WO2005119328A1

WO2005119328A1 - Improved scanning microscope source

Info

Publication number: WO2005119328A1
Application number: PCT/GB2005/002077
Authority: WO
Inventors: Gail Mcconnell
Original assignee: University Of Strathclyde
Priority date: 2004-06-03
Filing date: 2005-05-25
Publication date: 2005-12-15

Abstract

A light source (1, 23) that is ideal for use within a scanning microscope (10, 28) is described. The light source (1) comprises a laser source (2) coupled to a photonic crystal fibre (5) so as to generate a continuum spectrum from the fibre (5). Thereafter, selection apparatus, such as a bandpass filter (7) or a grating (24), is employed to select a wavelength range of the generated continuum spectrum. By matching the wavelength range to the peak absorption wavelengths of a particular fluorophore makes the light source ideal for use with the scanning microscope e.g. a confocal laser scanning microscope (10), a multiphoton laser scanning microscope or a fluorescence lifetime imaging microscope (28).

Description

Improved Scanning Microscope Source

The present invention relates to the field of laser scanning microscopy. In particular the present invention relates to an improved light source for a laser scanning microscope e.g. a confocal laser scanning microscope (CLSM) or a fluorescence lifetime imaging microscope (FLIM) .

In recent years confocal laser scanning microscopy has become one of the most widely applied and versatile microscopic tools in the fields of biology, medicine, engineering and physics. For example, within the life sciences CLSMs provide a tool that enables minimally intrusive optical sectioning of fluorescently prepared cells and tissue at sub-micron resolutions, as described in J.B. Pawley's text entitled "Handbook of Biological Confocal Microscopy" 2^nd edition (Plenum Press, New York 1995) .

The majority of CLSMs known to those skilled in the art employ gas-based laser light sources (e.g. a helium-neon or argon ion laser) in order to provide the required visible or ultraviolet excitation radiation required to excite many of the widely used fluorophores. However, these laser sources exhibit several shortcomings, including stringent maintenance requirements, limited lifetimes, heat generation, large scale and high noise levels .

Alternative light sources known to those skilled in the art comprise solid state lasers, as described by J.M. Girkin et al "Confocal Microscopy Using An InGaN Violet Laser Diode At 406nm" Optics Express, Volume 7, Page 336- 341 (2000) . Generally, solid-state laser sources are more robust than gas laser sources, however they are not wavelength flexible and hence are suitable for use with only a limited range of useful fluorophores that are available within the art.

Multi-photon laser scanning microscopy (MPLSM) is an alternative technique that is increasingly being employed by those skilled in the art as a complimentary method to confocal laser scanning microscopy techniques. Typically, with MPLSM an ultra-short pulsed infrared emitting laser source provides the high peak powers required to instigate simultaneous excitation of multiple electrons within the fluorophore, as taught by D.L. Wokosin et al "All Solid State Ultrafast Lasers Facilitate Multiphoton Excitation Fluorescence Microscopy", IEEE J. Sel Top. Quantum Electronics Volume 2, Page 1051-1065 (1996). For a truly comprehensive imaging workstation comprising multiphoton and confocal microscopy capabilities, several laser sources are therefore often required. Alternative laser scanning microscopes include those based on fluorescence lifetime imaging microscopy (FLIM) . FLIM is a burgeoning technique for exploiting intensity independent contrast. In particular these techniques enable the quantitative determination of protein-protein interactions via measurement of the fluorescence resonance energy transfer (FRET) between donor and acceptor, fluorescently tagged, proteins.

To perform time correlated single photon counting (TCSPC) FLIM, a pulsed excitation source is required whose excitation wavelength should ideally be well-matched to the absorption wavelength (s) of the sample. Currently, the preferred options are either to employ pulsed laser diodes or via multi-photon excitation. In the former case, only a very limited number of discrete, fixed wavelengths are available. Although multi-photon excitation inherently provides optical sectioning capability, the lower excitation efficiency, limited tuning range (particularly the 1000-1200 nm range) and simultaneous excitation of multiple fluorescent molecules are significant limitations. Optical parametric oscillators have been used to perform both single and multi-photon excited TCSPC FLIM, however, these sources are complex and difficult to operate.

