GB2529719A - Tracer - Google Patents

Abstract

A method of non-invasively imaging a dispersion of particles in time and space, where each particle comprises a coating of a fluorescent material over an inert mineral core and the particles range between 0.5 -5 micron diameter, the method comprising illuminating the area of particle dispersion with spectrally selected excitation light of substantially uniform intensity to excite fluorescence, and recording a time series of images of emitted light from the area of dispersion using at least one imaging device such as a camera. The method is used for monitoring environmental transport of pollutants in soil for example.

Description

TRACER

The present invention concerns methods of tracing particulate materials.

Particulate matter (such as clay) is an important vector for the environmental transport of pollutants such as nutrients, pesticides, effluents and metals. The transport and fate of many chemicals in soil systems is closely linked to that of particulates. Understanding such mechanisms has been hampered by the lack of a suitable tracer.

Of particular interest are particles of the order of a few microns in size. Such particles may be transported by air or water or by mechanical means, and the pattern and range of their dispersion is of interest. Equally such dispersion may represent an erosion process where the rate of erosion and the fate of eroded material are important.

The small size of the particles involved in chemical transport, and their physical properties have hitherto made development of a suitable tracer very challenging.

For larger particles (the size of grains of sand; 62.5 microns to 2,000 microns) there has been success in mixing a dye with a binding agent and then applying this mixture to the surface of the particles. However this technique cannot be applied to particles with a diameter of a few microns, because the dye coating significantly enlarges the particle and alters the density.

It is known to use microspheres, plastic magnetic beads, ceramic prills, and rare earth oxide particles ("REOs") which strictly speaking are fine silt particles. ISO 14688 grades clay particles as being smaller than 2 microns and silts larger. It is also known to use particles with similar physical and chemical properties to those of interest, but additionally comprising labels enabling subsequent detection. Such labels may include radioactive components and/or dyes. All of these techniques have a number of disadvantages: When such materials are used in experiments, they require sampling of the soil after the experiment, and subsequent chemical processing to obtain results. Sampling during such experiments is impractical as it impacts on the processes being studied. In addition there are significant density differences between the particles used and native clay particles, impacting on transport.

There is therefore a need for a novel method of particle tracing to overcome existing deficiencies and to offer improved means of tracing micron sized particles.

Such a method requires particles with essentially the same properties as the native particles whose dispersion it will trace, and able to be manufactured easily. For best understanding of dispersion processes, the tracer material should be able to be analysed using non-invasive, non-destructive and in-situ techniques, with the ability to repeat analysis often during the course of an experiment.

The modification of clay particles by a coating of fluorescent materials ("fluorophores") offers a means of satisfying these requirements. Fluorescence is the phenomenon of emission of (usually) visible light by a substance following irradiation by excitation" photons of higher energy (usually shorter wavelength light or gamma photons).

Fluorescence enables experimental illumination at one wavelength and detection at a longer wavelength, allowing highly sensitive detection. This in turn permits minimal usage of fluorescent materials in tracer production, resulting in insignificant modification of the coated particle.

When using fluorescence to detect labelled particles, it is essential to distinguish between on the one hand emitted light (signal) and on the other hand scattered excitation light and light emitted by naturally fluorescent material present in the medium.

This distinction may be achieved by selecting a fluorophore with different photoluminescent properties to the background material, as will be explained.

The difference in wavelength between absorbed excitation light and subsequently emitted light is called the Stokes shift'. Materials with a larger Stokes shift make it easier to select appropriate filters to detect emitted light and attenuate excitation light and so obtain a good signal to noise ratio. For example, rhodamine B has a larger Stokes shift (about 40 nm in solution for the emission peak) than most soil luminescence and gives a sharp line emission profile.

Another useful photoluminescence characteristic is luminescence lifetime, which is a property of how long the electron stays in the excited state before emission of light.

Where there is a difference in luminescence lifetime between two materials, a pulsed excitation system (for example a pulsed laser) may be used and a time-delay built in before image capture, so that the presence is detected of only the material whose luminescence matches that time delay. Scattered light (excitation light) has a lifetime of zero, and so vanishes after even a short delay.

Autofluorescence in soil systems normally originates from polyaromatic species which show the typical photoluminescent properties of an organic fluorophore, in that emission occurs from singlet excited states. Emission from singlet states involves only loss of vibrational energy (or other changes) rather than electronic spin transitions, so usually gives a small Stokes shift. It is a rapid process so the luminescence lifetime is short.

