This work describes a hydrogel fluorescence microsensor for prolonged stable temperature measurements. Temperature measurement using microsensors has the potential to provide information about cells, tissues, and the culture environment, with optical measurement using a fluorescent dye being a promising microsensing approach. However, it is challenging to achieve stable measurements over prolonged periods with conventional measurement methods based on the fluorescence intensity of fluorescent dye because the excited fluorescent dye molecules are bleached by the exposure to light. The decrease in fluorescence intensity induced by photobleaching causes measurement errors. In this work, a photobleaching compensation method based on the diffusion of fluorescent dye inside a hydrogel microsensor is proposed. The factors that influence compensation in the hydrogel microsensor system are the interval time between measurements, material, concentration of photo initiator, and the composition of the fluorescence microsensor. These factors were evaluated by comparing a polystyrene fluorescence microsensor and a hydrogel fluorescence microsensor, both with diameters of 20 µm. The hydrogel fluorescence microsensor made from 9% poly (ethylene glycol) diacrylate (PEGDA) 575 and 2% photo initiator showed excellent fluorescence intensity stability after exposure (standard deviation of difference from initial fluorescence after 100 measurement repetitions: within 1%). The effect of microsensor size on the stability of the fluorescence intensity was also evaluated. The hydrogel fluorescence microsensors, with sizes greater than the measurement area determined by the axial resolution of the confocal microscope, showed a small decrease in fluorescence intensity, within 3%, after 900 measurement repetitions. The temperature of deionized water in a microchamber was measured for 5400 s using both a thermopile and the hydrogel fluorescence microsensor. The results showed that the maximum error and standard deviation of error between these two sensors were 0.5 °C and 0.3 °C, respectively, confirming the effectiveness of the proposed method.
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.