It is an object of at least one aspect of the present invention to provide a light source for a laser scanning microscope that provides the microscope with more flexible operating characteristics than those described in the prior art. Summary of Invention

According to a first aspect of the present invention there is provided a light source for use with a scanning microscope, the light source comprising a coherent light source coupled to a photonic crystal fibre so as to generate a continuum spectrum from the fibre and selection apparatus for selecting a predetermined wavelength range of the generated continuum spectrum.

According to a second aspect of the present invention there is provided a scanning microscope comprising a coherent light source coupled to a photonic crystal fibre so as to generate a continuum spectrum from the fibre, selection apparatus for selecting a predetermined wavelength range of the generated continuum spectrum, a microscope and scanning means for scanning the spectral output of the^" light source over a field of view of the microscope.

Preferably a first lens couples the coherent light source into the length of photonic crystal fibre.

Preferably a second lens collimates the continuum spectrum generated by the photonic crystal fibre.

The first and second lenses may comprise aspheric anti- reflection coated lenses.

Optionally the selection apparatus for selecting a predetermined wavelength range of the generated continuum spectrum comprises a bandpass filter. Alternatively, the selection apparatus for selecting a predetermined wavelength range comprises an optical dispersion means.

Preferably the optical dispersion means comprises an element selected from the group comprising a grating, an electro-optic modulator, an acoustic-optic modulator, a spatial light modulator, a monochrometer, a prism, a fibre, a grism, adaptive optics or one or more chromatic dispersion lenses .

Optionally the light source further comprises an optical isolator located between the coherent light source and the photonic crystal fibre.

Preferably the light source further comprises a power control means for controlling the output power of the device.

Most preferably the coherent light source comprises a pulsed single mode laser suitable of generating an output at a wavelength between 720 and 890 nm.

Preferably the photonic crystal fibre exhibits a zero dispersion wavelength below 720 nm. Optionally the photonic crystal fibre exhibits a zero dispersion wavelength of 670 nm.

Optionally the microscope comprises a confocal laser scanning microscope (CLSM) . Alternatively the microscope comprises a fluorescence lifetime imaging microscope (FLIM) . - ^• According to a third aspect of the present invention there is provided a method of generating light for a scanning microscope comprising the steps: 1) Generating a continuum spectrum; and 2) Selecting a portion of the continuum spectrum that corresponds to an optical characteristic of a sample to be tested.

Most preferably the step of generating a continuum spectrum comprises coupling a coherent light source to a photonic crystal fibre wherein the photonic crystal fibre exhibits a zero dispersion wavelength of λ₀ while the coherent light source operates at a wavelength greater than λ₀.

Alternatively the step of generating a continuum spectrum comprises coupling a coherent light source to a photonic crystal fibre wherein the photonic crystal fibre exhibits a zero dispersion wavelength of λo while the coherent light source operates at a wavelength of λ₀.

Preferably the step of selecting a portion of the continuum spectrum comprises the selection of portion that corresponds to an excitation wavelength of a flurophore located within the sample.

Brief Description of Drawings

Aspects and advantages of the present invention will become apparent upon reading the following detailed description and upon reference to the following drawings in which:

Figure 1 provides a schematic representation of a light source, suitable for employment with a scanning microscope, in accordance with an aspect of the present invention;

Figure 2 presents a sample unfiltered spectral output of the laser source of Figure 1;

Figure 3 presents a schematic representation of a confocal laser scanning fluorescence microscope in accordance with an aspect of the present invention;

Figure 4 presents a: a) fluorescence image; and b) transmission image of a guinea pig detrusor labelled with anti-PGP 9.5 and Alexa 488 obtained employing the confocal laser scanning fluorescence microscope of Figure 3.