Thus autofluorescence has a small Stokes shift and a lifetime typically less thanlO ns.

Transition-metal and lanthanide dyes typically emit light from triplet electronic states, in which the electron spins are parallel. The extra energy loss in the excited triplet states leads to larger Stokes shifts, and emission from triplet states is characterised by long lifetimes. Such metal dyes have advantageous Stokes shifts and photoluminescence lifetimes, while organic dyes may give greater signal intensity. Selection of an appropriate fluorophore thus depends in detail on the application.

Traditionally, fluorescence measurements are taken using a fluorometer which gives detailed spectral information about a small physical area. However it is also known to use a visible-light film camera, for example monitoring the movement of silt sized glass particles (with diameters from 44 to 2,000 microns) labelled with fluorescent uranium salts (Young & Holt, Tracing soil movement with fluorescent glass particles. Soil Sd Soc Am Pro 1968, 32, (4), 600-602).

The present invention comprises a method of non-invasively imaging a dispersion of particles in time and space, where each particle comprises a coating of a fluorescent material over a mineral core, and substantially all the particles have a maximum diameter between 0.5 microns and 5 microns inclusive. The method further comprises illuminating the area of dispersion with spectrally selected excitation light of substantially uniform intensity to excite the fluorescent material to emit light of at least one emission wavelength and recording a time series of images of emitted light from the area of dispersion using at least one imaging device The spectrally selected excitation light may be substantially monochromatic, and its uniformity may be provided by passing it through an optical diffuser At least one imaging device may be equipped with an optical filter to attenuate the excitation light At least one emission wavelength may be within the visible spectrum The imaging device may comprise a camera Each particle may comprise a core of clay such as montmorillonite, bentonite, chlorite, kaolinite, smectite or illite Substantially all the particles may have a maximum diameter between 1 micron and three microns inclusive. Substantially all the particles may have a maximum diameter between 1.5 microns and 2.5 microns inclusive The fluorescent material may comprise a chemical derivative of xanthene such as fluorescein, an eosin, or a rhodamine. The fluorescent material may comprise an organo-metallic compound, such as a lanthanide or a transition metal dye, such as a ruthenium tris (2,2'bipyridyl) compound or a rhenium-fac-tricarbonyl-2,2'bipyridyl compound. The fluorescent material may comprise a derivative of acridine such as acridine orange The spectrally selected excitation light may be provided by a laser and/or LED, which may be operated in continuous mode or in pulse mode. The spectrally selected excitation light may be provided by a broad spectral source and a suitable filter to attenuate light at wavelengths including at least the emission wavelength(s) At least one imaging device may be controlled by a means of computing Figure 1 shows dispersion of tracer particles in a rainfall experiment.

Figure 2 shows derived area containing tracer in the same experiment.

Method of manufacture of tracer pad/c/es The manufacturing process consists of selecting and preparing suitable mineral particles and coating them with a suitable fluorophore material.

A suitable type of mineral is clay. A suitable type of clay is montmorillonite (also known as bentonite) available commercially from Sigma Aldrich (catalogue number 69904).

The clay may be chosen to reflect the environment in which it is to be used. For example in soil tracing studies the clay may be the type of clay present locally in the natural soil environment. Such clays may include chlorite, illite, kaolinite, montmorillonite and smectite. In other studies, a type of clay may be selected according to its density to match the density of the particles whose dispersion is being studied. Tables of properties of different types of clay are available in the literature and/or from suppliers.

The clay is ground so that its average particle size is less than the maximum required for the study. A typical size used by the present inventors is 2.0 microns. Particles of this size may be obtained by use of a planetary mill.

The clay is made into slurry with minimal water and sonicated for thirty minutes, and the fluorophore is prepared as follows: A suitable solution of the fluorophore is made, preferably an aqueous solution. For

example:

For rhodamine B (from Acros Organics (132311000)) and other rhodamines dissolve 200 milligrams of the fluorophore into one litre of water.

For ruthenium tris (2,2'bipyridyl) (available as hexafluorophosphate ("Ru(bpy)3(PF6)2" from Sigma Aldrich) and its derivatives add one gram of the fluorophore (per twenty grams of clay) to minimal acetone or DMSO (dimethyl sulphoxide) to form a solution. Rapidly add one litre of water ensuring that the fluorophore remains in solution (and if any particulates result, filter them off).

Other derivatives may require other solvents as is well known to those skilled in the art.

