EuroTracker Dyes: Synthesis and Application in Functional Cell Imaging

The visualisation of certain living cells or microbes by optical microscopy requires the introduction of bright, luminescent dyes or stains, that can light up selected parts of the cell effectively.

We will develop bright complexes of the rare earth element, europium, (EuroTracker dyes) as optical probes that emit light efficiently and report information back to the observer about their chemical or biological environment.

These probes have been designed to allow them to be excited by the light sources that are available in microscopes used by physical scientists, biochemists and cell biologists, to study the interior composition of cells or related small compartments. By examining series of closely related probes, we shall define systems that locate to a particular compartment of a cell, allowing it to be visualised selectively.

The optical probes that are currently used are based either on organic compounds, on large man-made proteins or on so-called quantum dots; each of these probes suffer from problems of low chemical stability, a tendency to fade and, in certain cases, they are rather toxic to the living cell. Thus we aim to change the thinking of the microscopy community and to pave the way for such emissive metal complexes to be used as stains and probes in biology. These metal complex probes emit light over longer timescales than conventional probes, and so their is more time to collect the emitted light after pulsed excitation and only short exposure times to the light that is used to excite them. The long emission lifetime avoids problems in data acquisition that are associated with light scattering and background autofluorescence.

In parallel, we shall develop instrumentation methods that allow the spectral signature of these probes to be recorded quickly using fast imaging methods. We shall work with microscope manufacturers and with optics specialists to this end. This will allow changes in chemical composition within a cell to be tracked on a 10 second timescale. As a first step, we shall develop probes that can report on pH change and on the amount of sodium, magnesium and zinc within a specific cell compartment, allowing changes in the amount of these important metal ions to be tracked in living cells in real time. By adopting a modular approach to probe design, different metal ions can be examined subsequently, by introducing the appropriate metal-binding moiety into the probe structure.

The impact of this work will be felt among the following cohorts:

1. Physical, biological and clinical research scientists will benefit from this work by gaining access to new optical probes for time-resolved microscopy to accompany the introduction of better instrumentation for such studies, leading to enhanced levels of sensitivity and improved signal/noise performance. In addition, the introduction of sensitive pM probes will enhance the study of metal ions in cell biology, improving our understanding of their role and importance in the health and food sectors, for example.

2. Industrial companies will benefit who are either involved in selling probes to the microscopy community (dominated currently by Invitrogen) or who develop and sell microscopy and spectral imaging instrumentation (e.g. Leica - the preferred partner; Nikon, Olympus, Zeiss, PicoQuant) or optical instrumentation for portable use, e.g. based on fibre optics, LEDs and CCD detectors (e.g. Ocean Optics, Nichia).

3. Commercial companies will benefit who sell products to the pharmaceutical sector for time-resolved bio-assays, (e.g. CISbio Bioassays - our preferred partner; Perkin Elmer, GE Healthcare; Brahms).

4. In the longer term, multinational companies in the food sector (e.g. Unilever, Syngenta) may benefit from the higher yields emanating from precise control of key essential metal ions (e.g. Zn) in fermentation products. In addition, bio-processing companies, e.g. Lonza and MSD Biologics, now Fujifilm Diosynth Biotechnologies - based in the North East of England), seek to to optimize metal availability to recombinant proteins. The ratio of available Zn versus less competitive metals (such as Mg) is significant for product quality. Probes are needed to optimise these parameters in cell models.

5. Industrial companies in human healthcare and beauty products (e.g. Procter and Gamble) seek to understand the control of metals in healthy and bacterial cells, allowing the development of more effective antimicrobial agents that should enhance safety profiles, lengthen product lifetimes and conform to recent European and US environmental legislation that is limiting or will further restrict the use of many current products.