Figure 5 presents a superimposed fluorescence and transmission image of a guinea pig smooth muscle cell containing flo-4 obtained employing the confocal laser scanning fluorescence microscope of Figure 3 ;

Figure 6 presents mean fluorescent intensity signals taken from a control (unloaded) , and ten loaded, smooth muscle cells obtained employing the experimental set up of Figure 5;

Figure 7 provides a schematic representation of an alternative embodiment of the light source suitable for employment with a scanning microscope;

Figure 8 presents a sample non-dispersed spectral output and two dispersed spectral outputs obtained from the laser source of Figure 7 ;

Figure 9 presents : a) a fluorescence image of a guinea pig detrusor labelled with anti-PGP 9.5 and Alexa 488; b) a fluorescence image of a guinea pig detrusor labelled with anti-smooth muscle myosin and Alexa 594; and c) a merged representation of the fluroscence images of (a) and (b) obtained employing a confocal laser scanning fluorescence that incorporates the laser source of Figure 7;

Figure 10 presents a schematic representation of a fluorescence lifetime imaging microscope in accordance with an aspect of the present invention; and

Figure 11 presents an Intensity and a TPSPC FLIM image of a mouse fibrosarcoma stained with haematoxin and eosin obtained by employing the fluorescence lifetime imaging microscope of Figure 10. Specific Description

Referring initially to Figure 1 a schematic representation of a light source 1 is presented in accordance with an aspect of the present invention. The light source 1 can be seen to comprise a commercially available Ti : Sapphire laser 2 (namely a Coherent, Mira 900-F) that exhibits an operating range of λ = 720 - 890 nm in the form of a 76 MHz train of pulses, with a pulse width of approximately 250 fs . In the presently described light source 1, the single-mode laser 2 is arranged to provide an emission wavelength of λ = 750nm. The output radiation generated is then propagated through a Faraday isolator 3 employed to reduce feedback from the subsequent surfaces within the light source 1.

On being transmitted through Faraday isolator 3 the output radiation is focused by a first aspheric anti- reflection coated lens 4, of focal length f=+4.5mm and numerical aperture of 0.4, into a 38cm length of photonic crystal fibre (PCF) 5.

PCF 5 is microstructured fibre, known to those skilled in the art of fibre optics. Radiation incident on the PCF 5 is guided by periodically arranged air holes that surround a solid silica core that extends the length of the fibre. This fibre design produces a photonic bandgap in the transverse direction that results in a fibre that is continuously single-mode throughout the visible range, see J.C. Knight et al "All-silica single-mode optical fibre with photonic crystal cladding" Optics Letters, Volume 21, Pages 1547-1549 (1996) . A result of the improved guiding properties of the PCF 5 is that it enables a reduction in the core diameter, down to a few microns, to be achieved. This results in a significant increase in the peak intensity of a propagating field that is enhanced with the application of ultra-short pulsed radiation. An increase in the peak intensity is of obvious general benefit in the study of nonlinear effects, and also is significant in the fact that the exact nature of the icrostructure determines the group velocity dispersion (GVD) of the fibre.

Typically, the zero-dispersion point λ₀, in a l-2μm core diameter PCF 5, is shifted from the bulk silica value of around λo = 1270nm down to the value of around λo = 600- 800nm. In the presently described embodiment the PCF 5 comprises pure silica and exhibits a hexagonal structure of air holes so as to guide the light within a 1.7 μm core. This arrangement gives rise to a zero dispersion wavelength at λ₀ = 670nm. It will therefore be appreciated by those skilled in the art that the fibre exhibits a low and positive dispersion throughout the normal Ti: Sapphire operating range (λ=720-890nm) and so generates a white light continuum for pump wavelengths within this range transmitted through the PCF 5.

