement method proves to be quite reliable. In addition, it is noteworthy that the glass substrate is more appropriate to be used as thermally-insulating locally-heating heater in micro thermal devices. This fact is identified in the temperature measuring experiment on the locally-heating heaters made on the wafer of silicon and glass substrates. Accordingly, this technique is capable of accurate and non-intrusive high-resolution measurement of temperature field in microscale.

Combined planar laser-induced fluorescence-particle image velocimetry technique for velocity and temperature fields

Combined use of particle image velocimetry (PIV) with planar laser-induced fluorescence (PLIF) was developed to measure both velocity and scalars such as temperature and concentration in thermo-fluid flow. PLIF, which uses a temperature-sensitive fluorescent dye excited by a laser light sheet, is a whole-field temperature diagnostic. The technique was applied to a thermal stratified flow and a plane impinging jet. The time evolutions of vortical and thermal structures were successfully obtained in order to understand the mechanism of the turbulent heat transfer in these flows.