This invention relates to the field of fluorescence imaging and tunable spatially coherent light sources. More particularly, this invention relates to tunable spatially coherent light sources with a high intensity output at visible, ultraviolet and near infrared wavelengths and their application, for example, to fluorescence imaging.

Tunable spatially coherent laser light sources are desirable for many imaging applications, including microscopy and endoscopy, for display applications and for spectroscopy. It is known to filter a thermal white light source to provide almost arbitrary wavelengths. However, the spatial spectral brightness of such systems is too low for many applications, including projection scanning displays and confocal microscopy. Such applications typically require a laser radiation source because of the requirement for high intensity spatially coherent radiation.

There are only a limited number of low-cost and convenient lasers which operate in the visible spectrum. This has the result that most imaging and display systems are constrained to use a disadvantageously limited number of gas laser or solid-state laser lines which are available within the required spectral range. These problems limit the information to be gained in imaging systems and can compromise displays. This limitation has a particularly significant impact upon fluorescence microscopy since many biological fluorophores absorb light in the UV and visible range. The limitation has a particular impact on fluorescence microscopy-particularly when one considers most biological fluorophores exhibit UV to visible features.

High peak powers associated with ultra-short pulses can be exploited to use non-linear optical effects so as to generate tunable radiation. For example, continuum radiation may be generated from a quasi-monochromatic source and

then tunable radiation can be realised by selecting a desired portion of the continuum. Tunable radiation can then be conveniently realised by selecting a desired portion of the continuum. The selected portion of the continuum then can be amplified using a further gain medium or parametric amplifier to produce a useful intensity of tuned radiation. Such systems are typically complex and expensive being based upon dye or Ti: Sapphire laser systems.

Viewed from one aspect the present invention provides a fluorescence imaging apparatus comprising : a laser operable to generate pulses of laser radiation at variable wavelengths, including throughout the visible spectrum; a continuum generating medium through which said pulses of laser radiation are transmitted, said pulses of laser radiation having an intensity such that non-linear optical interaction between said pulses of laser radiation and said continuum generating medium generates pulses of continuum radiation extending across a continuum of wavelength values; a wavelength selector operable to select one or more spectral components of said pulses of continuum radiation to form pulses of tuned radiation; and an optical pathway operable to guide said pulses of tuned radiation to a subject to be imaged and to capture fluorescence radiation from said subject.

The invention recognises that the technique of continuum generation and wavelength selection can be extended to provide tunable radiation in the visible and shorter wavelengths, e. g. less than 800 nm, despite the prejudice in the field which would point away from deliberately rejecting a large portion of the radiation present within the continuum pulses (use of a wavelength selector in such short wavelength environments where the optical powers available are normally too low would point away from rejecting any of the available radiation). The development of high average power lasers that provide sufficiently high peak powers means it is possible to obtain a useful power output of tuned radiation without any need for the expense and complication of a further gain medium or parametric amplification.

Furthermore, the alteration of the temporal characteristics of the pulses by the

continuum and spectral tuning does not have any significant effect upon fluorescence imaging since this is relatively insensitive to the pulse temporal characteristics.

Whilst it will be appreciated that the continuum generating medium could take a variety of forms, including liquids, such as ethylene glycol, CS2, and solids, such as fused silica or sapphire, it is more efficient to use optical fibres where the light guiding behaviour maintains a long non-linear interaction length so improving the efficiency of continuum generation.

The use of microstructured optical fibres further enhances the continuum generation by making it easier to transmit the pulses of laser radiation and the pulses of continuum radiation within a single spatial mode (or modes) of the optical fibre and to arrange for the optical fibre to exhibit an appropriate group velocity dispersion.

The efficiency of the non-linear interaction generating the continuum may be further enhanced when the pulses of laser radiation have a wavelength close to a wavelength of zero chromatic dispersion within the continuum generating medium.

This feature may not always be desirable or appropriate.

Whilst it will be appreciated that the wavelength selector could take a variety of different forms, preferred embodiments include a dispersive [this could be a refractive prism or a diffractive grating or even a combination of both] element operable to spatially separate different wavelength components of the continuum radiation. Preferably this is used with a spatial light modulator, which may be reflective or transmissive.

In preferred embodiments a feedback system may be provided which is responsive to at least one detected spectral characteristic of the tuned pulses to control the action of the wavelength selector. In this way the wavelength selector can be accurately adjusted to produce an actively controlled spectral characteristic

within the pulses of tuned radiation as well as enabling the system to provide an advantageous degree of stability.

