Abstract
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.
Highlights
Both the measurement and control of the cellular environment with high spatial resolution are essential for investigating cellular conditions systematically, defining normal cell-to-cell variation, quantifying the influence of environmental perturbations, and understanding cellular responses in the tissues and complex environments
The hydrogel fluorescence microsensor consisted of poly (ethylene glycol) diacrylate (PEGDA) 575 (Sigma-Aldrich), deionized water, and Omnirad 1173 (BASF Japan Ltd., Tokyo, Japan), which is a photo initiator
The results show that the measured temperature values of the hydrogel fluorescence microsensor and thermopile exhibited good agreement
Summary
Both the measurement and control of the cellular environment with high spatial resolution are essential for investigating cellular conditions systematically, defining normal cell-to-cell variation, quantifying the influence of environmental perturbations, and understanding cellular responses in the tissues and complex environments. In addition to pH measurement [2], the use of optical sensors to determine various environmental parameters—such as temperature [3,4,5,6] and oxygen concentration [7]—with high spatial resolution is promising for biological investigation. Optical sensors using fluorescent dyes have significant potential in non-contact measurement for microscale applications. Environmental parameters such as temperature [8,9], oxygen concentration [10,11,12], pH [13,14], and organophosphate compounds [15] can be measured by utilizing fluorescent dyes that are sensitive to these parameters. Fluorescence sensors have paved the way for the investigation of cells, tissues, and the environmental conditions of culture systems, amongst other parameters
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