For acridine orange (N,N,NN'-Tetramethylacridine-3,6-diam me, obtained from Magnacol) add 200 milligrams of the fluorophore (per twelve grams of clay) to water. Filter off any particulates remaining.

For derivatives of rhenium-fac-tricarbonyl-2,2'bipyridyl (X) (Re(CO)s(bpy) (X) where for example is X is bromide, chloride or 4-hydroxypyridine or another suitable moiety, use water. These materials were synthesised by the inventors using techniques well known to those skilled in the art. Other types of moiety X may require other solvents as is well known to those skilled in the ad.

The solution containing the fluorophore is added promptly to the clay slurry (avoiding delay helps to prevent unwanted precipitation of the fluorophore). This is then sonicated for 45 minutes, and stirred for another two hours.

Then the particles are allowed to settle and recovered from the liquid, for example using vacuum filtration and a Whatman number 5 filter.

The recovered clay is washed using a washing solution suitable for the chosen fluorophore: For rhodamine B use a mixture of equal parts of saturated brine (sodium chloride in water) and ethanol.

For Ru(bpy)3 the use of saturated brine (sodium chloride in water) is preferred.

Alternatively other solvents such as acetone or DMSO may be used.

For acridine orange, use water.

In the cases above where brine is used, other highly ionic solutions may be substituted as convenient.

The clay particles are washed repeatedly until very little fluorophore is coming off.

The particles are air-dried in a desiccator while importantly protecting them from light.

If the particles have aggregated, they are ground to disaggregate them.

Those skilled in the art will appreciate that other fluorophores may also be used with suitable choices of solvent and wash, to create additional types of fluorescent particles.

Many other metal based fluorophores exist for example containing d or f block elements (referring to the periodic table), such as platinum or iridium.

The finished particles should be kept cool and light excluded to avoid unwanted photobleaching which is the photo-degradation of fluorophores via reaction with ambient oxidising chemical (such as oxygen), to give non-emissive compounds. Photobleaching may be severe in bright sunlight and may result in loss of all emission. Metal-based dyes may be more resistant to photobleaching. a

Imaging The principles of the method of imaging are the same for the range of particles.

Suitably spectrally filtered light that is substantially uniform in intensity across the region of interest is used to excite the fluorophore, with the wavelength(s) of illumination chosen depending on the fluorophore.

The filtered excitation light may comprise a range of wavelengths or may be substantially monochromatic.

The filtered excitation light may be provided by a broad spectral source and a filter selected such that wavelengths shorter than the emission wavelength are provided to the fluorophore. Preferably wavelengths at and around the emission wavelength(s) are attenuated, The filtered excitation light may be provided by a broad spectral source and a monochromator such that substantially monochromatic light is provided to the fluorophore at a suitable wavelength shorter and distinct from the emission wavelength(s), The illumination may suitably be provided by a laser or LED with the wavelength(s) chosen depending on the fluorophore.

Suitable uniformity of illumination may preferably be achieved by the use of a diffuser.

The diffuser may preferably be rotated to remove artefacts.

For rhodamine B the excitation wavelength is preferably around 532 nm. This may also be used for other rhodamines (such as rhodamine 6G, rhodamine 123, rhodamine 101, rhodamine 19).

For Ru(bpy)3 excitation illumination around 445 to 450 nm is preferred.

For Re(CO)3(bpy) excitation illumination around 532 nm is preferred The more intense the illumination that can be provided, the better are the images obtained. Monochromatic excitation may give a better signal to noise ratio for the detection of emission.

For excitation light at 532 nm a green laser diode may be used. For example such a laser from Shanhai Industrial provides 75 milliwatts.

For excitation light at 445 to 450 nm a blue laser diode may be used. For example such a laser from Shanhai Industrial provides 50 milliwatts.

Images may suitably be obtained using one or more imaging devices sensitive to the emission wavelength of the fluorophore. Suitable devices may consist of a matrix of pixels. Suitable imaging devices may include without limitation CMOS devices (complementary metal oxide semiconductor) or CCDs (charge coupled device) Light arising from fluorescence is captured by the imaging device, Conveniently a fluorophore may be selected whose emission wavelength is in the visible spectrum.

The image may be obtained using a standard digital camera sensitive to visible wavelengths.

Preferably a filter is used so that light of the illumination wavelength is attenuated.

For example, when using blue light illumination a long-pass filter (for example with 50% cut-off at 470 nm) may be used, and when using green light illumination a long-pass filter (for example with 50% cut-off at 570 nm) may be used. A long-pass filter here means a filter that attenuates shorter wavelengths more than longer wavelengths.

