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This invention concerns a modular multi-band fluorescence excitation system. More specifically, the invention concerns a multi-band fluorescence excitation system suitable to collect together light from multiple LEDs (Light Emitting Diodes), hence combining light bands of the same, and/or partly superimposed, and/or different well determined wavelength intervals, to provide a mixed light beam of high intensity, and comprising the different wavelength intervals as required, such system being modular so as to be flexibly used.

Fluorescence based methods are used in the bio-medical field to observe or reveal a variety of natural, genetically introduced, or exogenous fluorescent molecules (or fluorochromes), the latter two being used to label cell and tissue components directly or by means of specific antibodies or other molecular probes.

Fluorescence microscopes and related equipment (such as stereo- microscopes or other fluorescence visualization or imaging systems) are used to reveal fluorochromes within cell and tissue preparations, from very low (life size) to high magnification, and to record and/or analyse the relevant images. In today's applications, and in parallel with the increasing use of multiple-analyte methods (e.g. gene arrays, protein- and antibody- arrays), the possibility of labelling and observing or revealing at the same time multiple (i.e. more than 2 or 3) different fluorescent molecules or labels in one and the same tissue or cell preparation is under increasing demand, since it provides a unique amount of information as to the simultaneous presence and spatial relationships between multiple cellular components and molecules of interest.

Most fluorochrome labels presently used are excited and then emit at different wavelengths. Therefore, in principle, the advantage of fluorescence visualization and/or imaging methods is that: (i) an appropriate selection of the fluorescence excitation wavelength intervals used results in the selective excitation of the wanted one/s within a mixture of different fluorochromes, and: (ii) an appropriate selection of the emission wavelength ranges transmitted to the eye or imaging system results in the visualization or imaging of the wanted one/s within said mixture of different fluorochromes. In applicative terms, however, all fluorochromes show extended spectral regions of low-level excitation and/or emission (excitation and emission tails), so that their effective separation requires carefully chosen wavelength ranges on both the excitation as well as the emission side.

Furthermore, though many fluorochromes of different excitation and emission maxima are available or are being introduced, when more than

4-5 such fluorochromes are combined, at least partly overlapping spectral regions of excitation and emission will result. Hence, their selective visualization or imaging, i.e. ensuring that each of them is seen or detected independently of all others, may require that only some but not all of the simultaneously present fluorochromes are visualized or imaged at the same time, only to be "switched off' when the other ones present are to be seen or imaged. This way, the overall profile of simultaneously used fluorochromes may be divided in appropriate sub-groups, each group being seen/imaged at once, and all groups being seen/imaged in sequence.

The distribution and concentration of the molecules of interest is a further crucial issue, multiple simultaneous visualization (or imaging) being easy to use when the items or molecules of interest are present in similar local concentrations, thus resulting in labelling signals of consistent and comparable intensities (e.g. certain FISH analyses). However, most biological molecules are found in very different and widely variable amounts (e.g. proteins and post-translational products such as peptide hormones), such differences and changes even within one and the same preparation being often the major scope for investigation. Thus, a working system for simultaneous multiple fluorescence visualization or imaging ought to be able not only to provide the highest possible sensitivity, but also to handle highly varied signal intensities in connection with concentration differences spanning several orders of magnitude (e.g. 1 to 1 million, or 1 billion fold difference, or higher), both between as well as within preparations. A crucial contribution would be that excitation light intensities were finely tunable, from the highest to very low levels. The latter need is further underlined during the simultaneous visualization/imaging of multiple flurochromes, when multiple emissions and colours (plus infrared bands) are seen and/or imaged at the same time. In view of their varied and varying local concentrations, as well as detection efficiencies, each of the various labels used may show as very bright to very weak signal/s, so that one or a few fluorochromes may dominate and virtually obscure all the others. Hence, it will be necessary that all and each of the excitation light bands is independently and finely tunable in intensity, and can be rapidly switched on-and-off, so that even the weakest detectable signals can be shown, distinguished and discriminated in their co-distribution/s and co-localization/s compared to all other labels.