Whilst various different laser sources may be used, it is preferred to use a diode-pumped solid state laser or diode-pumped fibre laser as these are able to provide high power pulses of laser radiation, which produce a large degree of non- linear interaction to enable continuum generation, with high average power.

It is also possible and in some circumstances desirable, to provide a wavelength selector which is able to adjust the relative phases between different spectral components of the pulses of tuned radiation, such as by varying the different optical path lengths of different spectral components that have been spatially dispersed or other such techniques.

Whilst the tuned radiation produced by the present techniques has a wide variety of different uses it is particular well suited for use with spectrally-resolved fluorescence imaging and fluorescence lifetime imaging.

Viewed from another aspect the present invention provides a tunable source of electromagnetic radiation, said tunable source comprising: a laser operable to generate pulses of laser radiation at wavelengths, including throughout the visible or near infrared spectrum ; a continuum generating medium through which said pulses of laser radiation are transmitted, said pulses of laser radiation having an intensity such that non-linear optical interaction between said pulses of laser radiation and said continuum generating medium generates pulses of continuum radiation extending across a continuum of wavelength values; a wavelength selector operable to select one or more spectral components of said pulses of continuum radiation to form pulses of tuned radiation; and

a feedback system operable to control said wavelength selector in dependence upon at least one of : one or more detected spectral characteristics of said pulses of tuned radiation; and one or more desired outcomes of an experiment or procedure.

The feedback control of the tunable source provides improved accuracy and usefulness to the operation of the tunable source.

Various other aspects of the invention are set out in the appended claims.

Examples of the present invention will now be described, by way of example only, with reference to the accompanying drawings in which: Figure 1 schematically illustrates a tunable source of electromagnetic radiation in accordance with one example embodiment; Figures 2 and 3 schematically illustrate more detail of how tuning may be performed; Figure 4 schematically illustrates a second example embodiment; and Figure 5 schematically illustrates a further example embodiment using feedback to control a spatial light modulator.

Figure 1 illustrates a tunable source of radiation 2 which includes a diode- pumped solid-state laser 4 generating pulses of laser radiation. The laser 4 may be a passively mode-locked diode-pumped solid-state laser producing pulses in the range of approximately 100 fs to lOps duration at a repetition rate of approximately 20 to 100 MHz with average powers in the range of 100's mW tolO's of Watts and with a wavelength of approximately 1. 06, um. fIarmonic generation using these lasers can then produce pulses with up to multi-watt average power at 355nm and

multi-watt powers at 532nm. These visible wavelengths are suited for visible continuum generation so as to provide a tunable source of visible radiation but continua extending down to the visible and ultraviolet spectral regions may also be obtained using near infrared radiation, for example at 1064 nm or using radiation at two or more different wavelengths, for example 532 nm and 1064 nm.

The laser pulses output from the laser 4 are coupled into a microstructured optical fibre 6 so as to propagate within predominately a single spatial mode along the optical fibre 6. The high intensity of the pulses within the optical fibre 6 results in non-linear optical effects, such as the optical Kerr effect, stimulated Roman scattering, and others which have the result of transforming the wavelength of the laser pulses into a continuum of both longer wavelengths and shorter wavelengths.

In some favourable situations, the characteristics of the optical fibre 6 may be chosen such that it has a wavelength of zero chromatic dispersion close to the wavelengths of the input laser pulses or with an optimal relationship to the wavelength (s) of the input pulses.

The pulses of continuum radiation output from the optical fibre 6 are collimated by a lens 8 and then diffracted by a diffraction grating 10 and focused by a further lens 12 onto a spatial light modulator 14. In this way, wavelengths of a different value are focused at different points upon the spatial light modulator 14.

Thus, by controlling the transmission (or in other embodiments the reflection) of the spatial modulator 14 at different points a desired spectral characteristic can be imposed upon the pulses of radiation. The radiation thus selectively modulated which is transmitted through the spatial modulator 14 is collimated again with a lens 16 and forms pulses of tuned radiation having a spectral profile imposed upon them as controlled by the spatial modulator 14.

