WO2009001390A1 - Adjustable multi-band excitation and visualization / imaging system for simultaneous viewing multiple fluorescence - Google Patents

Abstract

In the bio-medical field, fluorescence visualization or imaging systems and microscopes are used to observe and/or image fluorescent molecules within cell or tissue preparations. A device is described, which receives light from one or more light sources, such as wide spectrum lamps and/or light emitting diodes (LEDs), separates ultraviolet (UV) from visible and near infrared light components, selects multiple excitation bands including at least one in the UV range, each of which is independently switchable and adjustable in intensity, hence recombines excitation bands into a single multiple-band excitation light beam which goes to the excitation path of a reflected fluorescence visualization or imaging system or microscope.

Description

ADJUSTABLE MULTI-BAND EXCITATION AND VISUALIZATION / IMAGING SYSTEM FOR SIMULTANEOUS VIEWING MULTIPLE FLUORESCENCE

BACKGROUND AND FIELD QF THE INVENTION

Fluorescence based methods are used in the bio-medical field to reveal and analyse 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 the relevant images. In today's applications, the possibility of labelling and observing at the same time multiple (i.e. more than 2 or 3) different fluorescent molecules or labels in one and the same 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.

A major aspect in multiple simultaneous fluorescence is that each label remains recognisable, hence it is to be ensured that each label results in an easily distinguished colour, different from those produced by the other labels present. The distribution and concentration of the molecules of interest are also important issues, multiple simultaneous visualization being easy to use when the items/molecules of interest are present in similar local concentrations, thus resulting in labelling signals of consistent and comparable intensity. However, most biological molecules are found in very different and widely variable amounts, such differences and changes 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 multiple orders of magnitude, both between as well as within preparations.

The^' present invention relates to a device and method, to be used in fluorescence visualization and/or imaging and microscopy, which permits the simultaneous excitation, observation and/or imaging of multiple fluorescent molecules excited and emitting at different wavelength ranges within a preparation. More particularly, the invention relates to a means to obtain multiple, simultaneous, well determined, selective wavelength intervals of excitation light (excitation bands), including at least one UV band (at any suitable wavelength), several visible light bands, and near infrared light band/s, which are combined in a mixed excitation light beam, and are then reflected by appropriate multi-band dichroic/s through the microscope's lens and onto the specimen to excite the relevant fluorescent molecules. Each excitation band can be rapidly and independently adjusted in intensity, or switched on-and-off entirely, by use of a band-selective rotating notch-type rejection filter which leaves all other bands unaffected, and/or by the relevant driver circuitry for LED operated excitation bands. When such excitation bands are used to excite appropriate fluorescent molecules, or fluorochromes in the preparation/s, the resulting multiple fluorescence emission bands are made visible and/or imaged by means of appropriate multi-band emission filter/s, and emission band rejection filter/s as needed, in such a way as to allow their highest sensitivity visualization (or imaging, or detection), but also to permit their viewing as clearly distinct colour labels in the blue, cyan, green, yellow, orange, red, violet/purple, and/or white ranges, plus infrared emission band/s. Appropriate combinations of single- and multi-band excitation and related filters, selective excitation-rejection filters and/or the relevant LED- driving circuitry, multi-band dichroics, multi-band emission and emission band rejection filters permit a multiplicity of visualization and/or imaging approaches, including simultaneous multi-label, as well as comparative sequential single- and multi-label visualization and/or imaging.

DESCRIPTION OF THE RELATED ART

A reflected (or incident) 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 fluorescent 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; (f) 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 imaging or recording apparatus. Nowadays, virtually all 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, one would want to be able to see and 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 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 make the most of the powerful approach of simultaneous viewing / imaging multi-labelling methods in fluorescence microscopy and related areas. The central issue can probably be defined as the need to maintain each fluorochrome's signal fully separate and independent from each of the others (i.e. without cross-talk of any of the labels in any other labels' excitation and emission intervals), while distinctly visualizing or imaging each label in its full dynamic range (i.e. from weakest to brightest localization signal), as well as permitting the distinct recognition and detailed analysis of each label, in each and every of its locations, in comparison with each other label used at the same time. Relevant issues include:

(i) recognizing each fluorochrome independently as a distinct colour signal;

(ii) inclusion of UV excitation/s, for blue-emitting fluorochromes or else;

(iii) separating "combination" versus "single label" colour signals; (iv) adjusting, as well as rapidly blocking, each and every excitation band;

(v) use of wide and/or of narrow wavelength spectrum light sources.

Recognizing fluorochromes as distinct colour signals.

When using multiple fluorochromes together, the starting issue is that none of the labels "cross-talks" with any of the other labels' chosen

- excitation and/or emission interval, especially with high intensity labelling. In general, both excitation and emission intervals must be properly restricted, in order to minimize the effect of the overlapping excitation and emission "tails" between fluorochromes with adjacent excitation and/or emission spectra.

A further, immediately related issue is that labelling ought to result in distinct colours such as blue, cyan, green, yellow, orange, red, violet-purple, or white, so that each labelling can be easily and reliably distinguished from the others, be it visually or upon digital imaging and/or recording. Conversely, it would be impervious to distinguish between multiple shades of one or a few colours, short of using a spectroscopic approach. In double fluorescence, such issue was addressed previously, to the effect that a distinct green colour signal ought to be combined with a distinct red (i.e. not yellow/orange) signal, in order to ensure that where the two labels coincide a distinct yellow co-localization signal will show (Ferri et al., 1995).

In the present invention, the latter issue is addressed in several ways. Firstly, the invention permits the general use of UV-excited, blue- emitting fluorochromes in simultaneous visualization or imaging with any other colour label, thus increasing the number of distinct colour signals which can be used. Secondly, "colour band rejection" filters are used to optionally "remove" appropriate portions of the emission range/s, thus allowing the operator to rapidly switch between: (i) maximum signal and maximum sensitivity conditions tailored to the fluorochrome/s in use; (ii) "distinct colours" visualization to address co-localization questions. In addition, the sensitivity of most digital sensors to infrared light permits imaging and recording of near infrared emitting fluorochromes as further distinct signals, in combination with appropriately selected fluorochromes emitting in the visible range. The precise "colour" assigned to the infrared fluorochrome/s in the imaging system will depend on the spectral sensitivity of the elementary sensors in the digital camera or imaging system used. In general, sensors responding to red and/or to blue visible light are most responsive to near infrared light, so that the use of a near infrared-emitting and a green-emitting fluorochrome can result in distinctly imaged "colours" for both the two separate labels and their combination.

UV excitation for blue-emitting fluorochromes.

An especially convenient, presently hardly usable label colour in simultaneous viewing or imaging multiple fluorescence is blue, both as single label, as well as in combination with green, yellow or red labels, hence resulting in cyan, combination-green, and purple-violet labelling, respectively. Unfortunately, blue-emitting fluorochromes commonly used for labelling of antibodies and other probes (such as AMCA and similar ones) require UV excitation in the 340-375 nm range, well below the lowest, approximately 390 nm excitation bands presently provided by state-of-the- art double- and triple-fluorescence excitation filters.

In fact, so far available optical coating materials used to produce multi-band excitation filters severely interfere with UV transmission below such limit (~390nm). A major scope of our invention is to provide one (or more) UV excitation band/s, in whatever required wavelength interval below 390 nm, and operating at the same time as the other excitation bands. This is achieved by separating UV light, selecting the wanted UV excitation band/s, hence re-combining UV and other excitation bands in one and the same multi-band excitation beam. Patent DE4221063 described the splitting of light from a wide spectrum source, using a dichroic mirror, into beams of different wavelengths, which are then carried through further dichroics, optical lenses (to compensate for optical aberrations) and excitation filters, to select a UV and a green excitation bands which are then recombined into a single excitation beam. The present invention makes use of two optical pathways of equal length, thus requiring no optical lens component and resulting in a simpler system of higher optical efficiency. In addition, it involves the selection of two, three or more excitation intervals using one and the same optical pathway, within which each excitation band is also independently modified without affecting the other excitation intervals carried through the same optical pathway.

"Combination" versus one-label colours: more distinct labels.

Certain perceived, or imaged colours (e.g. green, yellow, orange, violet/purple) can result from light containing either one wavelength range only (e.g. ~575nm light, yellow), or two spectrally distant wavelength ranges, each of which on its own would result in another different colour (e.g. ~520nm and ~620nm light, green and red on their own, showing together as yellow). Both the human eye and digital imaging systems largely handle colours by use of elementary sensors most sensitive in the blue, green and red ranges, so that single- and combined- wavelength light can stimulate the same two types of sensitive elements and result in the same visual perception, or recorded colour image. The present invention permits distinction of single-wavelength colour labelling versus apparently identical, dual-wavelength colour labelling, by means of comparative, differential visualization or imaging. This way, a further multiplicity of labels can be used, while retaining complete label distinction.