Altogether, multiple fluorescence visualization and/or imaging requires the availability of a multiplicity of highly optimised excitation light intervals (excitation bands).

Said excitation light intervals will need to be: (i) highly discreet and separate from each other, i.e. showing high intensity in the wanted excitation interval, said intensity very rapidly decreasing at the extremes of the wanted interval (e.g. to 10exp(-6) or lower, within a few nm from the wanted band limit); (ii) available in multiple different intervals, including closely adjacent or partly overlapping ranges as may be required for specific fluorochromes; (iii) each rapidly switchable on-and-off, so that the selective visualization of single, as well as specific groups of fluorochromes can be accomplished at ease and repeatedly during observation or imaging sessions; (iv) each finely tunable in intensity, from maximum to very low intensity, in a way entirely independent from all other excitation bands. It is perhaps needless to say that any system providing such multiple, independently adjustable, simultaneously operating, rapidly switchable excitation bands must be easily suitable for the introduction and use of novel excitation bands and wavelength ranges, as required by the introduction of novel fluorochromes and/or labels. Hence, a modular implementation in which only a limited sub-portion of the system will have to be changed or adapted to satisfy the above need will be necessary.

A reflected (or incident) fluorescence visualization or imaging system, such as a fluorescence microscope, is essentially composed of: (a) a light source; (b) optics to convey light into an excitation light pathway, generally as parallel (collimated) light upon reaching the excitation filter; (c) an excitation filter to select one or more excitation wavelength bands to be conveyed to the object; (d) a dichroic mirror, i.e. a device which will reflect the excitation wavelength band/s to the object, while transmitting the fluorescence emission wavelength/s from the object to the emission optical pathway; (e) optics to focus excitation light onto the object and to collect fluorescence emission light from said object; (T) an emission, or barrier, filter to block any stray excitation light wavelength/s transmitted into the emission optical pathway, while letting through the fluorescence emission light; (g) optics to convey the fluorescence emission wavelength/s to the eye and/or to a camera or other recording apparatus. Alternatively, especially for low-magnification applications such as stereo- microscopy, the excitation light can be projected directly onto the specimen, while the emission light follows the same pathway as above, hence is collected by optics, and is transmitted through an emission filter to the visualization or imaging optics.

Nowadays, virtually all reflected fluorescence microscopes are provided with a multiplicity of rapidly switchable "filter cubes", each filter cube being dedicated to specific fluorochrome/s and including a matched set of excitation filter, dichroic mirror, and emission filter. Hence, the separate and sequential visualization of different fluorochromes (such as fluorochromes A, B, C, D) can easily be accomplished by switching through the appropriate four, selective single-band filter sets one-at-a-time in succession.

Ideally, however, it would be convenient to be able to see and/or record two, three, four and more different distinct fluorochrome signals (A+B+C+D...) at one and the same time, in order not only to acquire more information as to the expression and localization of the molecules of interest, but also to analyse their detailed spatial relationships within the cell and tissue components of interest. In parallel with the growing use of other "parallel multiple-analyte" approaches, such as array technologies for the parallel analysis of expression and changes of hundreds or thousands of genes or proteins, there is a rapidly increasing need for effectively "multiple" methods in microscopy, namely in fluorescence microscopy, and/or in fluorescence visualization and/or imaging.

In recent years, improvement in optical surface coating technology by the use of stacks of optically interfering layers has made it possible for dedicated producers to provide excitation and emission filters, as well as dichroics, with multiple spectral regions of alternating high transmission and high reflection/rejection. Numerous "filter sets" have thus been made available, which permit the simultaneous excitation and viewing of two or three fluorochromes at the same time. This is obtained by: (a) selecting two or three suitable excitation bands, using one and the same excitation filter; (b) reflecting these towards the object, by means of a matched multiple-band dichroic; (c) letting through the corresponding two, or three emission bands, using a multi-band emission filter matched to the above excitation filter and dichroic. With the availability of such "multi-band sets", however, serious problems and issues have become apparent, which needed and still need addressing in order to enable scientists and technologists to make the most of the powerful approach of simultaneous viewing/imaging multi-labelling methods in fluorescence microscopy and related areas.

