Investigators at the University of Sheffield and Durham University in the UK have developed photostable platinum(II) complexes that are highly emissive and synthetically versatile and enable time-resolved emission imaging microscopy on a microsecond time scale.
According to the researchers, the fluorescence lifespan of the platinum complexes, also called Pt(II) complexes, is several orders of magnitude longer than currently used fluorophores. The combination of TREM, two-photon excitation, and Pt(II) complexes could be a powerful tool for investigating intracellular processes in vivo, because the long lifetimes allow discrimination for autofluorescence, they said.
“It would depend on what particular system you would use, and whether you were looking at any labeled sites,” said John Haycock, a senior lecturer in the department of engineering materials in the University of Sheffield.
In basic, fundamental studies in which researchers target a particular site or a particular protein and its expression, “I think, generically, if you are using such a molecule to identify the presence of that protein by whatever means, such as an antibody, then it would definitely have an advantage there,” he said.
In general, if researchers can use a technique such as emission imaging microscopy to follow events occurring inside a cell, they can start to design molecules that inhibit different cell activities. They can use the Pt(II) complexes as probes of cellular processes that may be linked to different types of disease.
“For example, if we had a probe that was responsive to [reduction] potential, that may tell us about oxidative stress within a cell, which is related to aging,” said Gareth Williams, a senior lecturer in the department of chemistry at Durham University.
The idea, which is outlined in a study published online last week in PNAS, came from the use of the platinum-based fluorophores, said Haycock. Two of his colleagues, Williams and Julia Weinstein, a lecturer and research fellow in the department of chemistry at the University of Sheffield, who were studying these complexes, realized that they had very long stability and fluorescence illumination.
At the time, Weinstein and Williams were wondering if there was any biological use for their platinum-based fluorophores. “So initially, we simply started by seeing if they had any intrinsic cellular targets on their own, in very much the same way that other dyes, such as diamidinophenylindole, might,” Haycock said.
“We put [the Pt(II) complexes] onto cells, and we found that they are readily uptaken, and that they bound specifically to DNA, and we think to a lesser extent to the RNA, inside the cell,” he added.
“Since they are relatively flat molecules, one could actually envision them sort of intercalating between the DNA base pairs.”
Of particular note was the fact that the fluorophores were not toxic at the concentrations at which the investigators used them to image the cells.
Additionally, the lifespan of the fluorophores was much longer and the molecules more stable than any of the other biological fluorophores, such as fluorescein — “certainly more than the ones I have used,” said Haycock.
The study had two goals, Haycock said: First, the researchers wanted to see where in the cells the Pt(II) fluorophores bound.
“Since they are relatively flat molecules, one could actually envision them sort of intercalating between the DNA base pairs,” said Haycock.
Secondly, the investigators wanted to determine how long these molecules fluoresced after being flashed by a laser.
“That sort of took us into doing some time results experiments, which is what the paper is all about. It actually details microsecond lifespans, which are orders of magnitude greater than the lifespans of other types of fluorophores,” said Haycock.
The scientists would see autofluorescence associated with a particular cell or tissue, and once the autofluorescence would decay, they would see the extended fluorescence of the fluorophore.
“We were hoping to create a long-lived probe so that we can get rid of autofluorescence and do imaging on a longer timescale,” said Weinstein.
The investigators also wanted to develop the technology in order to sense emission inside of a cell, and target small molecules and reactive oxygen species such as oxygen and nitric oxide.
“This is much easier to do with a long-lived probe because one has to measure much larger changes,” said Weinstein.
The idea is that if researchers are looking at these compounds in biological media that contain a fluorescent background, they can specifically see the emissions after a certain time interval has elapsed, Williams said.
“During that time interval, all of the other emission has decayed, and only the stuff that you are looking at is still emitting,” he said.
Chemically, what is needed for fluorescence imaging are compounds that emit light on a long time scale, and that means using metal complexes because their emission comes from a triplet state, in which two electrons share a common spin, compared with the opposite spins associated with a singlet state.
The platinum complexes accumulate inside the cells under diffusion control. “It literally takes five minutes to put the probe complexes inside the cells, and after that, we put the cells under the microscope and we excite the complexes inside the cell with a near-infrared light,” Weinstein explained.
The complexes are very photostable and can simultaneously absorb two photons of near-infrared radiation, which is harmless to tissues but excites the complexes, which then emit light on the microsecond time scale.
“The next step would really be to identify applications for this technology,” said Haycock. This may involve taking the fluorophores, conjugating them to antibodies, and seeing whether first of all, the excitation of the fluorophore is maintained, and then seeing whether the researchers can observe a really long fluorescence lifespan on the order of microseconds.