Adjusting and switching-off single excitation bands.

In most settings, the molecules of interest can vary enormously in overall as well as in local concentrations, hence resulting in a very wide range of labelling intensities, both across specimens and conditions, and within specimens. The detection and analysis of such inter- and intra- specimen differences themselves is often the major scope for investigation. Hence, while analysing the expression and spatial relationships of different labelled items or molecules, one ought to be able to adjust signal intensities to a fairly wide extent, easily and rapidly switching each between its maximum, whatever intermediate "optimum" and a virtually "zero" signal intensity. This way, the user will not only be able to balance signal intensities in such a way as to produce an appropriately descriptive visualization or recorded image, but will also be in a position to ascertain whether any, however weak, signal of "fluorochrome A" coincides with the location of even the strongest "fluorochrome B" or "C" etc, and vice-versa. Understandably, such adjustments ought to be carried out largely on the excitation side, in order to minimize the pointless loss of the fluorochromes' ability to fluoresce (progressive fading.due to exposure to excitation light).

Patents US5371624 and US5710663 describe fluorescence microscopes endowed with two and up to four excitation bands, respectively, both of which permit to affect each of two excitation bands, in turn, out of a plurality of narrow-band excitation bands provided by the microscope, by means of rotating interference filters. More specifically, each of such filter elements will affect two excitation bands only in an alternative way (i.e. will affect their intensity ratio), by being tilted at any angle between a limit position at which it will affect one of the excitation bands at a pre-determined degree of suppression ("low transmittance"), to the alternative limit position, at which it will affect the other excitation band at the corresponding predetermined degree of suppression. While complete suppression of either excitation band is not ensured in the latter invention, only one excitation band will be reduced while the other one will be less affected, or entirely unaffected when the first undergoes the pre- set degree of suppression. According to said invention, several highly specific, essentially dedicated, excitation tuning filters appear to be required for each multi-band filter set, resulting in further complexity, and in the need to change both multi-band filter sets and their own excitation tuning filters whenever one is to switch to a differing combination of excitation / emission wavelengths. Conversely, the present invention in which each excitation band is adjusted by "its own" selective rejector, introduces at least three major advantages: (i) maximum rejection can in any case be effected at a normal angle of light incidence, at which optical interference of each rejector's surface coating is at its maximum efficiency; (ii) hence, very deep, virtually complete suppression of each excitation band can be obtained, as is often required with high intensity and/or high sensitivity labelling; (iii) each rejector can be designed to provide a comparatively broad rejection band in its selective range (e.g. "blue excitation rejector", or "green excitation rejector"), compatible with a range of different multi-band excitation filters, thus resulting in a far simpler and more flexible system.

Patents US6747280, WO0036451 and related ones describe a method to independently regulate the intensity of each of 2 or 3 excitation bands, by variably sliding into the excitation pathway a wavelength- selective filter per each band, which will absorb (or reflect) a variable portion of the excitation light. This is obtained by means of either surface regions arranged in a graded transition between high and low transmission, or absorbing surface elements gradually changing in size or distance as one moves in the direction of sliding of the filter. Patent US203227674 describes a similar use of filters having a plurality of zones with different notch-like spectral transmission characteristics capable of blocking a specific narrow wavelength band and transmitting the remainder of wavelength bands, arranged in such a way that the specific narrow wavelength band varies continuously along the direction of sliding of the filter. While production of such graded interference filters is complex, differences in attenuation across the light beam diameter are not unlikely, and would result in uneven specimen excitation. In Patent US6594074, light from each of several laser sources (of un-specified wavelength not comprising UV) is transmitted to a variable degree by two coupled, identical selective interference filters rotating in opposing directions, so that any shift in the light beam due to passing through said filters at a varying angle of incidence is also compensated. In the present invention, beam shift is not a significant problem, and far wider-spectrum, wide notch-type selective rotating rejection filters are used including at least one operating in the UV range. At variance with previous art, the present invention involves the use of band ^" selective, wide notch-type rotating rejection filters of comparatively wide blocking regions (e.g.: matching the majority of blue- light excitation filters by operating in the whole 450-495nm range, with blocking OD>3 or higher), with no need for critical matching to each multi- band filter set, nor any uneven effect on the excitation light beam. Furthermore, such single-band rejection filters are designed to reach deep suppression (up to OD 3 to 4, or possibly further), as required depending on the excitation band and fluorochrome used, as well as on the detector (eye or camera) sensitivity to the fluorochrome's emission band and its transmitted portion. An additional consideration concerns spectral changes in interference filters, when they are rotated in such a way that light hits them at a decreasing angle, compared to the normal (90°) incidence. The relevant rejection interval not only shifts to lower wavelengths, but also tends to broaden and to decrease in its degree of rejection (optical density decreases). Hence, wherever high blocking is required, one ought to operate with a rejection filter which "blocks" at the normal angle, while progressively lets through the corresponding excitation light with progressive rotation toward 40-45° (the blocking interval shifts to progressively lower wavelengths). It is only when a lower degree of blocking is deemed sufficient, that a reverse approach may also be appropriate, in which the relevant excitation band is fully transmitted at a normal angle, and progressively blocked with rotation, maximum blocking being achieved at 35-40° or thereabout.

LEDs as light sources for fluorescence visualization and microscopy.

In recent years, the increase in output power of Light Emitting

Diodes, or LEDs, has made these suitable as light source/s in microscopy. This is especially the case in fluorescence microscopy, in view of the narrow emission spectra of single-colour LEDs, and of their being easily controlled and finely tuned by electronic driving circuitry. However, even assuming a perfect match between each fluorochrome and its chosen LED-operated excitation interval, more than one or multiple identical LEDs may be required per each wavelength in many cases. In fact, with simultaneous viewing multiple fluorescence, emission intervals, too, need to be properly restricted, in order to maintain complete separation between spectrally adjacent fluorochromes. Thus, reduced efficiency in the visualization of fluorochromes' emission may need to be compensated by using higher excitation intensity/ies, such as can be obtained by summing the light emitted by two or more LEDs operating at the same wavelength. Only for LEDs emitting at wavelengths sufficiently separate from each other, light can easily be collected using dichroic mirrors, or prisms with appropriate, wavelength specific dichroic reflecting surfaces (as in Patent JP2005215522). Alternatively, Patent JP2006038947 proposed to input the light emitted by several LEDs to the incident ends of optical fibers, which were then randomly bundled to form a larger fiber bundle to guide the output light to the microscope, and therethrough to the specimen.

On the whole, several important points ought to be pointed out: (i) dichroic mirrors or surfaces cannot collect together light from multiple LEDs emitting at the same, or adjacent wavelength/s; (ii) collection of light from LEDs emitting at either the same and/or diverse wavelengths ought to result in the even distribution of the light emitted by each LED throughout the final mixed light beam, so that an even field illumination is obtained at each wavelength in the easiest possible way; (iii) LEDs emit light almost instantly upon being powered (<100ns or faster), hence they can be switched on and off at will, and finely controlled by high frequency switching; (iv) LEDs can be flash operated at high current, to provide very intense 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.

According to the present invention, each LED's emission is captured into one each of multiple "primary" bundles of optic fibres. Such primary bundles are joined together at their output end to form a larger "secondary" bundle, in such a way that the fibres derived from each primary bundle are orderly distributed throughout the cross-section of the secondary bundle. Hence, light coming from each LED will be evenly distributed throughout the mixed, output light beam in a very simple way. In addition, multiple LEDs emitting at sufficiently separate wavelengths can be mounted per each "primary" bundle, by means of simple dichroic mirrors. Hence, it becomes possible to effectively collect light from multiple LEDs emitting at the same, as well as emitting at diverse wavelengths, as needed. At the same time, any number of such LEDs can be flash operated synchronously or sequentially. As part of the present invention, electronic driving circuitry is provided, to control each LED operation and emission intensity, and to permit their alternative, synchronized or simultaneous use, including high current short-time-period or flash operation/s, as well as their regulation, control and/or programmed operation by interfacing to a computer and the relevant software.