A standard consumer camera may be used as the imaging device. Such devices usually contain CMOS imaging arrays requiring the emitted fluorescent wavelength to be within the visible range (roughly 400 to 700 nm).

Because imaging may take place in situ, it is possible to use a standard video camera to obtain real-time images of the movement of particles during the course of an experiment.

It is equally possible to use a still-image imaging device to take multiple images. This may be under the control of a repeater unit, or under the control of a connected means of computing such as a personal computer or a single board computer or micro-controller such as a Raspberry Ri® or Arduino®, running suitable software. The said connection may be by wire or wireless.

In some cases multiple imaging devices may be used.

In some cases it may be useful to make use of the luminescence lifetime of the fluorophore. In such cases a pulsed laser may be used and the timing of image capture linked to the timing of the laser pulse using a shared mechanism of control (preferably electronic). Such a mechanism of control preferably operates in real-time mode to ensure that low-tolerance time-dependent processing is correctly handled in order to allow correct capture of each image.

It is possible to view captured images directly, but much can be gained by post-processing. A wide range of techniques is well known to those skilled in the art. For example well-known techniques may be used to generate false colour images.

Certain colour imaging devices record light intensity at each pixel in multiple channels.

Such channels may be wavelength dependent (for example red, green and blue colour channels). In such cases it may be found that the light emitted by a particular fluorophore falls mainly within the wavelength range of one colour channel (for example the green channel), and it may be advantageous to form an image using only the information in this colour channel.

Equally it may be advantageous to form an image from the information in the selected colour channel but to use the other colour channels in selection algorithms (for example use the green information if more intense than both the red and blue). Those skilled in the art will appreciate that many such algorithms may be devised as appropriate.

Other well known computational techniques may be used to derive quantitative metrics describing the particle dispersion process.

A sequence of still images may be put together to synthesise a time-lapse film of the (accelerated) progress of the experiment. By such means a long dispersion experiment may be reviewed qualitatively in a short time, for example one minute.

The present invention may be used for real-time monitoring of dispersion experiments.

The particles whose manufacture and application have been described may be used in a wide range of studies in the laboratory and in the field, including studies of the mobility, dispersion and/or fate of: soil and sediment particles, particles discharged into watercourses and/or estuarine, littoral and/or marine environments and/or the atmosphere.

The particles may be used dry or wet, as a powder, suspension, solution or colloid. The particles may be diluted with suitable solvents such as water. The particles may be diluted by mixing with liquids or solids. The particles may be mixed with a sample taken from the test environment and subsequently replaced.

The particles may also be used in studies into losses of colloidal material, for example from road surfaces and/or other elements of the built environment, Suitable such particles may also be used in medical, biological and/or ecological studies to follow the transport, dispersion and/or fate of particulate material in living organisms.

The present invention may be used in a laboratory environment to measure the dispersion properties of materials and/or devices. It may equally be used in the field to make measurements of dispersion in situ.

Note that where the chosen fluorophore is liable to photobleaching, the present invention may preferably be used in dark or blackout conditions. In the field this may require erection of a temporary structure and/or operation under low levels of ambient light (for example at night).

Example -Soil experiment An experiment by the inventors using the present invention for tracing soil particles (montrnorillonite) will now be described as an illustration of material selection and use.

Soil may auto-fluoresce, for example due to aromatic acids that are naturally present.

These typically have excitation around 465 nm and emission around 517 nm. So to minimise interference, a fluorophore that excites outside this range is desirable. A well characterised fluorophore with a high quantum yield, good photo stability and emission in the visible spectrum is also desirable.

For this experiment, rhodamine B was selected as the fluorophore. It has maximum emission at around 590 nm. It is commercially available and inexpensive, and is known to bind to montmorillonite.

Particles of montmorillonite were coated with rhodamine B using the method detailed above.

The size range of particles was measured using a Malvern Mastersizer 2000. About fifty five percent of the tracer particles had a size of less than 2 microns compared to fifty one percent of the clay used to produce the tracer. The physical sizes of the clay particles were not significantly altered by the addition of fluorophore to the surface.

A Leica confocal microscope was used to record images of clay and tracer particles.

Images were taken using a 63X optical lens under oil, and show that the particles retain their irregular sizes and shapes, and that the fluorophore appears to be uniformly spread over the surface of the particles without disturbing surface texture.

