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<p>The two-colour laser-induced fluorescence (2c-LIF) method is capable of time-dependent, spatially resolved temperature and pH measurements, respectively. In the present study, temperature and pH are investigated in separate measurement series, using the same optical principle and dye system under different experimental conditions. Together with a still uninterrupted growth in the life science field—especially molecular biology and diagnostics—the adaptation of this method might play a key role in improving future assays. 2c-LIF is providing a new level of process verification within temperature-controlled microplate wells under realistic operating conditions, i.e. in automated laboratory workflows for both high-throughput and high-content. Specifically, within each microwell the confined liquid volumes and the imposed vertical temperature gradients lead to thermally induced flow structures that influence scalar transport and desired mixing of the liquid. Within this paper, a Fluorescein/Sulforhodamine 101 dye system in water enables combined spectroscopic and imaging-based thermometry in the temperature range 283–343&#xa0;K under realistic conditions. Temperature sensitivities of 2–6%/K by using a spectrometer and 2–3.5%/K for an image-based measurement are achieved, yielding a statistical precision of approximately 0.23–0.50&#xa0;K. Absorption-corrected dual-camera imaging is providing quantitative full-field temperature maps and also resolving the temporal evolution of thermally driven flow patterns during a heating ramp. In buffered solutions (pH 6–11), the fluorescence ratio exhibits a linear response between pH 6 and 9, allowing spatially resolved visualisation of biochemical mixing processes. Time-dependent isothermal droplet-injection experiments are conducted to quantify homogenisation dynamics in the absence of external mechanical stimuli. A normalised mixing index based on the coefficient of variation is introduced to characterise mixing times and to assess the relative contributions of advective and diffusive transport, which can be interpreted in terms of local Péclet numbers. Thus, it can be demonstrated that 2c-LIF is providing a quantitative diagnostic framework for investigating coupled heat and mass transfer in confined microplate systems and for analysing transport-dominated mixing processes in small-scale liquid volumes.</p> Graphical abstract <p></p>

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Spatially resolved and time-dependent temperature and pH measurements in microplate wells using two-dye two-colour laser-induced fluorescence

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  • 发表日期：

    2026年7月10日

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  • Peter Felbinger,
  • Michael Straußwald,
  • Peter Foehr,
  • Lars Zigan

摘要

The two-colour laser-induced fluorescence (2c-LIF) method is capable of time-dependent, spatially resolved temperature and pH measurements, respectively. In the present study, temperature and pH are investigated in separate measurement series, using the same optical principle and dye system under different experimental conditions. Together with a still uninterrupted growth in the life science field—especially molecular biology and diagnostics—the adaptation of this method might play a key role in improving future assays. 2c-LIF is providing a new level of process verification within temperature-controlled microplate wells under realistic operating conditions, i.e. in automated laboratory workflows for both high-throughput and high-content. Specifically, within each microwell the confined liquid volumes and the imposed vertical temperature gradients lead to thermally induced flow structures that influence scalar transport and desired mixing of the liquid. Within this paper, a Fluorescein/Sulforhodamine 101 dye system in water enables combined spectroscopic and imaging-based thermometry in the temperature range 283–343 K under realistic conditions. Temperature sensitivities of 2–6%/K by using a spectrometer and 2–3.5%/K for an image-based measurement are achieved, yielding a statistical precision of approximately 0.23–0.50 K. Absorption-corrected dual-camera imaging is providing quantitative full-field temperature maps and also resolving the temporal evolution of thermally driven flow patterns during a heating ramp. In buffered solutions (pH 6–11), the fluorescence ratio exhibits a linear response between pH 6 and 9, allowing spatially resolved visualisation of biochemical mixing processes. Time-dependent isothermal droplet-injection experiments are conducted to quantify homogenisation dynamics in the absence of external mechanical stimuli. A normalised mixing index based on the coefficient of variation is introduced to characterise mixing times and to assess the relative contributions of advective and diffusive transport, which can be interpreted in terms of local Péclet numbers. Thus, it can be demonstrated that 2c-LIF is providing a quantitative diagnostic framework for investigating coupled heat and mass transfer in confined microplate systems and for analysing transport-dominated mixing processes in small-scale liquid volumes.

Graphical abstract