The recent introduction of an Olympus IX71 inverted microscope in a chemistry laboratory at the University of Durham is bringing scientists a step closer to unravelling dynamic cellular processes that may have been undetectable before.

The group, led by Dr Andy Beeby works alongside colleagues in Durham’s newly formed Centre for Bioactive Chemistry. They are using the Olympus’ IX71 in a method called time-resolved luminescence microscopy to develop and introduce new molecular probes for bio-imaging. The Centre brings together a range of expertise from chemistry, biology and engineering with the aim of facilitating interdisciplinary research programmes in biological chemistry and bioengineering in the fields of bioimaging, redox biochemistry, biocatalysis and protein engineering.
The intention of Beeby’s group is to enhance the luminescence imaging of biological systems, and in particular develop a means of viewing samples without the interference caused by autofluorescence.

Time Gating

The new alternative approach to overcome autofluorescence being developed by Dr Beeby involves using a time filtering rather than wavelength filtering technique. The fluorescence lifetime of the autofluorescence from a specimen is typically of the order of a few nanoseconds, that is if a sample is excited by a very short pulse of light the intensity of the fluorescence decays over a period of nanoseconds. The Durham group are developing luminescent probes which have much longer decay times, allowing the emission form the background and probe to be differentiated.

One simple way to accomplish this is to use a 'gated' detector synchronised to the excitation source. A gated image intensifier is used as a fast shutter that is capable of being opened or closed in 5 nanoseconds, allowing the emission form a well-defined time-period to be acquired. If a similar measurement is made without time gating, that is the intensifier is permanently turned on, then all emission is seen. The intensified image is recorded using a low cost, cooled CCD camera which is interfaced to a PC for image acquisition and analysis.
Using this technique Beeby et al.1  have demonstrated that a long-lived luminescent probe or label can be detected with high sensitivity against a highly fluorescent background, and is illustrated in figure 1.

  Figure 1 illustrates the power of time-resolved imaging. Europium complexes are one of the luminescent dyes incorporated into this 10 EURO note for security purposes. In this example a small area is imaged with the microscope and the image recorded following excitation by a Nd:YAG laser (355nm): the image on the left is with a delay of 0ns, and a gate of 1µs and shows the strong fluorescence from the ‘blue’ background, while that on the right was recorded using a delay of 10µs with a gate of 100µs. Significantly this shows only the phosphorescence from the europium chelate (the halo around the star is real).

Dr Beeby’s group is working with long lifetime luminescent probes based upon lanthanide chelates. Dr Beeby said ‘A characteristic of the lanthanide ions is their long luminescent lifetimes. The lifetimes of fluorescent probes and auto-fluorescence from biological systems are very short, normally less than 100 nanoseconds. But the emission lifetime of the lanthanides is about a millisecond. By time-gating our detection system we can effectively cut out autofluorescence.’

Figure 2 The spectra below were obtained from the blue area of the flag in the EURO note of Figure 1 (green line) and one of the yellow stars (red line) using a mercury lamp with a WU excitation filter. The latter shows the characteristic spectrum of a europium complex.
Samples incubated with a luminescent agent are placed on the stage of the Olympus IX71 microscope and are excited either with a pulsed Nd:YAG laser, xenon lamp or even a UV-LED. The luminescence images are regularly collected at tiny time intervals at well-defined delay times after the excitation pulse. The CCD camera captures these intensified images and displays a visible intensity and/or luminescence lifetime map of the sample on the computer screen. The IX71 microscope system is also equipped with a fibre-coupled miniature spectrometer that can record the emission spectrum from a small area of the sample.



Figure 3 Photograph showing the Olympus IX71 microscope and the PC. The gated image intensifier and CCD camera are mounted on the left-hand port, in front of this on the bench is the fibre optic mount for spectroscopy. There is a video camera mounted on the trinocular tube for monitoring the image in real time.

‘We selected all items on a stand alone basis for maximum versatility and performance’: said Dr Beeby. ‘We already had the lasers and lamps from previous experiments. But we chose Olympus’s IX71 because we were familiar with their performance and they had excellent optics and competitive prices. In combination with the gated image intensifier, manufactured by Photek Ltd (UK), we found that our modular unit is simple and versatile’.
‘We had to marry all of the components in the microscopy system together. This includes the engineering, electronics and optics. We are fortunate that in Durham, our engineering and electronic workshops provide us with excellent technical support.  Ultimately the entire system will be controlled by a single PC using software written in LabVIEW.’ Drivers for the control of Olympus BX2 and IX2 microscopes by LabView are provided on the National Instruments website.

Probes

A key factor behind the success of this system is the choice of luminescent agents or molecular probes. Lanthanide ions, because of their poor ability to absorb light, are difficult to directly excite. But when the ions are combined with an organic ligand containing an antenna, for example acridone, they can be efficiently sensitised to emit luminescence.

With high-resolution microscopy free of background fluorescence, the group plan to obtain emission maps of lanthanide-based probes, allowing them to trace even small cellular events such as sub-cellular organisation.
‘The system is already showing promise in biology’; said Dr Beeby. ‘My colleague Professor David Parker, in the Chemistry Department here at Durham has been designing responsive lanthanide probes which will allow us to map the local concentration of ions such as bicarbonate and other important cellular components. In conjunction with our colleagues in the Biology Department they have demonstrated that the complexes do penetrate into cells’. Importantly, Professor Parker found that the lanthanides were not toxic to their live cells, opening avenues for other biologists to use the same method of molecular imaging.

Tracking molecular changes

Alterations in bicarbonate ion concentrations in mammalian cells have been linked to a variety of tumours, mental retardation, renal tubular acidosis and osteoporosis. Yet, there are very few methods than can explain what triggers the concentration changes and how.
In a bid to find new methods of measuring the component in intracellular and extracellular environments, Professor Parker designed the lanthanide complexes to have a selective response to bicarbonate ions in the cell.  The presence of this analyte in the environment would be indicated by a change in the complex which gives rise to a change in the emission intensity, spectrum and lifetime.
Monitoring the ratio of lanthanide emission at up to three wavelengths, it is hoped that the method will allow them to deduce the concentration of bicarbonate against the other competing ions such as lactate, citrate and phosphate.

Future trends

The Durham group hopes to extend their collection of lanthanide-based probes, to carry out several assays simultaneously. Studies exploring the use of lanthanides for clinical chemistry, particularly in drug testing, are also underway.

1 Beeby A, Botchway SW, Clarkson IM, Faulkner S, Parker AW, Parker D, Williams JA.
Luminescence imaging microscopy and lifetime mapping using kinetically stable lanthanide(III) complexes. J Photochem Photobiol B. 2000 Sep; 57(2-3):83-9.

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