Multiple-band excitation filters are especially critical, since they must change from high transmission (>80-90%) to very high rejection (<10exp(-6) transmission or so) within a few nm, at each and every interface between a transmission region (which lets through light in a wanted excitation interval) and the adjacent high rejection regions. Such high-rejection spectral regions in the excitation filter correspond to high- transmission regions in the dichroic mirror and emission filter, for collection and visualization of the far weaker light emitted by the fluorochrome/s being studied. While such degree of spectral separation between transmission and rejection bands are relatively easy to obtain for a state- of-the-art single-band filter, the association of two, three or more such alternating excitation-emission intervals implies major trade-offs, hence reduced efficiency of excitation bands, and the transmission of significant, visible/detectable amounts of excitation light within the "emission" intervals. Since the emission light intensity is comparatively very weak, compared with the excitation light intensity required (e.g. emission/excitation light intensity ratio 10exp(-6)/1 , or lower), even minor degrees of undue transmission of excitation light will result in a much reduced signal-to-noise ratio, hence in a poor outcome of the wanted fluorescence visualization or imaging. In addition, present state-of-the art optical surface coating technology does not permit the inclusion of any effective UV excitation band (below approximately 380-390 nm) in a multiple-band excitation filter, due to poor UV transmittance of materials presently in use.

Due to their structure, light collected and collimated from LED sources will contain at least partly non parallel light beams. In addition, if multiple LEDs are mounted side by side and their light beams are made to converge, light beams deriving from centrally located versus laterally located LEDs will reach any interposed optical surface at differing angles of incidence. Hence, an issue relevant to the present invention is that the spectral behaviour of interference filters changes with the angle of light incidence, transmission and rejection intervals moving to lower wavelengths when the angle of light incidence deviates and decreases compared to the normal (90 degree) incidence. Such changes, however, are negligible for small deviations from the normal incidence (-10-15 degrees), and can be further minimized using an appropriate filter design according to established technology. To solve the above problems and needs, one would ideally aim at combining together multiple "single excitation band" light sources, each providing one of the wanted excitation bands so that a single-band excitation filter is used, hence combining high transmission with high spectral resolution at the band edges. Light Emitting Diodes (LEDs) are in principle especially suited for such purpose, in view of their intense emission in comparatively narrow wavelength ranges (e.g. from 5-10, to

30-60 nm). In addition, LEDs are easily controlled and finely tuned by electronic driving circuitry, and can be switched on and emit light very rapidly (<100 nsec). Also, LEDs can be flash operated at high current, high light output for short to very short time periods, hence a set-up comprising parallel LEDs would permit exposure of the specimen to multiple coincident or synchronized pulses of high-intensity light of controlled wavelength/s.

In spite of the recent introduction of LEDs of ever increasing output power, a single LED may none the less provide an insufficient light intensity for the purpose at hand. For instance, a very narrow excitation band may be required, or the excitation interval required may not exactly superimpose the available LED emission, so that only a portion of the LED emission light power can be profitably used. In addition, while the spectral variety of high-power LEDs has greatly increased, none the less the spectrum of interest for fluorochrome excitation is far from evenly covered (e.g. little narrow band LED emission light is available in the green-yellow range around 550-580 nm, as would be profitable for certain commonly used fluorochromes such as lissamine Rhodamine and others.

Especially for use with fluorescence visualization and/or imaging systems or microscopes, LED based light sources have been proposed as follows: (i) multiple, different wavelength/colour LEDs arranged in groups or arrays, to permit regulation of colour balance in the overall light output (WO2007/111735A2, WO2006/136406A1 , CN2630877YY);

(ii) a LED with its individual optics, or with its individual optics and its band selection filter (excitation filter), or multiple, interchangeable such combination of LED, optics and filter, is/are used as a single-band source, or as multiple, interchangeable single-band sources for fluorescence microscopy, using either transmitted light excitation (WO2006/072886; US2007/0211460), or incident/reflected light excitation (WO2004/088387, US2007/0053058), or is sent to the specimen via an independent optical pathway (JP2006301523A, US2008/0043324A1, US2008/0013166A1 , WO2007/090591A1 , EP1528421 B1 , US20G4/0061070A1);