SUMMARY QF THE INVENTION

The present invention has been made in order to address and solve the difficulties and problems described above, with the object of providing a means to:

(i) operate one or more UV excitation band/s, of any suitable wavelength below 390 nm, together with other excitation bands in the visible and/or near infrared range/s in multiple reflected fluorescence visualization or imaging;

(ii) make each and every of the multiple excitation bands in use independently "on-off switchable, as well as finely tunable in intensity;

(iii) reveal a multiplicity of fluorescent molecules of appropriately differing spectra simultaneously, i.e. at one and the same time;

(iv) reveal multiple, simultaneously visualized fluorochromes in easily distinguished visualisation or detection colours (including the detection and imaging of infrared band/s);

(v) reveal single- versus mixed-fluorochrome labelling of the same apparent colour in such a way, that the two are reliably distinguished;

(vi) reveal complex multi-label profiles as organised sequential, comparative profiles of easily distinguished colours, in such a way that such profiles can be easily and rapidly revealed in a determined sequence and association. According to the present invention, a multiple band excitation set-up is provided, to be used in connection with any optically suitable reflected fluorescence microscope, or reflected fluorescence visualization or imaging system. It is an object of the present invention to provide a system which can be incorporated into, or implemented onto a variety of reflected fluorescence microscopes, or reflected fluorescence visualization or imaging systems, by providing an article and method to collect light from a light source, such as a microscope's light source, as well as from additional or alternative light source/s, hence to convey a single beam of excitation light comprising said combination of selective excitation light bands, into the excitation path of said microscope or visualization system in the same or similar way as a (or the) microscope's standard light source would be intended to do.

It is another object of the present invention that each and every of such excitation bands is rapidly switchable (i.e. can be blocked as deeply as to induce no visible nor imaged emission of the relevant fluorochrome) and tunable in intensity, in such a way as not to interfere with any of the other simultaneously operated excitation bands.

It is another object of the present invention that multiple band excitation light from the above set-up is sent to the specimen by means of matched multiple band dichroic mirror/s, while the resulting fluorochromes' emission is let through by said dichroic mirror/s and further selected by means of matched multiple band emission filter/s, and is optionally corrected in its spectral profile as required by means of selective emission band rejection filter/s.

It is another object of the present invention that a multiplicity of 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 another object of the present invention that multiple light emitting diode/s (LEDs) of narrow light spectrum/a may be used as light source/s, the combination and even distribution of their emitted light onto the specimen being ensured by a system of orderly arranged optic fibres and bundles. It is also a part of the present invention that electronic driving circuitry is provided to switch each LED on and off, as well as to regulate its light output intensity by high frequency switching. It is also 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. It is also part of the present invention that such system and computer are interfaced with the interchangeable excitation and emission band selection elements (especially the single- and multi-band dichroics, single- and multi-band emission filters, excitation and/or emission band rejection filters, etc) for integrated system control, programming and/or automation.

In general, the present invention comprises:

• a "first light input port", to which a wide-spectrum light source (such as a standard microscope's mercury, xenon or other lamp, or other sources) with its relevant optics is connected, so that a beam of parallel light is collected into the system.

• an "first" dichroic mirror, which receives the incoming parallel light beam, and reflects its UV portion (<400nm, or at any other wanted selection wavelength) at an angle (such as 90°) into an "UV light path", while transmitting visible and near infrared light (>400nm, or as above) towards a "visible+near infrared light path", or vice-versa transmits the UV portion to the "UV light path", while reflecting visible and near infrared light to the "visible+near infrared light path".

^• a UV light path, starting at the above first dichroic, along which are provided:

.. a UV excitation filter, such as a 365-375 nm band pass filter, or otherwise as required by the fluorochrome/s or else to be excited, the whole UV light spectrum provided by the light source being fully available and usable as far as compatible with the microscope's optics UV transmission. Such filter is mounted in such a way as to be easily changed or replaced with other/s of different spectra, as required; .. a "second" UV-reflecting dichroic mirror, identical to the first one above, which reflects the selected UV excitation band at an angle (such as 90°);

.. a UV rejecting rotating filter, which either fully transmits the selected UV excitation band, or rejects a more or less extensive portion of it, up to complete blockade (transmission below approximately 10^"3 or lower). The filter is made in such a way, as to: (i) preferentially, block at a normal light incidence angle and progressively transmit when rotated (the rejection spectrum coincides with the selected UV excitation band, at normal angle, while moving to lower wavelengths with rotation); or, alternatively: (ii) transmit at normal angle and block when rotated (the rejection spectrum is at wavelengths longer than the selected UV excitation band, at normal angle, while moving to lower wavelengths and progressively overlapping and blocking the selected UV excitation band, with rotation). Said rejection filter is mounted in such a way as to be easily changed or replaced with other/s of different spectra, as required. When mounted, it will be arranged in such a way, that a handle well away from the light path will permit the filter's rotation (on an axis perpendicular to that of the UV light reaching it) from a normal angle (i.e. the angle at which the UV light band reaches the filter's surface at 90°) to an approximately 35° to 45° angle;

• a "visible+near infrared light path", starting at the above "first" dichroic, along which are provided: .. a one, two or more band/s visible light excitation filter, such as one providing at least a blue and a green excitation band, appropriate to the relevant fluorochromes to be observed or imaged;

.. a green-excitation rejecting rotating filter, in general similar to the one described above for the UV-excitation range, which either fully transmits the selected green excitation band, or rejects a more or less extensive portion of it, up to complete blockade (transmission below 10^'3~ 10^'4, or lower). This filter is preferentially made to block at a normal light incidence angle and progressively transmits when rotated. At any angle between the normal angle and the maximum angle of tilt (35° to 45°), such filter will transmit at high efficiency the blue excitation range, as well as any other relevant excitation band used (including those entering the system through the light input port two, as relevant: see below); .. a blue-excitation rejecting rotating filter, in general similar to the ones described above for the UV- and green-excitation ranges, which either fully transmits the selected blue excitation band, or rejects a more or less extensive portion of it, up to complete blockade (transmission <10^"3- 10^"4 or lower). This filter is preferentially made to block at a normal light incidence angle and progressively transmits when rotated. At any angle between the normal angle and the maximum angle of tilt (35° to 45°), such filter will transmit at high efficiency the above green, as well as any other excitation band used (including those entering the system through the light input port two, as relevant: see below);

.. a "third" mirror, in the form of either: (i) a wide-spectrum mirror reflecting the above selected visible light excitation bands onto the "fourth" dichroic (see below), in which case the "second light input port" will not be used; or: (ii) a dichroic reflecting the above selected visible light excitation bands, while transmitting longer wavelength light coming from the "second light input port".

.. a "second light input port", to which a second, optional light source with its relevant optics is connected, in such a way as to send its output parallel light beam through the above "second light input dichroic mirror", to superimpose with the excitation bands obtained from the first light source;

.. a second, additional light source, aimed at providing additional excitation band/s preferentially at wavelengths longer than those above. Since complex excitation filters, with more than 2 or 3 excitation bands involve severe trade-offs, the combination of multiple excitation bands from light sources one and two will be in a position to significantly increase riot only the precise spectral separation between the wanted excitation bands, but also the signal-to-noise ratio and especially the suppression of unwanted light in-between excitation bands. Such second light source is either: (i) a wide-spectrum light source with its one- two- or multiple-band excitation filter and relevant band-selective rejection rotating filter/s, so that one, two or more additional, adjustable excitation bands are obtained preferentially at wavelength/s longer than those selected from the first light source; or: (ii) an array of LEDs, each provided with its selective excitation filter and relevant optics, emitting at the same and/or at several different wavelengths (or, alternatively, an array of LED groups arranged with intervening dichroics, where both output light power and wavelength diversity are an issue), each LED or group sending its emission light into one each of multiple "primary" bundles of optic fibres. Such primary bundles are joined together at their other (output) end, to form a larger "secondary" bundle, in such a way that the fibres derived from each primary bundle are orderly distributed throughout the cross-section of the secondary bundle. Hence, an even distribution of light coming from each single LED is obtained in a simple way throughout the mixed, output light beam.

• a "fourth" dichroic mirror, in the form of either: (i) a UV- reflecting, visible and near-infrared transmitting dichroic, or: (ii) a UV- transmitting, visible and near-infrared reflecting dichroic. In either case, such dichroic mirror receives the light output from the "UV light path" and from the "visible+near infrared light path", and transmits either while reflecting the other, thus re-combining and superimposing all excitation bands.

• an output port for connection to the excitation path of the fluorescence visualization system or microscope, so that the mixed excitation output beam reaches the microscope's excitation path and dichroic, much the same way as light from a standard light source would do.