Tests were undertaken to investigate the stability of the particles. Over forty hours, negligible loss of fluorophore was detected with the particles mixed with water. Over the same period a loss of about 0.2% was observed from particles mixed with artificial seawater. It is believed that the binding of the fluorophore to the clay is via electrostatic forces, hence there is more disruption in the more ionic environment. In either case the loss of fluorophore is insignificant for experimental purposes.

The experiment consisted of monitoring the movement of particles in a soil box under simulated rainfall.

A clear plastic soil box (350mm by 500mm), with drainage holes in the base was filled with 40 mm depth of fine gravel, landscape fabric membrane, 30 mm of sand and 40 mm of soil (screened to 4 mm) to simulate natural infiltration conditions. A section of soil x Sax 5 mm was removed, mixed with 4 grams of tracer and then replaced. To approach saturation the soil box was then placed in water, to a depth 10mm above the soil-sand interface for 22 hours. The box was then drained for one hour immediately before being exposed to simulated rainfall.

The rainfall simulator used hypodermic needles to form raindrops, and was gravity fed with distilled water with the intensity kept constant by maintaining a constant hydraulic head. The rainfall intensity was 42 mm per hour. The box was placed on a 4 degree slope.

The experiment was maintained in a dark environment, and illumination was provided by a 75 milliwatt laser outputting 532 nm wavelength (green) light that was passed through a rotating diffuser. Images were acquired using a Canon 500-D DSLR camera fitted with a EF 50mm f/i.8 II lens and mounted on a tripod approximately 2 metres directly above from the soil surface. A coloured glass filter (Optical Knight 570nm 570FC5550 long-pass) was attached tapped to the UV filter ring. The camera was autofocused under artificial light then switched to manual focus to prevent the camera from refocusing under low light conditions. Images were acquired without flash and the following setting: exposure time 6s, f-stop 1.8, ISO 800, RAW + JPEG at highest quality.

An intervalometer (Shoot) with an interval of 7 seconds was used.

The laser beam was passed through a diffuser (Thorlabs ED1 -S20-MD). This diffuser is efficient (-90%) while providing near-Lambertian diffusion. The diffuser was mounted on a bearing and rotated using an electric motor (Maplin -sn35q) and a pulley system. The diffuser was rotating at roughly 100 revolutions per minute. This avoided the interference patterns often seen in laser light.

Achieving uniform illumination is critical to producing accurate images, and visual and photographic assessment showed a high degree of uniformity of illumination.

The results of the experiment are shown in Figure 1. To avoid edge effects the 25 mm adjacent to the sides of the box were discounted. Each of the three images in Figure 1 is derived from an image taken by the camera. Figure 1A shows the distribution of tracer particles at the start of the experiment. The black pixels show the replaced soil sample (approximately 150mm by 50 mm) revealed by the presence of tracer particles.

Figure 1 B shows the dispersion (black pixels) of the tracer particles after 262 seconds of simulated rainfall, and Figure 1 C shows the dispersion (black pixels) after 2252 seconds of simulated rainfall.

Using the programming language [r], the raw images were converted into false colour images with noise suppressed. These images are easy to analyse visually because they show presence of tracer as colour (black in Figure 1) and its absence in white. The file size of the resulting images is roughly 100 times smaller than the raw images. These images were then converted (Cinepak codec by Radius, quality 100) into a time-lapse video using VirtualDub (version 1.10.4) allowing the whole dispersion experiment to be reviewed in about one minute.

The images in the Figures were converted for the purposes of this document to monochrome bitmap images using Microsoft Paint.

Figure 2 shows a plot of the area of the soil box over which tracer could be detected as a function of time. The x-axis represents the elapsed time of the experiment, and runs from zero to 2500 seconds with intervals between tick marks of 500 seconds. The y-axis represents area in units of square millimetres where the lowest tick mark represents zero, the interval is 20,000 and the highest tick mark represents 80,000.

Importantly, Figure 2 illustrates how the present invention may be used for real-time monitoring of dispersion experiments.

While the present invention has been described in terms of several embodiments and examples, those skilled in the art will recognize that the present invention is not limited to the embodiments and examples described, but can be practised with modification and alteration within the scope of the appended claims. The Description is thus to be regarded as illustrative instead of limiting.