(iii) light from one or multiple LEDs is focussed to the entry of an optical rod, to yield an even light beam, which is then sent to the excitation pathway of a microscope (JP2006337925A, US2007/0253733A1 , WO2006/059900A1);

(iv) light from one or multiple LEDs and their individual optics, with/out the relevant excitation band selection filter, is sent to bundles of optical fibres, hence multiple such bundles are collected together at random (JP2004212469, JP2006038947, DE10339618A1), or in an orderly fashion (PCT/IT2007/000470), and the resulting light beam is sent to the fluorescence visualization/imaging apparatus, or is further carried to an optical rod, hence to said fluorescence visualization/imaging apparatus, such as a microscope (JP2004212469);

(v) light from multiple LEDs emitting at different, separate wavelengths is collected together by means of dichroic mirror/s (JP20050101296) or optical/dichroic prisms (JP2005215522) hence is sent to a fluorescence visualization or imaging system, such as a microscope; (vi) several recently introduced commercially available set-ups for fluorescence microscopy rely on single-band LED arrays, excitation light from different arrays (of differing wavelength) being collected together by means of dichroic mirrors. Such an arrangement works well when the relevant LED emission intervals are sufficiently separated in their spectra (e.g. by 30 nm or more), while rapidly decreasing in efficiency for closer wavelength ranges. Hence, only a small number of LED excitation modules can be effectively combined by means of dichroic mirrors, so that the latter approach can address the simultaneous visualization or imaging of an equivalent number of fluorochromes. (vii) individual LEDs emitting at different wavelengths, their optics, and the relevant wavelength specific excitation filter, dichroic mirror, as well as emission filter are assembled together in further commercially available set-ups, to compose interchangeable sets for use in a fluorescence visualization/imaging system, such as a fluorescence microscope, much the same way as "fluorescence filter cubes" are commonly used and interchanged.

In light of the above, it is object of the invention to provide a multiple

5 band excitation set-up to be used in connection with any optically suitable reflected fluorescence microscope, or fluorescence visualization or imaging system, or incorporated into, or implemented onto a variety of reflected fluorescence microscopes, or fluorescence visualization or imaging systems, suitable to collect light from a multiple LED operated

1.0 light source, to select the wanted wavelength ranges, in order to convey a single beam of mixed excitation light comprising said combination of selective excitation light bands to a specimen, or to the excitation path of said microscope or visualization or imaging system in the same or in a similar way as the microscope's standard light source would be intended 5 to do.

It is another object of the present invention that multiple band excitation light from the above set-up is sent to the specimen directly by means of appropriate optics, or by means of matched multiple band dichroic mirror/s, hence appropriate optics (such as the microscope's0 lens/es), while the resulting fluorochromes' emission is let through by said dichroic mirror/s and/or selected by means of appropriate multiple band emission filter/s, and is optionally colour-corrected as required by means of selective emission band rejection (colour correction) filter/s.

It is another object of the present invention that a multiplicity of5 fluorochromes of appropriately differing spectra are either revealed simultaneously, or are differentially revealed and compared in selected, sequentially visualized or imaged groups of fluorochromes.

It is also an object of the invention to provide electronic driving circuitry to switch each LED on and off, as well as to regulate its light0 output intensity by high frequency switching. Moreover, it is part of the present invention that LED driving electronic circuitry is arranged in such a way as to be able to provide high current, short to very short duration power pulses to any number of wanted, simultaneously, synchronously or sequentially operated LEDs. It is also part of the present invention that said driving electronic circuitry is interfaced to a computer, for system operation, control and/or programming. Hence, it is an object of the present invention that each and every of said excitation wavelength 5 ranges (excitation bands) is rapidly switchable on-and-off and tunable in intensity, in such a way as not to interfere with any of the other simultaneously operated excitation bands.