• one or more combination/s of dichroic/s and emission filter/s, as well as excitation band/s rejection and/or emission band/s rejection filter/s, mounted in the fluorescence visualization system, or reflected fluorescence microscope, is such a way as to be easily switchable, or slidable into / out of their relevant optical pathway, including:

.. at least one, or more dichroic/s of appropriate spectrum, hence reflecting the selected UV excitation band, as well as the blue, green and/or any additional excitation band/s, while transmitting the relevant fluorescence emission bands. Or a multiplicity of dichroics of appropriate spectra, for the selective, alternative and easily switched excitation and visualization or imaging of the relevant fluorochrome/s, by making use of the relevant excitation bands provided or part thereof;

.. one or more "excitation rejection" filter/s, aimed at rejecting one or more of a multiplicity of excitation bands selected above, so that a group of excitation bands can be used simultaneously, while others are not, and vice-versa, by simply switching between two or more alternative excitation rejection filters

.. at least one, or more emission filter/s of appropriate spectrum, hence fully rejecting the excitation bands, while letting through the relevant fluorescence emission bands, matched to the above dichroic/s. Or a combination of emission filters of appropriate spectra, matched to the relevant dichroics, for the selective, alternative and easily switched visualization or imaging of the relevant fluorochromes; .. one or more "emission band/s rejection" filter/s, such as rejecting one or more portion/s of the fluorochrome's emission let through by the emission filter, which may cause confusion when observing the co- localization of two (or more) fluorochromes as a combination colour. For instance, one such filter will remove any "yellow" light (such as the 570- 600nm range), when the combination of a green- and a red-emitting fluorochrome is observed, their coincidence in the preparation been perceived by the eye or camera as yellow, and indicating co-localization of the items/molecules of interest.

Such filter will transmit at high efficiency any other portion of the emission spectra let through by the emission filter, and will be mounted in such a way, as to be easily moved into the emission pathway of the microscope such as to observe co-localization/s, but also to be moved out of it when the full emission interval is required for the highest possible visualization or imaging sensitivity.

• a LED driver circuitry, to switch each LED used on and off, as well as to regulate its light output intensity by high frequency switching. Such LED driving electronic circuitry is arranged in such a way as to also be able to also provide high current pulses, of short to very short duration, to any number of wanted, simultaneously, synchronously or sequentially operated LEDs. Such LED driving electronic circuitry is interfaced to a computer, for system control and/or programming of its operation and adjustments. Such LED driving electronic circuitry is also interfaced to hardware components in the fluorescence microscope or visualization or imaging system, such as especially the single- and multi-band dichroics, single- and multi-band emission filters, excitation and/or emission band rejection filters, etc, for integrated system control, programming and/or automation.

It is specific subject matter of this invention a system for simultaneously revealing multiple fluorescent molecules or items, said system being connectable to a fluorescence visualisation or imaging apparatus and comprising a light source providing a main light beam, of a spectrum comprising an ultraviolet wavelength range and other wavelength components; a first dichroic mirror to receive said main light beam, suitable to separate from said light beam said ultraviolet wavelength range, which is carried to a first optical path, and said other wavelength components, which are carried to a second optical path; and, arranged along said first optical path, at least a one-band selective excitation filter, capable of selecting at least one or more ultraviolet wavelength excitation band/s, and at least one or more rotating notch-type rejection filters, each corresponding to one excitation band selected by said selective excitation filter and capable of selectively affecting said excitation band, by transmitting its wavelength range to a varying degree depending on the rejection filter's rotation at any angle between a first angular position, at which it fully transmits said excitation band, and a second angular position, at which it blocks said same band; and, arranged along said second optical path, at least a one-band or a multiple-band excitation filter, capable of selecting one or more excitation bands, and one or more rotating notch-type rejection filters each corresponding to one of the excitation bands selected by said excitation filter, and capable of selectively affecting said one excitation band each, by transmitting its wavelength range to a varying degree depending on the rejection filter's rotation at any angle between a first angular position, at which it fully transmits said excitation band, and a second angular position, at which it blocks said same band; and a second dichroic mirror which receives light from both of said first and second optical pathways, and is suitable to superimpose said first and second light beams into a single output beam, so that said output beam is directed to said fluorescence visualisation or imaging apparatus. Alwais according to the invention, said first optical path and said second optical path could be of the same length. Further according to the invention, said first dichroic mirror could reflect said ultraviolet wavelength components of the light beam and transmits longer wavelength components of said spectrum, or viceversa.

Still according to the invention, said first optical path could further comprise a reflecting mirror, inserted between said first dichroic mirror and said second dichroic mirror, to re-direct said first light beam.

Always according to the invention, said second optical path further comprises a reflecting mirror inserted between said first dichroic mirror and said second dichroic mirror, to re-direct said second light beam. Further according to the invention, said system could comprise a further light source providing a further light beam, and a further dichroic mirror onto which the light beams from said second light pathway and from said further light source are both directed, so that one is reflected and the other is transmitted, resulting in a superimposed mixed light beam. Still according to the invention, between said further light source and said further dichroic mirror said system could comprise at least a one- band or a multiple-band selective excitation filter, capable of selecting one or more excitation bands, and a number of rotating notch-type rejection filters corresponding to the number of excitation bands selected by said excitation filter, each of said rotating notch-type rejection filters being suitable to adjust the overall intensity of one of said selected excitation bands obtained from said second light beam, each of said rotating notch- type rejection filters being rotatable at any angle between a first angular position at which it fully transmits the corresponding selected band, and a second angular position at which it blocks the corresponding selected band.

Always according to the invention, said excitation filter arranged along said second optical path, could select at least two excitation bands within the visible wavelength range, or at least one excitation band within the visible wavelength range and at least one excitation band within the infrared wavelength range.

Preferably according to the invention, said excitation filter, arranged along said second optical path, could select one or more band/s within the visible wavelength range; and said excitation filter, arranged between said further light source and said further dichroic mirror, could select at least one excitation band in the near infrared range or in the visible range, or at least one excitation band in the near infrared wavelength range and at least one in the visible range.

Still according to the invention, said excitation filters and the corresponding rotating notch-type rejection filters could be removable and interchangeable.

Always according to the invention, said angular positions between which each of said rotating notch-type rejection filters rotates, and at which it either rejects, or transmits, respectively, the wavelength band for which it is selective, could correspond to: a 0-15° deviation from a normal light incidence, and: a 30-50° deviation from a normal light incidence, respectively; or viceversa.

Further according to the invention, said system could comprise at least two or more of said rotating notch-type rejection filters, each of which either fully transmits, or partially transmits, or blocks said excitation band for which it is selective without affecting the other/s.

Always according to the invention, said light sources could provide a main parallel or collimated light beam.

Still according to the invention, said further light source could comprise: a plurality of LED units (LED1 , LED2,..., LEDn), each LED unit being provided with its selective excitation filter (20) and relevant optics

(19) to convey the LED's light beam onto a bundle of optical fibres; a plurality of optical fibres grouped in a plurality of primary bundles (22), each one of said bundles (22) collecting light from one of said LED units

(LED1 , LED2,... , LEDn), and optical fibres composing said primary bundles (22) being further re-arranged in a secondary bundle (23), so that the optical fibres composing each primary bundle (22) are orderly arranged throughout the cross section area of said secondary bundle (23); and optics (24) adapted to collimate the light output from said secondary bundle of optical fibres into a single output beam of parallel light of suitable diameter.

Preferably according to the invention, said LED units could comprise a single LED each.

Further according to the invention, said LED units could comprise a plurality of LEDs, the light beams from the various LEDs in the unit being collected together and superimposed by means of dichroic mirrors. Always according to the invention, said further light source could comprise a driver circuitry capable of controlling the light emission of each LED within each LED unit.

Still according to the invention, said driver circuitry could supply power to each LED within each LED unit, so that each LED and/or group/s thereof can be regulated in their light output, switched on and off entirely, as well as flash-operated for short to very short time periods, either individually or in a synchronized fashion and/or in groups or sequence/s of LEDs of the same or of different wavelengths. Preferably according to the invention, said system could comprise interface means to connect said driver circuitry to a computer for operation control and/or programming.

Further according to the invention, said system could comprise means to interface said driver circuitry and said computer to the interchangeable excitation and emission band selection elements in the system, for integrated system control, programming and/or automation.

Still according to the invention, said fluorescence visualisation or imaging apparatus could be a microscope, which could comprise at least a multiple-band dichroic mirror, or multiple interchangeable one- and/or multiple-band dichroic mirrors, adapted to reflect said selected excitation band/s, provided by said system, into an excitation optical path toward a specimen to be excited, and to transmit the emission light from said specimen to the microscope's emission optical path; and, arranged along said excitation optical path, at least one objective lens to focus the excitation light from said dichroic mirror onto said specimen and collect the specimen's fluorescence emission; and, arranged along said emission optical path, after said dichroic mirror, at least a multiple-band emission filter, or multiple interchangeable one- and/or multiple-band emission filters, hence optics to permit visual observation and/or photography or imaging.

Further according to the invention, said microscope could comprise at least one, or more, input excitation rejection filter/s to select and transmit to the microscope's dichroic one or multiple excitation bands among those provided by said system. Still according to the invention, said microscope could comprise an emission band rejection filter, which can be inserted into the emission optical path, between said multi-band emission filter and the visualization or imaging optics.