Figures 2 and 3 schematically illustrate in more detail how the tuning may be performed. The input to the tuning section is assumed to be spatially coherent broad-band (white) light source _ for instance as generated in the core of a mono- mode fibre. For the tuned source to be most useful it should be possible for that too

to be delivered through a pinhole or the core of a mono-mode fibre. The essential mechanism for achieving this is to project an image of the input source onto a spatially selective element (such as a spatial light modulator) but to include in that imaging section a dispersive element such as a prism or grating that spreads the image of the source across the selective element. In this way a different wavelength component of the source then falls on each pixel of the selective element which can then alter its amplitude and/or phase. To reconstruct a tuned point source the light from the selective element must be imaged onto the output point again through a dispersive element, which this time operates in reverse to recombine the spread spectrum into a single tuned point.

Figure 2 shows such a system which needs only a linear selective element to function. Of course, when the selective element is reflective then the input section can, to some extent, serve as the output section and hence the number of components may be reduced.

In a second example embodiment as schematically illustrated in Figure 3, the system can be arranged with cylindrical optics which in one direction (the x- direction say) operates identically to that shown in Figure 2. However in the other direction, (the y-direction) the light from the source is collimated across the selective element as shown in Figure 3.

In this arrangement the 2-dimensional array selective element actually displays a diffraction grating in the y-direction which is different for each wavelength (in the x-direction). The gratings can be used to steer the output light to different ports to give multiple outputs with different tuned spectral characteristics.

A further advantage of this system is that the device can operate at a high contrast ratio and offer both amplitude and phase modulation irrespective of the exact type of selective element (spatial light modulator) used as these are all controlled by the grating pattern written onto the element rather than the direct modulation imparted by the element. Again this system can operate in double pass to reduce the number of required elements.

Figure 4 schematically represents the system of Figure 1. The microstructured optical fibre is indicated as having a zero group velocity of dispersion in the visible spectrum so as to render it more efficient for continuum generation in this wavelength range.

Figure 5 illustrates a modified version of the system of Figure 4, but in this case including a feedback system comprised of a part silvered mirror 18, a radiation monitor 20 and a computer 22. A portion of the pulses of tuned radiation is directed by the part silvered mirror 18 to the monitor 20 where it is spectrally analysed in conjunction with the computer 22. The desired spectral characteristics of the pulses of tuned radiation is compared with those of the pulses of tuned radiation being generated and the spatial light modulator 14 adjusted by the computer 22 using electronic signals so as to match the desired and actual spectral characteristics.

It will be appreciated that the spatial light modulator can be considered to be formed of a collection of selectively opened or closed apertures through which a different spectral component may be allowed to pass with adjustable attenuation.

By adjusting the optical path length associated with each of these different apertures, the relative phases of the different spectral components may also be controlled in a manner that may be desired for the pulses of tuned radiation that are generated.

It will be appreciated that the present embodiments generate pulses of tuned radiations. Such a pulsed output is ideal for many applications, such as spectrally resolved fluorescence imaging and fluorescence lifetime imaging. The tunable source with feedback may also be used for multi-photon microscopy, coherent anti-Stokes Raman scattering, stimulated emission depletion etc. For other applications, such as confocal microscopy, display technology, cytometry, etc the main requirement is for tunable radiation and the system is often insensitive to whether this is continuous wave or pulsed radiation.

One particular application of this technique is to create pails (or larger numbers) of synchronised ultra short like pulses at different wavelengths for pump- probe spectroscopy and for realising microscopic imaging with super-resolution through the technique of stimulated emission depletion or similar techniques. This same approach can be applied to coherent anti-Stokes Raman scattering and to other imaging modalities. It may also be applied to coherent control applications.

The systems incorporating computer feedback control of the spatial light modulator can be configured such that the spectral profile of the output is iteratively changed to obtain an optimum output signal for a desired application.

As an example, the spectral characteristics of the output could be optimised to maximise fluorescence yield for microscopy or coherent control applications.

Alternatively the spectral optimisation could be used to maximise fluorescence contrast between different components of a sample, e. g. for clinical diagnosis using fluorescence or for fluorescence assays that may be used for high throughput screening. The spatial properties of the output beam may also be adjusted by the spatial light modulator, or an additional spatial light modulator, at the output. Such a spatially controlled tuned radiation is useful in various advanced microscopy techniques, including the imaging of polarization anistropy and stimulated emission depletion microscopy. The ability to produce multiple independently tunable excitation beams offers the possibility of performing excitation ratio imaging and the ability to separate different signals from different florescent molecules.