It is specific subject matter of this invention a multi-band fluorescence excitation system, said system having an overall reference

10 axis and comprising a light source providing light beams, means for filtering said light beams of said light source, and means for mixing said light beams coming from said filtering means, in order to obtain at least one output light beam having a combination of wavelength ranges, characterised in that said light source comprises one or more light source

15 modules, each light source module comprising a LED mounting support, onto which an array of LEDs are mounted, and being mounted in turn onto a module mounting support, each one of said LEDs being capable of emitting a light beam at a wavelength range, and in that said filtering means comprise one or more modular excitation band selecting filter

20 elements each one corresponding to one of said light source modules, each excitation band selecting filter element having a light receiving area and being capable to receive the light beam of each one of said LEDs of the respective light source module on a corresponding portion of said light receiving area, whereby selecting a band of the light beam wavelength

^• 25 range of each one of said LEDs.

Always according to the invention, said LEDs and said excitation band selecting filter elements could be arranged in such a way that the light beam emitted by each one of said LEDs has an angle of light incidence of substantially 90°, normal incidence, with respect to the30 corresponding portion of said light receiving area of said excitation band selecting filter element, said angle of light incidence being comprised within such a maximum deviation from said normal incidence such that the spectral selection operated by said excitation band selection filter elements is substantially superimposable for all light beams from any LED in each light source module.

Still according to the invention, said light source modules could be removably arranged side by side, so as to form a matrix composed of interchangeable light source modules, and said excitation band selecting filter elements, each one corresponding to one light source module, are removably arranged side by side, so as to form a matrix composed of interchangeable excitation band selecting filter elements.

Further according to the invention, said system could comprise one or more light beam separators for preventing the diffusion of stray light among LED light beams.

Always according to the invention, each one of said excitation band selecting filter element could be arranged at a respective first angle of tilt with respect to said overall axis of the system, so that the angle of incidence of each light beam from each one of said LEDs is of substantially 90° with respect to the corresponding portion of said light receiving area of the relevant excitation band selecting filter element.

Still according to the invention, each one of said LEDs could comprise its own collimating and projecting optics, each one of said LEDs being arranged at a respective, individual second angle of tilt with respect to the said LED mounting support in said light source module, so that each

LED's light beam is aligned with the LED's collimating and projecting optics, in such a way that the light beam from each one of said LEDs is collimated by said optics of each LED and projected to the corresponding portion of said light receiving area of said excitation band selecting filter element, therethrough converging onto the light mixing means.

Further according to the invention, said module mounting support could be so shaped that each light source module is interchangeably mounted at a respective third angle of tilt with respect to said overall reference axis, so that the light beams from all said LEDs in each light source module are collimated, projecting onto the corresponding portion of said light receiving area of said excitation band selecting filter element, therethrough converging together onto said light mixing means. Preferably according to the invention, the LEDs of each light source module emitting in substantially the same wavelength range could be mounted at adjacent locations, and the corresponding excitation band selecting filter element area portions are adjacent as well and/or compose a single homogeneous area of said filter.

Always according to the invention, said system could comprise first optics for collimating said output light beam coming from said mixing means.

Still according to the invention, said system could comprise second optics arranged between said filtering means and said mixing means, whereby reducing the overall length and size of said system, without increasing the maximum deviation from a 90° angle of light incidence, normal incidence, of said light beams at the entry of said mixing means.

Further according to the invention, said mixing means could comprise an optical rod and/or an optical fibre and/or a bundle of randomly arranged optic fibres or of optical rods, so that said at least one mixed output light beam is obtained by multiple internal reflections.

Always according to the invention, said system could comprise electronic driving circuitry connected to each one of said LEDs of said light source modules, said electronic driving circuitry being capable to switch on and off each LED and to regulate the light output intensity and the switching frequency, so as to switch on-and-off and tune the intensity of the light of each and any excitation band of each LED without interferences with any of the other excitation bands obtained from other LEDs, said driving circuitry is capable to be interfaced to a computer, for system operation, control and/or programming.

Still according to the invention, said LED mounting supports are heat-transmitting, said module mounting support is heat-transmitting, and the system comprises fins coupled to said module mounting support and/or forced ventilation means and/or cooling fluid means and/or heat pipes and/or Peltier.