Always according to the invention, said microscope could comprise a camera or other imaging and/or recording apparatus. Further according to the invention, said system could comprise two or more sets, each comprising an excitation selection filter, a dichroic

. mirror and an emission filter, and each set operating at different excitation and emission wavelength ranges, said sets being interchangeable with each other during imaging or. observation. Still according to the invention, said at least two sets could both include among others one common excitation and one common emission wavelength range.

Preferably according to the invention, said excitation filters could select a single or a plurality of excitation band/s. It is further subject matter of this invention a method for selection from a light source of multiple simultaneously operated excitation light intervals, comprising the following steps:

(a) providing a main light beam, whose spectrum comprises an ultraviolet wavelength range and other wavelength components; (b) separating said spectrum of said main light beam into a first and a second light beams, said first light beam including said ultraviolet wavelength range and said second light beam including other wavelength components;

(c) selecting one or more ultraviolet wavelength bands from said first light beam;

(d) adjusting the overall intensity of each such ultraviolet excitation band independently from the other bands running in the same pathway;

(e) selecting one or more excitation bands of said second light beam; and (f) adjusting the overall intensity of each such excitation band independently from the other bands running in the same pathway.

Still according to the invention, said method could comprise the following steps:

(g) superimposing said first and second light beams to get a single output beam; and

(h) directing said output beam toward a fluorescence visualisation or imaging apparatus. Always according to the invention, said method could comprise the following steps:

(f1) providing a further light beam for inclusion of further excitation band or bands; (f2) selecting one or more excitation bands from said further light beam;

(f3) adjusting the overall intensity of each such excitation band, independently from other bands running in the same pathway; and

(f4) superimposing said further light beam and its derived excitation bands, with the excitation bands provided within the second light pathway, to get a mixed light output beam;

(g) superimposing said mixed light output beam with the one provided from the first optical pathway, to get a single output beam; and

(h) directing said output beam toward a fluorescence visualisation or imaging apparatus.

It is further subject matter of this invention a light source for simultaneously revealing multiple fluorescent molecules or items, such light source connectable to a fluorescence visualisation or imaging apparatus, and comprising: a plurality of LED units, each LED unit being provided with its selective excitation filter and relevant optics; a plurality of optical fibers grouped in a plurality of primary bundles, each one of said bundles collecting light from one of said LED emission unit, optical fibers from said primary bundles being further collected to form a secondary bundle, so that the optical fibers of each primary bundle are orderly arranged and distributed throughout the cross section area of said secondary bundle; and optics adapted to collimate the output light beam from said secondary bundle of optical fibres.

Still according to the invention, said LED units could comprise a single LED or a plurality of LEDs, the plurality of light beams from such plurality of LEDs within each unit being collected together and superimposed by means of dichroic mirrors.

Always according to the invention, said light source could comprise a driver circuitry capable of controlling the light emission of each LED within each LED unit and said driver circuitry supplies power to each LED within each LED unit, so that each LED and/or group/s thereof can be regulated in their light output, switched on and off entirely, as well as flash- operated for short to very short time periods, either individually or in a synchronized fashion and/or in groups or sequence/s of LEDs of the same or of different wavelengths.

Further according to the invention, said light source could comprise interface means to connect said driver circuitry to a computer for operation control and/or programming.

Still according to the invention, said light source could comprise means to interface said driver circuitry and said computer to the interchangeable excitation and emission band selection elements in the system, for integrated system control, programming and/or automation.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will now be 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 an arrangement of the excitation and emission pathways of the excitation system and reflected fluorescence microscope according to a first embodiment of the present invention; figure 1B shows a slightly differing arrangement of the intial part of the embodiment shown in figure 1A; figure 2 shows an example of transmission spectra of a set of spectrally selective components (interference filters and dichroics) of an excitation system and reflected fluorescence microscope according to the first embodiment shown in figure 1A (up to three excitations and emissions, in the example provided; transmittance spectra); figure 3A shows a schematic view of an arrangement of excitation and emission pathways of an excitation system and reflected fluorescence microscope according to a second embodiment of the present invention; figure 3B shows a slightly differing arrangement of the initial portion of the embodiment shown in figure 3A; figure 4 shows a composition and arrangement of a "primary" and a "secondary" bundles of optic fibres, for light collection and merging from multiple LEDs; figure 5 shows a schematic view of an arrangement of the excitation and emission pathways of an excitation system and reflected fluorescence microscope according to a third embodiment of the present invention; figure 6 shows a schematic view of an arrangement of the excitation and emission pathways of an excitation system and reflected fluorescence microscope according to a fourth embodiment of the present invention; figure 7 shows an example of transmission spectra of a set of spectrally selective components (interference filters and dichroics) of an excitation system and reflected fluorescence microscope according to either the third or the fourth embodiment of the present invention, to permit multiple simultaneous visualizations of multiple fluorochromes (up to five simultaneous labellings, in the example provided; transmittance spectra).

Figure 8 shows an example of two matched sets of interference filter components, to operate as rapidly switchable sets for sequential, correlated and comparative visualizations according to either the third or the fourth embodiment of the present invention (up to seven versus three simultaneous labellings are possible, in the example provided; transmittance spectra).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

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

First Embodiment

Figures 1A (or 1 B) is an outline of the first preferred embodiment of the invention, in the form of a one-light-source system, comprising a UV and a visible to near infrared-excitation path, the relevant filter combinations and reflected fluorescence microscope or fluorescence visualization system, resulting in a multiple excitation, multiple simultaneous visualization system, with independent adjustment of each single excitation band, and optional emission colour-correction.

A wide spectrum light source, such as mercury or other lamp, with optics to collect light into a collimated beam ("light source") projects light onto a first dichroic mirror. Such first mirror is in the form of either: a UV-reflecting dichroic mirror 1 (figure 1A), which reflects UV light to a "UV light" excitation pathway, while transmitting the visible to near-infrared spectrum to a "visible & near-infrared light" excitation pathway; or (figure 1 B), a dichroic, which transmits UV to the "UV light path", while reflecting the visible and near infrared light to the "visible+near infrared light path". Hence, the "light source" will be located at different angles to the two excitation paths according to arrangements of figures 1A and 1 B, respectively, while the further components in the present embodiment will remain unchanged.

For the UV excitation pathway, at least a single-band excitation filter is provided 5, which selects the wanted excitation interval or UV excitation band, and is followed by a rotating rejection filter 7 in the form of a surface-coated interference filter of appropriate spectrum, which either fully transmits, or partially transmits, or blocks said selected excitation interval. The UV beam is then reflected by a second UV-reflecting mirror 2 in the form of either a UV-reflecting dichroic (such as identical to 1), or of a wide-spectrum mirror. In the visible and near infrared excitation pathway, the light beam is reflected by a third mirror 3 in the form of a wide- spectrum mirror. A multi-band excitation filter 6 selects two or more excitation bands, and is followed by two or more rotating rejection filters 8 and 9 in the form of two or more surface-coated interference filters of different, appropriate spectra, each of which either fully transmits, or partially transmits, or blocks (OD>3 or higher) one only of said selected excitation bands without affecting the other/s.

By use of a forth mirror 4 in the form of a dichroic either: identical to item 1 above (see figure 1A), or identical to item 1", above (not shown), the UV- and visible to near-infrared beams are superimposed into one and the same mixed and collimated beam, Which is sent to the excitation pathway of the fluorescence visualization or imaging system, or microscope, hence to the relevant filter combination. In general, the mixed excitation light beam goes directly to the multi-band dichroic 11 , to be reflected to the microscope's lens/es 12 and excite the specimen S. Emission light is collected through said lens/es 12, to be transmitted through the dichroic mirror 11 and multi-band emission filter 13, hence to the eye-piece/s for visualization or imaging and/or recording. Optionally, one or more emission band rejection filter/s 14 can be slid into the emission pathway, for colour adjustment/s. Figure 2 describes one exemplary set-up (out of many possible) comprising the spectrally active components according to the first embodiment of the invention (for simplicity, the spectrum of dichroic 2, identical to 1 , is omitted, as is the one of the spectrally neutral, visible and near infrared wide spectrum light mirror 3). A collimated beam of wide- spectrum light is split into its UV-containing and its visible and nearlR components by means of a UV-reflecting dichroic (figure 2, item 1 , according to arrangement of figure 1A), or by means of a visible and nearlR light reflecting, UV-transmitting dichroic 1", according to the arrangement in figure 1 B, above (not shown in figure 2). While in the present example the UV-reflecting dichroic has 50% transmission ~435nm, such turning point can be set virtually anywhere required.