The specific mechanisms involved in the generation of the white light continuum generation are complex. However, it is appreciated by those skilled in the art that these mechanisms comprise the PCF 5 ability to combine the effects of soliton self-frequency shift and the shedding of energy to shorter wavelengths due to third order dispersion. Both of these effects act to broaden the input radiation which then combines with four-wave mixing mechanisms that act to fill in any remaining gaps in the output spectrum. Within the light source 1 the radiation transmitted by the PCF 5 is collimated by employing a second aspheric anti-reflection coated lens 6 that exhibits a similar specification to that of the first 4. The collimated light is then passed through a band pass filter 7 and a neutral density filter 8. Both the band pass filter 7 and the neutral density filter 8 are easily interchangeable. The function of the band pass filter 7 is to provide a means for wavelength selection of the output from the light source 1 while the neutral density filter 8 provides a means for controlling the output power of the device. Figure 2 presents an experimentally measured spectral output 9 for the light source 1 in the absence of the bandpass filter 7 and the neutral density filter 8. The spectral output 9 can be seen to comprise a visible - continuum spectrum that exhibits a measured average output power of ~51mW. The length of the PCF 5 has been chosen so as to maximise the generation of radiation within the visible wavelength region, thus matching the single-photon excitation wavelengths of many of the fluorophores employed in the art, as described in detail below. Figure 3 presents a schematic representation of the light source 1 incorporated within a CLSM 10. The CLSM 10 can be seen to further comprises a scan head 11 (namely a commercially available Bio-Rad 1024ES) and an inverted microscope 12 (namely a Nikon, TE300) . The function of the scan head 11 and the inverted microscope 12 is to allow the collimated spectral output 9 of the light source 1 to be incident upon a sample contained within the inverted microscope 12. An image of the sample is then obtained by employing the scan-head 11 to scan the spectral output 9 over the field of view of the inverted microscope 12. This optical set up offers great flexibility in image acquisition strategies as is known to those skilled in the art. In particular it enables the production of optical section images, that is images in which light from out-of-focus regions do not contribute to the image.

From Figure 3 it can be seen that light entering the scan-head 11 is initially reflected by a dichroic mirror 13 and thereafter manipulated by scanning mirrors 14 towards the inverted microscope 12. A 40x/1.3 numerical aperture oil-immersion microscope objective lens 15 is then employed to focus the radiation onto a chosen fluorescently stained sample 16.

Fluorescence resulting from confocal excitation of the sample 16 is then collected by the objective lens 15 and directed back through the scanning mirror system 14 and the dichroic mirror 13 towards a photomultiplier tube 17. A second optical bandpass filter 18 is then located in front of the photomultiplier tube 17, the function of which is to provide a means for rejecting any light from the light source 1 than may be reflected by the components of the CLSM 10 directly onto the photomultiplier tube 17. An electrical signal generated by the photomultiplier tube 17 is then relayed to a computer processor (not shown) operating image capture software so as to allow for the visualisation of the fluorescently stained regions of the sample 16.

To demonstrate the suitability of the light source 1 for confocal laser scanning microscopy a series of experimental results obtained from a number of biological samples containing fluorophores are now presented. It should be noted that the broad spectral output 9 is particularly advantageous in that the light source can readily be employed to stimulate simultaneous or sequential multiple fluorophore excitations, as are commonly required within in the field of confocal laser scanning microscopy. However, for simplicity of interpretation the follow experimental results employ fluorophore transitions that only required single excitations to take place within the samples.

In particular, Figure 4 presents a typical confocal fluorescence xy cross-section obtained from a thick fixed tissue sample of guinea pig detruser (bladder smooth muscle layer) labelled with anti-PGP 9.5 and Alexa 488. Figure 5 presents similar results for a guinea pig detruser loaded with 2 μM of Fluo-4 AM.

Alexa 488 and Fluo-4 AM are known to exhibit peak absorption wavelengths at 490 nm and 494 nm, respectively. Therefore, in order to excite the applied fluorophores in each of the samples used to obtain the results of Figure 4 and 5 a 488±5nm optical bandpass filter 7, with a transmission of 82%, is employed within the light source 1. With this set up the light source 1 provided an average power output of 1.26 mW across the spectral output 9 which was then attenuated with the neutral density filter 8, of attenuation value ND=0.4, before being coupled into the scan-head 11, as previously described. The average power measured at the samples 16 was thus recorded at 620 μW, which is of a comparable value to the average powers typically obtained employing standard Kr/Ar laser sources.