Further according to the invention, said system could be coupled to an observation or visualization device, or to a microscope, which includes relevant optics and/or multi-band dichroic mirror/s to reflect the excitation light beams onto a specimen, and/or multi-band emission filters to permit observation or imaging of the relevant excited fluorochrome labels.

This invention will be now described, by way of explanation and not by way of limitation, according to its preferred embodiments, by particularly referring to the attached drawings, in which: figure 1a shows a schematic view of a first embodiment of a modular multi-band fluorescence excitation system according to the present invention; figure 1b shows a schematic view of the system according to figure

1a in which several LED modules are arranged side-by-side; figure 1c shows details of excitation band selection filter/s; figure 2a shows a schematic view of a second embodiment of a modular multi-band fluorescence excitation system according to the invention; figure 2b shows a schematic view of the system according to figure 2a, in which several LED modules are arranged side-by-side; figure 3a shows a schematic view of a third embodiment of a modular multi-band fluorescence excitation system according to the invention; and figure 3b shows a schematic view of the system according to figure 3a, in which several LED modules are arranged side-by-side.

The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. First embodiment

Referring to figure 1a it is shown a modular multi-band fluorescence excitation system, which has an overall reference axis A and comprising a light source LED-module V, which in its turn comprises, by way of example, eight LEDs 3', each one coupled to a first heat-transmitting support 2'. Moreover, each LED 3' comprises its own light collecting, collimating and projecting optic 4'.

Light beams from said LEDs 3' are hence projected to an excitation band selection interference filter element 6'. The arrangement and the dimensions of the set-up are such that light from even the most lateral

LEDs 3' in the light source module V (in the figure the uppermost and lowermost LED 3', respectively) will reach the filter receiving area at an angle of about 90°, precisely at an angle different from 90° (normal incidence) within such a maximum extent as to ensure that the spectral selection operated by the excitation band selection interference filter will be superimposable for all light beams from any LED 3' of the light source module 1'. After the LED optics 4', before and adjacent to the excitation light selection filter 6', a light beam separator 5 is provided to prevent diffusion of stray light between the single-LED 3' light beams and other LEDs' filter 6' area portions.

Upon selection of the wanted excitation wavelength ranges (excitation bands), light beams from each LED 3' are projected to the entry of an optical rod or an optical fibre 7, within a proper angle, ensuring efficient light entry into said rod or fibre 7.

Single-LED 3' light beams are hence collected together to form a mixed light beam, and an even distribution of light from each LED 3' within the mixed beam is obtained by multiple internal reflections within said optical rod or optical fibre 7. Bending or arching of said optical fibre 7 onto at least two perpendicular planes may be aimed at increasing the effect of internal reflections and light mixing within said mixed light beam. At the exit of said optical rod or optical fibre 7, mixed light is collimated by optics 8 and is sent to a specimen (not shown in the figure), or to the excitation path of a fluorescence visualization or imaging system, such as a reflected fluorescence microscope.

Figure 1b shows a plurality of light source modules 1 '...1 ^π arranged side-by-side, so as to form a matrix light source. Said light source modules ^{^} 1'...1 ⁿ are coupled to a second heat-transmitting and/or heat-dissipating support 9. The excitation light selecting filters 6'...6 ⁿ (each one of said selecting filters 6'...6 ^π corresponds to one light source module 1'...1 ⁿ ) are mounted side-by side immediately after the light separator 5, which prevents stray light diffusion between individual LEDs 3, as well as between said light source modules 1 '...1 ⁿ .

The arrangement and the dimensions of the set-up are such that light from even the most lateral LEDs 3 in the most lateral light source modules 1 will reach the respective filter element 6 surface at an angle of substantially 90°, and within such an allowable maximum deviation from 90°, that the spectral selection operated by said filter 6'...6 ⁿ will be superimposable for all light beams derived from any LED 3 in the different light source modules 1.