Specific excitation bands are selected within the UV and the visible to near IR excitation pathways by means of the single- and dual- band excitation filters 5 and 6, respectively. The UV excitation rejector 7 described in the present example transmits its relevant UV excitation band at a normal (or 0°) angle of light incidence, while progressively reducing transmission with rotation, up to complete blocking at an approximately 40° rotation (maximum OD -2.5, in the present example). Conversely, in the present example the blue 8 and green 9 excitation rejection filters both operate in the opposite way. Either one blocks the excitation band for which it is selective at 0° (i.e. at a normal angle of light incidence; with OD >3), while progressively transmitting it with increasing rotation, up to full transmission (>90%) at ~40° rotation. Either rejection filter fully transmits the other's excitation band at any degree of rotation. The three excitation bands are superimposed into one and the same mixed excitation beam by means of a further dichroic mirror, either in the form of a UV-reflecting and visible to near-infrared light-transmitting dichroic (4, identical to 1), or in the form of a UV-transmitting and visible to near-infrared light reflecting dichroic (identical to 1": not shown). The mixed excitation beam is sent to a 3-band dichroic 11 mounted in (one of) the microscope's filter holders (for the purposes of the present example, position 10 is left empty), its contained excitation bands being reflected by the dichroic's reflecting regions (represented here by the low-transmission regions) through the lens and onto the specimen S. When appropriate fluorochromes in the specimen S are exposed to the 3-band excitation light, their emission captured by the microscope's lens is transmitted through the dichroic's 11 transmission regions, then through the 3-band emission filter 13.

For maximum emission signal intensity in the yellow-orange-red region, no further spectral intervention is made in the example provided, hence item 14 is removed from the microscope's emission-and- visualization pathway, resulting in the full emission bands indicated as: "blue", "green" and "yellow-orange-red". The latter wide emission band is especially tailored to "high sensitivity" visual observations, since the human eye has highest sensitivity in the green-yellow region of the visible spectrum, with a rapid and progressive decrease in the longer (red) and lower (blue) wavelength regions. For comparative localization and co- localization studies, the yellow rejection filter 14 is moved into the emission pathway, so that the third emission band is restricted to its red component, resulting in three distinct colours for non-colocalized labelling (blue, green, and red), while the coincidence of either two or three labels will show as new, equally distinct colours (cyan = blue+green; purple/violet = blue+red; yellow = green+red; white = all three).

The set-up described is compatible with the use on the same microscope of additional single-band filter combinations, provided (as is commonly the case) their included excitation filter 10 selects excitation light within one of the excitation bands transmitted by the system. Such single-band filter sets permit visualization of single fluorochromes, in turn, at maximum efficiency, since spectra of single-band emitters and dichroics do routinely reach far higher efficiency and signal-to-noise ratios than the corresponding multiple-band filters.

Second embodiment

The second embodiment is similar to the first embodiment, except that a second light source is included, in order to provide additional, individually adjustable excitation bands. By using an independent, second light source, and its dedicated one- two- or three- band excitation filter, up to several excitation bands can be added, without the need of a highly complex and critical 4-, 5- or 6-band excitation filter. In fact, the increase in spectral complexity of such elements, with over 2 or 3 "cycles" of alternating regions of high reflectivity and high transmission severely hampers their capacity to produce clear-cut spectral separation, as well as greatly reducing signal-to-noise ratios. In the present exemplary embodiment, such additional excitation bands are operated at wavelengths longer than the excitation bands selected by the excitation filter 6 located along the visible to near IR excitation pathway. Figure 3A (or figure 3B) is an outline of said second preferred embodiment of the invention, in the form of a two-light-source system, comprising a UV-, and a visible plus near infrared-excitation pathways, the relevant filter combinations and reflected fluorescence microscope or fluorescence visualization system, resulting in a multiple excitation, multiple simultaneous visualization system, with independent adjustment of each single excitation band, and optional emission colour-correction. As described for the previous embodiment, two of the dichroic mirrors involved (items 1 and 4, respectively) can be (separately or at the same time) made to either reflect UV and transmit visible to near-infrared light spectra, or viceversa, both arrangements involved being part of the present invention. For the sake of simplicity, both are shown as UV- reflecting dichroics in figure 3A, while an example of the first one as a UV- transmitting visible to near-infrared reflecting dichroic (item 1") is provided in figure 3B. In either case, a wide spectrum light source L1 provides excitation light for the UV excitation band/s and for at least one or multiple excitation band/s in the visible range, such bands being selected by excitation filters, and adjusted/switched on/off by rotating rejection filters (for items 1 and 1"; 2; 4 and its alternative UV-transmitting dichroic; 5 to 9, see above: first embodiment).

In place of a wide-spectrum mirror (3, see above: first embodiment), the present embodiment comprises a dichroic mirror 3" reflecting the visible range excitation bands selected by exciter 6, while transmitting longer wavelength excitation light from light source L2, in the form of the excitation band/s selected by the relevant one- or multiple- band excitation filter 16 and regulated by one or multiple rotating rejection filter/s (17: while one rotating rejection filter is drawn here, it is intended to indicate "one or more" such filters, equal in number to the number of excitation bands provided by the excitation filter 16). Hence, four or more excitation bands are obtained, each being adjusted or switched on or off as needed, hence are carried to a multi- band dichroic 11 for reflection to the specimen S, the resulting emission bands being selected by the matched multi-band emitter 13. Depending on fluorochrome and colour combinations used, emission band rejection filter/s (14, to represent one or more such filters) are used to optimize colour separation, as described above (first embodiment). Compatibility with single-band filter sets is the same, as described for the first embodiment, except that a higher number of excitation and emission bands are accomodated.

Third Embodiment

The third embodiment is similar to the Second Embodiment, except that at least the second light source L2 is composed of an array of Light Emitting Diodes, or LEDs, emitting light of the same and/or of different wavelengths. Figure 5 is an outline of the third preferred embodiment of the invention, in the form of a two-light-source system, comprising a wide- spectrum lamp and a multiple-LED light source as light source L2, in which the emission of each LED is collected via bundles of optic fibres. A wide spectrum light source, as for previous embodiments, provides light for UV and visible range excitation bands, such bands being selected by excitation filters, and adjusted/switched on/off by rotating rejection filters, as described above (for items 1 - 2, 4 - 9, see: first embodiment). In place of a wide-spectrum mirror (3, above), this embodiment comprises a dichroic mirror 3" reflecting the visible range excitation bands selected by exciter 6, while transmitting longer wavelength excitation light from the second, LED operated light source L2. As described for the previous embodiments, two of the dichroic mirrors involved (items 1 and 4, respectively) can be (separately or at the same time) made to either reflect UV and transmit visible to near-infrared light spectra, or viceversa. Since no further item/s, part/s, nor arrangement/s is/are changed in the relevant portions of the system in the present embodiment, the present embodiment is shown and described here only according to the use of UV- reflecting visible to near infrared transmitting dichroics as items 1 and 4, under the explicit assumption that the alternative arrangements described above also fully apply to the present embodiment of the invention.

The LED operated light source described as part of the present invention (figure 4) is included in the present exemplary embodiment to provide light and excitation bands in the infrared and red wavelength bands (figures 5, 6 and 7). None the less, said LED operated light source can also provide excitation bands at lower-wavelengths down to the UV range, depending on the LEDs used, hence can also be used as the main or sole light source for the purposes of the present invention.

In the exemplary embodiment outlined in figure 5 (see also figure 4 for details of optical fibre arrangements), such a light source L2 is composed of single, high power LEDs each emitting light in a single wavelength interval, each followed by optics 19 to convey the LED's emission into a narrow angle beam, which goes through an excitation filter 20 for selection of the wanted excitation range, hence is carried to a "primary bundle" 22 and a "secondary bundle" 23 of optical fibres. As shown in figure 4, several primary bundles 22 converge to form a larger secondary bundle 23, optical fibres coming from each primary bundle being arranged within the secondary bundle in an orderly fashion, so that fibres originating from each primary bundle are regularly distributed throughout the secondary bundle. Hence, all parts of the secondary bundle are provided with a proportional contribution of optical fibres coming from each primary bundle, and light captured from each LED is proportionally distributed to all parts of the secondary bundle cross-section area. Optics 24 are provided to collimate the light output from the secondary bundle, and the resulting parallel light beam is transmitted through dichroics 3" and 4, to be superimposed to the mixed excitation light beam coming from light source L1. Alternatively, the collimated mixed light beam from the LED light source can be directly transmitted to the reflected fluorescence microscope or fluorescence visualization system, said LED light source operating as the main or sole light source for the purposes of the present invention. In either case, electronic driver circuitry 18a to 18n supplies power to each LED, so that each LED and/or group/s thereof can be regulated in their light output, switched on and off entirely, as well as flash- operated for short to very short time periods, either individually or in a synchronized fashion and/or in groups or sequence/s of the same or of different wavelengths. Such driver circuitry is also interfaceable to a computer, for operation control and/or programming. Such driver circuitry and/or computer are also interfaceable with the interchangeable excitation and emission band selection elements (especially the single- and multi- band dichroics, single- and multi-band emission filters, excitation and/or emission band rejection filters, etc) for integrated system control, programming and/or automation. By means of the present embodiment, four or more excitation bands are obtained, each of which can be adjusted or switched on or off as needed, or flash operated (limited to LED-provided excitation bands), hence carried to a multi-band dichroic 11 for reflection to the specimen S, the resulting emission bands being selected by the matched multi-band emitter 13. Examples of applications are attached below (see: fourth embodiment). The set-up can be compatible with the use on the same microscope of additional single-band filter sets/cubes, as described for the first and second embodiments.