The fluorescence image of Figure 4(a) was obtained at a sample depth of approximately 41 μM. Figure 4(b) presents the transmission image taken for the same region of the sample. Within the fluorescence image it is possible to observe the fluorescently labelled nerves 19 wrapped around a blood vessel 20 over a partial depth.

In practice the CLSM 10 is capable of operating over a total depth of 59 μm within this particular sample 16. Furthermore, over an image period of several hours, no photobleaching or tissue damage was observed within this sample 16, as is commonly experienced with conventional Kr/Ar laser confocal excitation. A high contrast ratio was also observed with a signal background ratio typically exceeding 100:1, which is again comparable to the excitation results achieved within Kr/Ar laser systems.

Figure 5 presents a superimposed fluorescence and transmission images of a guinea pig smooth muscle cell 21. It is noted that a dead cell 22 that also exhibits a fluorescent signal is present below the smooth muscle cell 21. Once more the continual exposure of this sample to radiation over several hours caused no cell damage i.e. membrane, blebbing or shape change or pronounced photobleaching to the sample. A high contrast ratio was again measured as indicated by the comparison of the mean fluorescence intensity signal from the Fluo-4 loaded and control (unloaded) cells, a can be seen in Figure 6.

An alternative embodiment of the light source 23 is now presented within Figure 7. The light source 23 can be seen to comprise common elements with the previously described embodiment presented in Figure 1, therefore for clarity purposes the same reference numerals are employed throughout, as appropriate.

The light source 23 again comprises the Ti : Sapphire laser 2 , the Faraday isolator 3 , the first and second anti reflection coated lenses 4 and 6, the length of photonic crystal fibre 5 and the neutral density filter 8 the function of which are as previously described. However, it should be noted that within the light source 23 a different PCF 5 is employed. In this source 23 the PCF 5 has an overall length of 29cm.

The light source 23 differs from the previously described embodiments in that, the bandpass filter 7 is replaced with a 600 lines/mm grating 24 positioned after the neutral density filter 8. Rotation of the grating 24 thus provides a means for changing the angle of dispersion, and hence the wavelength, of the output of the light source 23.

The light source 23 can again be employed with the scan- head 11 and the inverted microscope 12 so as to produce a CLSM. In practice a variable aperture is employed between the light source 23 and the scan-head 11 so as to provide a means for regulating the FWHM of the visible continuum propagating into the scan-head 11.

The spectral output from the light source 23 in the absence of the grating 25 (at 64mW) and for two separate grating angles is presented in Figure 8. The first grating angle was chosen so as to correspond to the absorption maximum of Alexa 488 (-490 nm) while the second to correspond to the absorption maximum of Alexa 594 (-564 nm) . In the absence of the grating 24 the continuum can be seen to extend from λ=400 nm to λ=710 nm. With the grating 24 located at the two separate grating angles the laser source output provides spectral coverage of 493 ± 7 nm 26 and 561 ± 8 nm 27, respectively.

To demonstrate sequential CLSFM using this light source 23, a multiple labelled sample was employed. A 20 μm- thick fixed tissue sample of guinea pig detrusor (bladder smooth muscle layer) was therefore labelled firstly with anti-PGP 9.5 and Alexa 488 to highlight the nerve structure and secondly with anti-smooth muscle myosin and Alexa 594 to determine the morphology of the smooth muscle layer. To sequentially excite both the Alexa 488 and Alexa 594 fluorophores, the grating and variable width apertures were orientated to deliver 859 μW and 782 μW to the sample alternatively, with spectral coverage again of 493 + 7 nm and 561 ± 8 nm, respectively. A 20x/0.75 numerical aperture air objective lens 15 was used to focus the radiation onto the labelled sample.