Said second heat-transmitting support 9 supports the light source modules 1 and dissipates heat into air by fins, with or without a forced ventilation. Alternatively, heat will be dissipated by means of a circulating coolant, or heat-pipe/s and an appropriate heat-exchanger, with or without an intervening element to allow heat exchange and to maintain LEDs 3 at a low temperature, such as a Peltier or similar elements. The set-up is intended to accommodate and collect together light from LEDs 3 emitting at different, as well as at more or less extensively superimposed, or at identical wavelengths. By way of example, one such light source module 1' could comprise four identical (i.e. having the same light spectrum), adjacent LEDs 3' (LEDs 3'a), and two further couples of identical, adjacent LEDs 3' (LEDs 3'b and 3'c, respectively). Hence, the relevant excitation band selection interference filter (6', the figure shows its light-receiving surface) comprises several distinct areas of differing surface treatment (hence of differing spectral behaviour), which in the above example are intended to select the same excitation band from the group of four identical LEDs 3a, i.e. "a" area portion of the filter 6' is arranged to receive the light from the four identical LEDs 3'a, while "b" and "c" area portions of the filter 6' are arranged to select the light from the other two groups of identical LEDs 6'b and 6'c respectively. In an alternative example, it is provided another filter 6' for the light source module 1'. This filter comprises one light selection area 6'd identical throughout the filter, being intended to work with multiple identical LEDs 3' to obtain a higher light output intensity. Each of said filter elements 6'...6 ⁿ , and/or each homogeneous excitation light selecting portion area of each said filter element 6'...6 ^π , will be designed and produced according to state-of-the- art technology to yield both high transmission as well as high spectral resolution at both edges of the desired transmission band.

As mentioned above, preferably the LEDs 3 emitting in one and the same wavelength range will be mounted at adjacent locations, so that the relevant filter element 6' areas required will also be adjacent, hence will be economically processed all at once. For specific purposes and needs, such as for the system set-up and experimental use, filter elements 6'...6 ⁿ each serving its intended light source module 1 '...1 ⁿ may be made to mount individual, removable and interchangeable single LED fιlter/s or filter areas (not shown).

Light collected and collimated from LED 3'...3 ⁿ sources will contain at least partly non parallel light beams, while light beams deriving from centrally located versus laterally located LEDs 3'...3 ⁿ and LED light source modules 1'...1", will reach the corresponding portion of the light receiving area of the filter element 6'...6 ⁿ at differing angles of incidence. While the overall system will be aimed at minimizing such variations in light incidence angles, filter elements will be designed according to available surface coating technology to minimize the spectral effects of such varied light incidence angle.

For the sake of simplicity, other elements and parts are not shown in the drawings. These include the use of a bundle of randomly arranged optic fibres, or of an optical rod, in place of a single optical fibre as depicted. Electronic driving circuitry is also part of the preferred embodiment, to switch each LED on and off, as well as to regulate its light output intensity by high frequency switching. In addition, such LED driving circuitry is to be arranged in such a way as to be able to provide high current, short to very short duration power pulses to any number of wanted, simultaneously, synchronously or sequentially operated LEDs. Said driving electronic circuitry is also intended to be interfaced to a computer, for system operation, control and/or programming. Hence, each and every of said excitation wavelength ranges (excitation bands) is rapidly switchable on-and-off, and tunable in intensity, without interfering with any of the other excitation bands being operated, or potentially available, nor requiring per se the interchange of optical or other parts in the visualization or imaging system or microscope.

Therefore, each and all excitation band/s can be rapidly and independently adjusted in intensity, or switched on-and-off entirely as required.

The resulting mixed excitation light beam can include any desired combination of wavelength ranges, including multiple adjacent, and/or partly or completely superimposed wavelength ranges, for simultaneous and/or sequential excitation of one, two, or multiple fluorescent molecules or probes, Said mixed excitation light beam is then sent to the object of interest by means of relevant optics, or reflected by appropriate multi-band dichroic/s, included in a further observation or visualization system, or in a microscope's excitation pathway, hence focussed onto the specimen, to excite the relevant fluorescent molecules.