Fourth embodiment

The fourth embodiment is similar to the third one above, except that at least the second light source L2 is composed of an array of groups of Light Emitting Diodes, or LEDs, each group of LEDs being arranged together by means of dichroic mirrors.

As mentioned above, the LED operated light source described as part of the present invention, and also including the use of groups of LEDs arranged together, by means of dichroic mirrors (figure 4, LED13 and LED14) is included in the present exemplary embodiment to provide light and excitation bands in the infrared and red wavelength bands (figures 5, 6 and 7). None the less, said LED operated light source can provide excitation bands also at lower-wavelengths down to the UV range, depending on the LEDs used. Thus, the collimated mixed light beam from the LED light source can be directly transmitted to the reflected fluorescence microscope or fluorescence visualization system, said LED light source operating as the main or sole light source for the purposes of the present invention.

Figure 6 is an outline of the Fourth preferred Embodiment of the invention, in the form of a two-light-source system, comprising a wide- spectrum lamp and a multiple-LED light source L2, in which the emission of each LEDs is first reflected and/or transmitted through an arrangement comprising one or more dichroic mirror/s of appropriate spectrum/a (21a; 21b), hence light from all LEDs in the group is collected into a "primary bundle" of optic fibres per each LED group 22. Light is then carried to a "secondary bundle" of optic fibres 23 and further as described for the Third Embodiment. Compared to the Third Embodiment above, the present embodiment can make use of two, three or more times the number of individual LEDs, hence permitting to obtain higher intensity excitation bands, without sacrificing and even increasing their spectral diversity.

As described for previous embodiments, two of the dichroic mirrors involved (items 1 and 4, respectively) can be (separately or at the same time) made to either reflect UV and transmit visible to near-infrared light spectra, or viceversa. Since no further item/s, part/s, nor arrangement/s is/are changed, the present embodiment is shown and described here only according to the use of UV-reflecting dichroics as , items 1 and 4, under the explicit assumption that the alternative arrangements described above also fully apply to the present embodiment of the invention.

Figure 7 describes one exemplary set-up (out of many possible) comprising the spectrally active components according to the third or fourth embodiment of the invention, and aimed at permitting the simultaneous visualization or imaging of up to five fluorochromes. For simplicity, the set-up which provides the "UV + Blue + Green" excitation bands is omitted (see figure 2, and relevant text, above).

Light from one or multiple red light- and near infrared light-emitting LEDs is selected from each by means of appropriate excitation filters 20_red, and 20JR, respectively, hence it is collected and transported through the primary and the secondary optical fibre bundles, and the relevant collimating optics. Alternatively, such two (red and near-infrared) excitation bands could be obtained according to the second embodiment (above), by means of a dual-band excitation filter selecting such bands from the light emitted by a wide-spectrum lamp (such as a Xenon, or metal halide lamp) followed by appropriate excitation band-selective rotating rejection filters. The red and near-infrared excitation bands shown are transmitted through the "third" dichroic 3", and further through the UV- reflecting dichroic 4, to join the mixed excitation light beam. directed to the microscope's excitation optical pathway. The five excitation bands obtained are reflected by the matched multi-band dichroic 11 towards to specimen S, and the fluorochromes' emission is collected and transmitted through the matched multi-band emission filter 13, for visualization as far as the four visible emission bands are concerned, as well as for imaging of all five emission bands.

Figure 8 describes one exemplary set-up (out of many possible) comprising the spectrally active components according to the third or fourth embodiment of the invention, and aimed at permitting the rapidly switchable, alternative comparative visualization of "three fluorochromes" versus "two (other) fluorochromes". For simplicity, the set-up which provides the five excitation bands is omitted (see figure 7 and relevant text, above).

The aim is here to label at the same time as many spatially separate tissue features, components or molecules as possible or convenient (up to seven), by means of single-labelling (blue, yellow, or red, in the example provided), as well as dual- and triple-labelling (green, orange, purple, and white, respectively). Such multiple "tissue tagging" is visible using the "BLUE + YELLOW + RED filter set", so that for instance up to seven separate cell types can be identified in the preparation.

By simply switching to the other filter set ("Green + Infrared filter set"), hence back and forth, the observer will be in a position to delineate in detail the precise location of one / two molecules of interest, labelled in

Green and infrared, respectively, in each and every cell type or tissue component labelled as above.

Understandably, such set-up is also very suitable to systematic and/or automated approaches based on paired images recording and image set analyses. As for all previous embodiments, the set-up is compatible with the use on the same microscope of additional single- or dual-band filter sets, for verification and control of the quality and reproducibility of each and every labelling, as required.

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.

Claims

1. System for simultaneously revealing multiple fluorescent molecules or items, said system being connectable to a fluorescence visualisation or imaging apparatus and comprising a light source (L1) providing a main light beam, of a spectrum comprising an ultraviolet wavelength range and other wavelength components; a first dichroic mirror (1 ; or 1 ") to receive said main light beam, suitable to separate from said light beam said ultraviolet wavelength range, which is carried to a first optical path, and said other wavelength components, which are carried to a second optical path; and, arranged along said first optical path, at least a one-band selective excitation filter (5), capable of selecting at least one or more ultraviolet wavelength excitation band/s, and at least one or more rotating notch-type rejection filters (7), each corresponding to one excitation band selected by said selective excitation filter (5) and capable of selectively affecting said excitation band, by transmitting its wavelength range to a varying degree depending on the rejection filter's rotation at any angle between a first angular position, at which it fully transmits said excitation band, and a second angular position, at which it blocks said same band; and, arranged along said second optical path, at least a one-band or a multiple-band excitation filter (6), capable of selecting one or more excitation bands, and one or more rotating notch-type rejection filters (8, 9) each corresponding to one of the excitation bands selected by said excitation filter (6), and capable of selectively affecting said one excitation band each, by transmitting its wavelength range to a varying degree depending on the rejection filter's rotation at any angle between a first angular position, at which it fully transmits said excitation band, and a second angular position, at which it blocks said same band; and a second dichroic mirror (4) which receives light from both of said first and second optical pathways, and is suitable to superimpose said first and second light beams into a single output beam, so that said output beam is directed to said fluorescence visualisation or imaging apparatus.

2. System according to claim 1 , characterised in that said first optical path and said second optical path are of the same length.

3. System according to any of the preceding claims, characterised in that said first dichroic mirror (1 , or 1") reflects said ultraviolet wavelength components of the light beam and transmits longer wavelength components of said spectrum, or viceversa.

4. System according to any of the preceding claims, characterised in that said first optical path further comprises a reflecting mirror (2), inserted between said first dichroic mirror (1 ; or 1") and said second dichroic mirror (4), to re-direct said first light beam.

5. System according to any of the preceding claims, characterised in that said second optical path further comprises a reflecting mirror (3) inserted between said first dichroic mirror (1 ; 1") and said second dichroic mirror (4), to re-direct said second light beam.

6! System according to any of claims 1 - 4, characterised in that it comprises a further light source (L2) providing a further light beam, and a further dichroic mirror (3") onto which the light beams from said second light pathway and from said further light source (L2) are both directed, so that one is reflected and the other is transmitted, resulting in a superimposed mixed light beam.

7. System according to claim 6, characterised in that between said further light source (L2) and said further dichroic mirror (3") it comprises at least a one-band or a multiple-band selective excitation filter (16), capable of selecting one or more excitation bands, and a number of rotating notch- type rejection filters (17) corresponding to the number of excitation bands selected by said excitation filter (16), each of said rotating notch-type rejection filters (17) being suitable to adjust the overall intensity of one of said selected excitation bands obtained from said second light beam, each of said rotating notch-type rejection filters (17) being rotatable at any angle between a first angular position at which it fully transmits the corresponding selected band, and a second angular position at which it blocks the corresponding selected band.

