Cellular physiological activity is markedly dependent on the physical properties of this medium. In particular, the dysregulation of intracellular parameters such as pH or microviscosity (understood as the localized viscosity experienced by molecules at the microscopic scale) may be indicative of pathology. As such, the monitoring of physical properties at the cellular level is very important for the study, and potentially for the diagnosis, of various diseases.

In this context, the use of fluorescent probes is a preferred approach for the observation and monitoring of biological media in biomedical applications. These probes are molecular systems whose photophysics allows the monitoring of environmental conditions through changes induced in their fluorescence, in terms of intensity or lifetimes. There is now a wide variety of fluorescent probes that can be used as microviscosity sensors, exploiting various photophysical mechanisms, as well as trans–cis photoisomerization.

Recently, a trans–cis photoisomerization process sensitive to environmental microviscosity was demonstrated in a natural p-hydroxycinnamate (sinapoyl malate, SM), in contrast to observations for similar molecules. At the same time, a new synthetic process for the production of SM derivatives was developed and patented by Prof. Florent Allais, a collaborator on this project. The accessibility of a wide range of these compounds, with easily tunable photophysical properties and low toxicity, enables a new class of fluorescent probes for intracellular microviscosity monitoring.

To address this opportunity, this project aims to explore the photophysics of several phenolic compounds inspired by SM. Some of these, including 4-methoxy coumaryl meldrum, caffeoyl meldrum, coumaroyl meldrum, feryloyl meldrum, and sinapoyl meldrum, are already available for study. However, many others can be synthesized through the protocol patented by Prof. Allais, including acids whose photophysics and dependence on environmental pH (in addition to microviscosity) may also be of interest.

This work will serve as a starting point for this new line of research, and as a proof of concept for the viability of these phenolic compounds for use as intracellular microviscosity probes.

The experimental work of this project will consist mainly of the extensive spectroscopic characterization of the phenolic compounds of interest. This work includes the use of steady-state spectroscopy techniques to obtain absorption and emission spectra (including fluorescence anisotropy studies), and complementary time-resolved spectroscopy techniques which, together, cover timescales from femtoseconds (10⁻¹² seconds) to seconds. Some of these techniques are accessible to the team through the services of the Warwick Centre for Ultrafast Spectroscopy (University of Warwick, UK), an external-use laboratory where the Principal Investigator (PI) has already worked several times.

Drawing on her experience in spectroscopy and molecular dynamics, and with the support of Prof. Berberan Santos and Prof. Ferreira, both also experienced spectroscopists, the PI will lead this spectroscopic work, with the assistance of a master’s-level researcher who will be hired specifically for this project.

Finally, the phenolic compounds whose photophysics prove to be sensitive to environmental microviscosity will advance to cellular microscopy studies that include experimental techniques such as confocal microscopy, phosphorescence lifetime imaging, and time-gated imaging, available at iBB. These studies serve several purposes, including: (i) identifying the cellular regions where these compounds accumulate, (ii) assessing the cytotoxicity of the compounds, and (iii) establishing their viability as microviscosity sensors.

This crucial stage of the project will be developed in close collaboration with Dr. Sandra Pinto, who has extensive experience in molecular and cellular biophysics.

Taken as a whole, the research plan of this project reflects a carefully designed combination of state-of-the-art methodologies, from the patented synthetic protocol for the production of the compounds of interest, to advanced spectroscopy techniques for the characterization of their photophysical properties, and fluorescence microscopy techniques to test their capabilities as intracellular microviscosity sensors.

The team proposed to work on this project has also been carefully selected to ensure that the collective expertise covers all aspects of the research plan, with adequate working time for each team member.

The success of this project will culminate in the development of a new class of fluorescent microviscosity probes, easily tunable and with low toxicity, constituting a potentially transformative step in biomedical imaging.