Figure 9 presents a typical CLSFM xy cross-sections of multiple-labelled guinea pig detrusor, obtained at a depth within the sample of approximately 13 μm, where 9(a) and 9(b) correspond to fluorescence from Alexa 488 and Alexa 594 respectively. The images were taken at a capture rate of 0.95 Hz with a 512x512 pixels box size and were averaged over six consecutive scans, leading to a 2.7 μs pixel dwell time. A merge of both 9(a) and 9(b) is shown in Figure 9(c) . This composite image clearly indicates the nerve structure and position relative to the smooth muscle layer. Complete optical sectioning of the 20 μm thick sample was readily achieved.

Over a typical imaging period of several hours, no photobleaching or tissue damage was observed with this thick sample. A high contrast ratio was routinely observed, with a signal-to-background ratio that typically exceeded 100:1. Increasing the width of the variable aperture and hence increasing the average power supplied to the sample resulted in no observable increase in fluorescence signal. Conversely, decreasing the width of the aperture from the values given previously resulted in a significant reduction in fluorescence signal from both fluorophores.

The flexibility of light source 1 is now demonstrated in relation to its employment within an alternative laser scanning microscope techniques. In particular Figure 10 presents a schematic representation of a fluorescence lifetime imaging microscope 28 that comprises the laser source 1 employed in conjunction with a time correlated single photon counting (TCSPC) module 29 (Becker-Hickl SPC830) . The bandpass filter 7 is arranged so that the light source 1 produces an output at a wavelength λ=540 ± 10 nm. This output is then employed within the fluorescence lifetime imaging microscope 28 so as to produce Intensity 30 and TPSPC FLIM 31 images of a mouse fibrosarcoma stained with haematoxin and eosin, as presented in Figure 11.

Alternatively, the laser source 1 or 23 can easily be adapted so as to be suitable for carry out multiphoton microscopy techniques . By taking advantage of the short zero dispersion wavelengths in the photonic crystal fibre 5 and by tuning the Ti : Sapphire laser 2 to the inherent zero dispersion wavelength of the photonic crystal fibre 5, the Ti: Sapphire laser 2 wavelength and pulse duration is unaffected and hence is ideal for MPLSM. Conversely, by tuning the Ti : Sapphire laser to anomalously pump the photonic crystal fibre 5, the resultant white-light supercontinuum source can again capably perform CLSM or FLIM, as previously described.

It will be readily appreciated by those skilled in the art that the described apparatus is generally modular in nature. This allows various component elements to be easily interchanged, for example: • the coherent light source is described as a Ti: Sapphire laser however any ultrashort pulsed laser source be alternatively be employed. • the bandpass filter can be replaced with an alternative filtration means such as a prism or a grating combined with a narrow slit; • the grating can be replaced with an alternative optical dispersion means for changing the wavelength of the output of the light source 23 e.g. an electro-optic modulator, an acoustic-optic modulator, a spatial light modulator, a monochrometer, a prism, a fibre, a grism, adaptive optics or chromatic dispersion lenses; • an inverted microscope has been described above however it will be readily apparent to those skilled in the art that an upright microscope may equally well be employed; • the light source has been described in connection with a confocal laser scanning microscope and a fluorescence lifetime imaging microscope that are based on detecting the fluorescence of one or more flurophores located within a sample. However, it will be appreciated by those skilled in the art that the same laser source could be used within a laser scanning microscope that is based on the detection light generated by the light source once it has been reflected from, or transmitted by, a particular sample; and • the microscope objective lenses were chosen because of their suitability for use with live cell imaging. However, other alternative lens systems may be employed so as to achieve the same function.

The present invention demonstrates a flexible laser source for a scanning microscope based on laser platform that is also ideally suited to carry out multi-photon laser scanning microscopy. In particular, a commercial ultra-short pulsed Ti : Sapphire laser has been employed as a pump source for a photonic crystal fibre so as to generate a visible continuum output spectrum. The wavelength range required to efficiently match the profile of a particular fluorophore is then selected from the visible continuum by either using a conventional and inexpensive optical bandpass filter or an optical dispersion means such as a grating.