8. System according to any of the preceding claims, characterised in that said excitation filter (6), arranged along said second optical path, selects at least two excitation bands within the visible wavelength range, or at least one excitation band within the visible wavelength range and at least one excitation band within the infrared wavelength range.

9. System according to any of claims 1 - 7, characterised in that said excitation filter (6), arranged along said second optical path, selects one or more band/s within the visible wavelength range; and said excitation filter (16), arranged between said further light source

(L2) and said further dichroic mirror (3"), selects at least one excitation band in the near infrared ligth range or in the visible ligth range, or at least one excitation band in the near infrared range and at least one in the visible wavelength range.

10. System according to any of the preceding claims, characterised in that said excitation filters (5, 6) and the corresponding rotating notch- type rejection filters (8, 9, 17) are removable and interchangeable.

11. System according to any of the preceding claims, characterised in that said angular positions between which each of said rotating notch- type rejection filters (8, 9, 17) rotates, and at which it either rejects, or transmits, respectively, the wavelength band for which it is selective, correspond to: a 0-15° deviation from a normal light incidence, and: a 30- 50° deviation from a normal light incidence, respectively; or viceversa.

12. System according to any of the preceding claims, characterised in that it comprises at least two or more of said rotating notch-type rejection filters (7, 8, 9, 17), each of which either fully transmits, or partially transmits, or blocks said excitation band for which it is selective without affecting the other/s.

13. System according to any of the preceding claims, characterised in that said light sources (L1 , L2) provide parallel or collimated light beams.

14. System according to any of claims 6 - 13, characterised in that said further light source (L2) comprises: a plurality of LED units (LED1, LJED2,..., LEDn), each LED unit being provided with its selective excitation filter (20) and relevant optics (19) to convey the LED's light beam onto a bundle of optical fibres; a plurality of optical fibres grouped in a plurality of primary bundles

(22), each one of said bundles (22) collecting light from one of said LED units (LED1 , LED2,... , LEDn), and optical fibres composing said primary bundles (22) being further re-arranged in a secondary bundle (23), so that the optical fibres composing each primary bundle (22) are orderly arranged throughout the cross section area of said secondary bundle (23); and optics (24) adapted to collimate the light output from said secondary bundle of optical fibres into a single output beam of parallel light of suitable diameter.

15. System according to claim 14, characterised in that said LED units comprise a single LED each.

16. System according to any of the claims 14 or 15, characterised in that said LED units comprise a plurality of LEDs, the light beams from the various LEDs in the unit being collected together and superimposed by means of dichroic mirrors (21a, 21b).

17. System according to any of claims 14 - 16, characterised in that said further light source (L2) comprises a driver circuitry (18) capable of controlling the light emission of each LED within each LED unit (LED1 , LED2,..., LEDn).

18. System according to claim 17, characterised in that said driver circuitry (18) supplies power to each LED in the LED units (LED1,

LED2,..., LEDn), so that each LED and/or group/s thereof can be regulated in their light output, switched on and off entirely, as well as flash- operated for short to very short time periods, either individually or in a synchronized fashion and/or in groups or sequence/s of LEDs of the same or of different wavelengths.

19. System according to any of claims 17 or 18, characterised in that it comprises interface means to connect said driver circuitry (18) to a computer for operation control and/or programming.

20 System according to any of claim 19, characterised in that it comprises means to interface said driver circuitry (18) and said computer to the interchangeable excitation and emission band selection elements in the system, for integrated system control, programming and/or automation.

21. System according to any of the preceding claims, characterised in that said fluorescence visualisation or imaging apparatus is a microscope.

22. System according to claim 21 , characterised in that said microscope comprises at least a multiple-band dichroic mirror (11), or multiple interchangeable one- and/or multiple-band dichroic mirrors (11), adapted to reflect said selected excitation band/s, provided by said system, into an excitation optical path toward a specimen (S) to be excited, and to transmit the emission light from said specimen (S) to the microscope's emission optical path; and, arranged along said excitation optical path, at least one objective lens (12) to focus the excitation light from said dichroic mirror (11) onto said specimen (S) and collect the specimen's fluorescence emission; and, arranged along said emission optical path, after said dichroic mirror (11), at least a multiple-band emission filter (13), or multiple interchangeable one- and/or multiple-band emission filters (13), hence optics (15) to permit visual observation and/or photography or imaging.

23. System according to any of the claims 21 or 22, characterised in that said microscope comprises at least one, or more, input excitation rejection filter/s (10) to select and transmit to the microscope's dichroic one or multiple excitation bands among those provided by said system.

24. System according to any of the claims 21 - 23, characterised in that said microscope comprises an emission band rejection filter (14), which can be inserted into the emission optical path, between said multi- band emission filter (13) and the visualization of imaging optics (15).

25. System according to any of the claims 21 - 24, characterised in that said microscope comprises a camera or other imaging and/or recording apparatus.

26. System according to any of claims 21 - 25, characterised in that it comprises two or more sets, each comprising an excitation selection filter, a dichroic mirror and an emission filter, and each set operating at different excitation and emission wavelength ranges, said sets being interchangeable with each other during imaging or observation.

27. System according to claim 26, characterised in that said at least two sets both include among others one common excitation and one common emission wavelength range.

28. System according to any of the preceding claims, characterised in that said excitation filters (5, 6, 16 and 20) select a single or a plurality of excitation band/s.

29. Method for selection from a light source of multiple simultaneously operated excitation light intervals, comprising the following steps:

(a) providing a main light beam, whose spectrum comprises an ultraviolet wavelength range and other wavelength components; (b) separating said spectrum of said main light beam into a first and a second light beams, said first light beam including said ultraviolet wavelength range and said second light beam including other wavelength components; (c) selecting one or more ultraviolet wavelength bands from said first light beam;

(d) adjusting the overall intensity of each such ultraviolet excitation band independently from the other bands running in the same pathway;

(e) selecting one or more excitation bands of said second light beam; and

(f) adjusting the overall intensity of each such excitation band independently from the other bands running in the same pathway.

30. Method according to claim 29, characterised in that it further comprises the following steps: (g) superimposing said first and second light beams to get a single output beam; and

(h) directing said output beam toward a fluorescence visualisation or imaging apparatus.

31. Method according to claim 29, characterised in that it comprises the following steps:

(f1) providing a further light beam for inclusion of further excitation band or bands;

(f2) selecting one or more excitation bands from said further light beam; (f3) adjusting the overall intensity of each such excitation band, independently from other bands running in the same pathway; and

(f4) superimposing said further light beam and its derived excitation bands, with the excitation bands provided within the second light pathway, to get a mixed light output beam; (g) superimposing said mixed light output beam with the one provided from the first optical pathway, to get a single output beam; and

(h) directing said output beam toward a fluorescence visualisation or imaging apparatus.

32. Light source for simultaneously revealing multiple fluorescent molecules or items, such light source connectable to a fluorescence visualisation or imaging apparatus, and comprising: a plurality of LED units (LED1 , LED2,..., LEDn), each LED unit being provided with its selective excitation filter (20) and relevant optics (19); a plurality of optical fibers grouped in a plurality of primary bundles (22), each one of said bundles (22) collecting light from one of said LED emission unit (LED1 , LED2,..., LEDn), optical fibers from said primary bundles (22) being further collected to form a secondary bundle (23), so that the optical fibers of each primary bundle (22) are orderly arranged and distributed throughout the cross section area of said secondary bundle (23); and optics (24) adapted to collimate the output light beam from said secondary bundle of optical fibres.

33. Light source according to claim 32, characterised in that said LED units comprise a single LED.

34. Light source according to any of the claims 32 or 33, characterised in that said LED units comprise a plurality of LEDs, the plurality of light beams from such plurality of LEDs within each unit being collected together and superimposed by means of dichroic mirrors (21 a, 21b).

35. Light source according to any of claims 32 - 34, characterised in that said light source comprises a driver circuitry (18) capable of controlling the light emission of each LED within each LED unit (LED1 , LED2,..., LEDn).

36. Light source according to claim 35, characterised in that said driver circuitry (18) supplies power to each LED within each LED unit

(LED1 , LED2,..., LEDn), so that each LED and/or group/s thereof can be regulated in their light output, switched on and off entirely, as well as flash- operated for short to very short time periods, either individually or in a synchronized fashion and/or in groups or sequence/s of LEDs of the same or of different wavelengths.

37. Light source according to any of claims 35 or 36, characterised in that it comprises interface means to connect said driver circuitry (18) to a computer for operation control and/or programming.

38. Light source according to any of claims 35 - 37, characterised in that it comprises means to interface said driver circuitry (18) and said computer to the interchangeable excitation and emission band selection elements in the system, for integrated system control, programming and/or automation.