Despite the low cross sections of the U VI O2 2+ species, experiments were carried out to discern whether 2PE microscopy could be used to image uranium in microbial systems. Due to its wide availability and a wealth of literature precedent Brewer's yeast (Saccharomyces cerevisiae) was chosen as a model system. In the absence of uranium, after being applied directly to a microscope slide without fixation, S. cerevisiae were observed as aggregates of multiple cells (Fig. , top left). Excitation at 405 nm resulted in intense autofluorescence which typically displayed a bi-exponential decay with a short-lived component of approximately 300 ps and a longer-lived component of 1200-1500 ps. These are comparable to literature reports of Brewers' yeast autofluorescence as is the steadystate spectrum obtained from this emission which revealed a broad peak with a maximum at 510 -520 nm (ESI). Weak phosphorescence was also observed with a lifetime centred at 300 ns. To the best of our knowledge phosphorescent lifetimes of this order have not been reported for S. cerevisiae, although some biological fluorophores such as tryptophan are known to be long-lived and spectroscopic studies on bulk samples of yeast in solution offer support for the existence of an emissive species with a lifetime on the order of hundreds of nanoseconds (ESI). Studies by Webb and co-workers have shown that several of the molecules responsible for autofluorescence have low but measurable two-photon cross sections. Indeed, following 2PE at 810 nm yeast samples showed emission with similar luminescence lifetimes to those observed following one-photon excitation. (Fig. ) For initial studies into uranium biosorption, yeast samples were exposed to a concentrated solution of uranyl nitrate (0.1 M, 10 mg yeast/10 ml, unadjusted pH ~ 2.5) as described by Popa et al. When these samples were viewed by PLIM long-lived emission (centred at 1900 ns) attributed to uranyl(VI) was observed. The emission was, however, rather weak (Fig. , left). Raising the pH to 4.5 (by addition of NaOH) and imaging under identical conditions (ESI) resulted in much stronger uranyl(VI) phosphorescence, with little change in lifetime (Fig. , right). Various studies carried out to optimise the conditions for uranyl removal form solution suggest that pH is the most important factor, with minimal accumulation occurring at low (2.5) and near-neutral pH (>6) and maximum accumulation taking place in mildly acidic conditions (pH 4-5). The increased intensity of long-lived emission associated with the surface of the S. cerevisiae cells suggests the adsorption of uranyl(VI) species, with the increased intensity at higher pH consistent with literature reports that highly acidic conditions are not ideal for biosorption. However, despite the relatively low determined two-photon absorption cross sections of uranyl(VI), these model studies clearly demonstrate that uranyl(VI) biosorption can indeed be imaged using two-photon fluorescence and phosphorescence lifetime image mapping. The ability to study uranyl adsorption by two-photon fluorescence microscopy was tested by adjusting the pH using a carbonate source (NaHCO3) to better emulate the U VI O2 2+ speciation in groundwater. Carbonate has a 8high binding coefficient and is known to have a rich coordination chemistry with U VI O2 2+ , with carbonate solubilising uranyl in high pH solutions in which oligomerisation would otherwise occur. Additionally, carbonate is known to quench U VI O2 2+ emission at room temperature, resulting in weakly emissive complexes with very short luminescent lifetimes. These factors suggest that carbonate may preferentially bind to and solubilise U VI O2 2+ , preventing biosorption while quenching emission and making imaging U VI O2 2+ absorption by luminescence microscopy difficult. Indeed, reduced sorption of uranyl onto clay minerals in the presence of carbonate has been observed. In fact, in these systems this was not the case, as PLIM revealed that as the pH is raised with NaHCO3 there is a significant increase in intensity of phosphorescence associated with the yeast cells (Fig. , centre and right). Unlike the carbonate-free system, in which the lifetime of the U VI O2 2+ phosphorescence showed little variation with pH, the samples which had been adjusted with sodium carbonate showed an increase in phosphorescence lifetime to ~ 2400 ns, potentially suggesting a change in speciation of U VI O2 2+ on the cell surface. While the effect of pH on the accumulation of uranium by yeast has been widely studied to the best of our knowledge the pH has not been adjusted using carbonate and further studies using better analogues for natural conditions are needed. While the previous examples demonstrate that 2PE FLIM/PLIM can be used to detect the adsorption of U VI O2 2+ to yeast, the concentrations are not representative of either the majority of sorption studies or the concentrations of uranium found in contaminated sites. In order to assess the viability of this technique to provide further insight on potential bioremediation techniques it was also applied to yeast which had been exposed to much lower concentrations of U VI O2 2+ (<3 mmol), according to the procedure reported previously by Omar and coworkers. 14 Although the wide range of two-photon cross sections and quantum yields of U VI O2 2+ species (cf. Table ) prohibit the exact quantification of a lower limit for the detection of U VI O2 2+ in biological systems by 2PE microscopy, the quantity of U VI O2 2+ removed from solution by yeast was measured in order to estimate the sensitivity of this technique. This was carried out by a well-established spectrophotometric technique using Br-PADAP dye. In accordance with the results obtained at high uranyl concentration the presence of carbonate did not show a significant adverse effect on the adsorption of U VI O2 2+ . Indeed, at the peak pH for uranyl sorption (~ pH 5) with 3 mmol U VI O2 2+ the carbonate-free system adsorbed slightly less U VI O2 2+ than systems in which the pH had been adjusted using NaHCO3. The yeast samples from these solutions were determined to have adsorbed approximately 1.5 -2 mmol uranium per gram of dry biomass. After separation by centrifugation these yeast samples were observed using 2PE microscopy and both showed significantly more phosphorescence than observed in the uranyl-free control sample. The system in which the pH had been adjusted using NaOH showed phosphorescence with lifetimes ranging from 1500 -3000 ns with a peak at 2500 ns, while the carbonate-containing system showed a broad range of phosphorescent lifetimes with a peak at 11500 ns (Fig. ).