A significant advantage of the present invention is that it provides a reliable, cost effective, modular system that can be employed to access the whole range of fluorophores currently employed within the field of laser scanning microscopy. The present invention has the added advantage that it employs a laser source that can also be used to perform multi-photon laser scanning microscopy thereby providing a complete laser scanning excitation solution. This has the advantages of reducing costs, maintenance requirements and complexity of the instrumentation while increasing the range of existing flurophores that can be excited.

The foregoing description of the invention has been presented for purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise form disclosed. The described embodiments were chosen and described in order to best explain the principles of the invention and its practical application to thereby enable others skilled in the art to best utilise the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, further modifications or improvements may be incorporated without departing from the scope of the invention as defined by the appended claims.

Claims

1) A light source for use with a scanning microscope, the light source comprising a coherent light source coupled to a photonic crystal fibre so as to generate a continuum spectrum from the fibre and selection apparatus for selecting a predetermined wavelength range of the generated continuum spectrum.

2) A light source as claimed in Claim 1 wherein a first lens couples the coherent light source into the length of photonic crystal fibre.

3) A light source as claimed in Claim 1 or Claim 2 wherein a second lens collimates the continuum spectrum generated by the photonic crystal fibre.

4) A light source as claimed in any of the preceding claims wherein the selection apparatus for selecting a predetermined wavelength range of the generated continuum spectrum comprises a bandpass filter.

5) A light source as claimed in any of Claims 1 to 3 wherein the selection apparatus for selecting a predetermined wavelength range comprises an optical dispersion means. 6) A light source as claimed in Claim 5 wherein the optical dispersion means comprises an element selected from the group comprising a grating, an electro-optic modulator, an acoustic-optic modulator, a spatial light modulator, a monochrometer, a prism, a fibre, a grism, adaptive optics or one or more chromatic dispersion lenses.

7) A light source as claimed in any of the preceding claims wherein the light source further comprises an optical isolator located between the coherent light source and the photonic crystal fibre .

8) A light source as claimed in any of the preceding claims wherein the light source further comprises a power control means for controlling the output power of the device.

9) A light source as claimed in any of the preceding claims wherein the coherent light source comprises a pulsed single mode laser suitable of generating an output at a wavelength between 720 and 890 nm.

10) A light source as claimed in any of the preceding claims wherein the photonic crystal fibre exhibits a zero dispersion wavelength below 720 nm.

11) A light source as claimed in any of the preceding claims wherein the photonic crystal fibre exhibits a zero dispersion wavelength of 670 nm.

12) A scanning microscope comprising a light source as claimed in any of claims 1 to 11, a . microscope and scanning means for scanning the spectral output of the light source over a field of view of the microscope.

13) A scanning microscope as claimed in Claim 12 wherein the microscope comprises a confocal laser scanning microscope. 14) A scanning microscope as claimed in Claim 12 wherein the microscope comprises a fluorescence lifetime imaging microscope (FLIM) .

15) A method of generating light for a scanning microscope comprising the steps: 1) Generating a continuum spectrum; and 2) Selecting a portion of the continuum spectrum that corresponds to an optical characteristic of a sample to be tested.

16) A method as claimed in Claim 15 wherein the step of generating a continuum spectrum comprises coupling a coherent light source to a photonic crystal fibre wherein the photonic crystal fibre exhibits a zero dispersion wavelength of λo while the coherent light source operates at a wavelength greater than λo .

17) A method as claimed in Claim 15 wherein the step of generating a continuum spectrum comprises coupling a coherent light source to a photonic crystal fibre wherein the photonic crystal fibre exhibits a zero dispersion wavelength of λ₀ while the coherent light source operates at a wavelength of λ₀.

18) A method as claimed in any of claims 15 to 17 wherein the step of selecting a portion of the continuum spectrum comprises the selection of portion that corresponds to an excitation wavelength of a flurophore located within the sample.