Claims (2)

1. CLAIMS1 A method of non-invasively imaging a dispersion of particles in time and space, where: each particle comprises a coating of a fluorescent material over a mineral core, substantially all the particles have a maximum diameter between 0.5 microns and 5 microns inclusive, the method comprises illuminating the area of dispersion with spectrally selected excitation light of substantially uniform intensity to excite the fluorescent material to emit light of at least one emission wavelength, and recording a time series of images of emitted light from the area of dispersion using at least one imaging device 2 A method as in claim 1 where the spectrally selected excitation light is substantially monochromatic 3 A method as in any previous claim where uniformity of excitation light is provided by passing the excitation light through an optical diffuser 4 A method as in any previous claim where at least one imaging device is equipped with an optical filter to attenuate the excitation light A method as in any previous claim where at least one emission wavelength is within the visible spectrum 6 A method as in any previous claim where the imaging device comprises a camera 7 A method as in any previous claim where each particle comprises a core of clay such as montmorillonite, bentonite, chlorite, kaolinite, smectite or illite 8 A method as in any previous claim where substantially all the particles have a maximum diameter between 1 micron and three microns inclusive 9 A method as in claim 8 where substantially all the particles have a maximum diameter between 1.5 microns and
2. 2.5 microns inclusive A method as in any previous claim where the fluorescent material comprises a chemical derivative of xanthene such as fluorescein, an eosin, or a rhodamine 11 A method as in claims 1 to 9 where the fluorescent material comprises an organo-metallic compound, such as a lanthanide or a transition metal dye, such as a ruthenium tris (2,2'bipyridyl) compound or a rhenium-fac-tricarbonyl-2,2'bipyridyl compound 12 A method as in claims 1 to 9 where the fluorescent material comprises a derivative of acridine such as acridine orange 13 A method as in any previous claim where the spectrally selected excitation light is provided by a laser and/or LED 14 A method as in claim 13 where the laser or LED is operated in continuous mode or in pulse mode A method as in claims 1 and/or claims 3 to 12 where the spectrally selected excitation light is provided by a broad spectral source and a suitable filter to attenuate light at wavelengths including at least the emission wavelength(s) 16 A method as in any previous claim where at least one imaging device is controlled by a means of computing Amendments to the claims have been filed as followsCLAIMS1 A method of non-invasively imaging a dispersion of particles in time and space, where: each particle comprises a coating of a fluorescent material over a clay core, substantially all the particles have a maximum diameter between 0.5 microns and 5 microns inclusive, application of the coating substantially retains the size, shape and surface texture of the particles, the coating material is selected to emit light by fluorescence of at least one emission wavelength distinct from wavelengths of fluorescence of materials present in the area of dispersion; the method comprises illuminating the area of dispersion with spectrally selected excitation light of substantially uniform intensity to excite the fluorescent material to emit light, and recording a time series of images of emitted light from the area If) of dispersion using at least one imaging device 2 A method as in claim 1 where the spectrally selected excitation light is 0 substantially monochromatic 3 A method as in any previous claim where uniformity of excitation light is provided by passing the excitation light through an optical diffuser 4 A method as in any previous claim where at least one imaging device is equipped with an optical filter to attenuate the excitation light A method as in any previous claim where at least one emission wavelength is within the visible spectrum 6 A method as in any previous claim where the imaging device comprises a camera 7 A method as in any previous claim where each particle comprises a core of montmorillonite, bentonite, chlorite, kaolinite, smectite and/or illite 8 A method as in any previous claim where substantially all the particles have a maximum diameter between 1 micron and three microns inclusive 9 A method as in claim 8 where substantially all the particles have a maximum diameter between 1.5 microns and 2.5 microns inclusive A method as in any previous claim where the fluorescent material comprises a chemical derivative of xanthene such as fluorescein, an eosin, or a rhodamine 11 A method as in claims 1 to 9 where the fluorescent material comprises an organo-metallic compound, such as a lanthanide or a transition metal dye, such as a ruthenium tris (2,2'bipyridyl) compound or a rhenium-fac-tricarbonyl- 2,2'bipyridyl compound 12 A method as in claims 1 to 9 where the fluorescent material comprises a derivative of acridine such as acridine orange LI") 13 A method as in any previous claim where the spectrally selected excitation light is provided by a laser and/or LED a) 14 A method as in claim 13 where the laser or LED is operated in continuous mode or in pulse mode (4 A method as in claims 1 and/or claims 3 to 12 where the spectrally selected excitation light is provided by a broad spectral source and a suitable filter to attenuate light at wavelengths including at least the emission wavelength(s) 16 A method as in any previous claim where at least one imaging device is controlled by a means of computing