Second embodiment

Referring to figure 2a e 2b, it is shown a second embodiment of the invention. The system is provided with additional optics 10 to reduce the overall length and size, without increasing the maximum angle of light incidence at the entry of the optical rod or fibre 7, hence maintaining such maximum angle of incidence within the required limits for an efficient entry into the optical rod or fibre 7, of light from each and all LEDs 3 and light source modules 1 '...1 ^π .

More in detail, light beams from the different LEDs 3 of light source modules 1'...1 ⁿ is converged by said additional optical element/s 10, hence collimated to the entry of said optical rod or fibre 7. This embodiment may also allow the use of a large number of LEDs 3 and/or light source modules 1'...1 ⁿ , so that the angle of light incidence for light beams from the most lateral LEDs 3 and light source modules 1'...1 ⁿ remains within an allowable range, without increasing the overall length and/or size of the system.

Third Embodiment

Figures 3a and 3b show a third embodiment of the invention, suitable to maintain the angle of incidence of the LEDs' light beams onto the selecting filter elements 6'...6 ⁿ closer to 90° (normal angle), as well as to make each LED's optics operate in axis with its LED light beam, or close to it. In particular, each single LED 3, each light source module 1 "...1 ^π , as well as each module's filter element 6'...6 ⁿ , or any of these, are mounted at their own proper angle of tilt each, with respect to said overall reference axis A of the system, said angle depending on the distance of said LED 3, light source module 1 '...1 ⁿ , and/or filter element 6'...6" from the overall optical axis of the system. This way, the following results are achieved:

- the angle of incidence of light from the most lateral LEDs 3 and light source modules 1'...1 ⁿ onto their respective portions of the light receiving area of the excitation light selecting filter will be made to be substantially close to 90° (normal incidence); and - single-LED optics will be made to work in axis to the LED 3 emission axis, or close to it.

In particular, the LED support element within each light source module 11 is, by way of example, so shaped, as to provide appropriately oriented mounting and heat transfer surfaces for each individual LED 3 in the light source module 1'. The heat-transmitting and/or heat-dissipating support 9 onto which the various LED modules 1'...1 ^π are mounted is also shaped in such a way, as to provide appropriately oriented mounting and heat transfer surfaces for each individual light source module 1'...1 ⁿ . In turn, each filter element 6'...6 ⁿ is mounted at an appropriate angle of tilt, always with respect to said overall reference axis A, adjacent to the light beam separator 5, or onto it.

The present system has many advantages, among which the following ones are recalled:

- it collects together light from multiple LEDs, hence combining light bands of the same, and/or partly superimposed, and/or different wavelength ranges, to provide a mixed light beam of higher intensity, and comprising the different wavelength ranges as required;

- it selects the desired excitation intervals from the light emitted by each LEDs, or by each group of adjacent LEDs emitting at the same wavelength, at high spectral resolution and high signal-noise ratio;

- it makes each and every one of said excitation bands independently "on-off" switchable, as well as finely tunable in intensity; - by means of said excitation band and mixed light beam, it is capable of exciting and revealing a multiplicity of fluorescent molecules of appropriately differing spectra, either singly or simultaneously (i.e. at one and the same time);

- it reveals the above simultaneously excited fluorochromes in easily distinguished visualisation or detection colours/wavelength ranges

(including relevant infrared band/s);

- it is capable of revealing complex multi-fluorochrome profiles as subgroups of distinct signals in the form of easily distinguished colours/wavelength ranges, in such a way that the visualization/imaging of each groups of flurochromes can be rapidly switched on-and-off in turn, so that the single flurochromes and fluorochrome groups are revealed in the wanted sequence/s and association/s;

- within said system, each light source module can be interchanged with others, or novel ones can be added or mounted, in a simple and quick way, hence providing an easily tailored system suitable to satisfy a wide diversity of requirements, as well as to permit easy and economical updating to new or novel requirements, e.g. new excitation wavelength intervals for novel fluorochromes.

This invention have been above described by way of illustration, but not by way of limitation according to its preferred embodiments and it should be understood that those skilled in the art can make other modifications and changes without departing from the scope of the invention as defined in the following claims.