WO2011099938A1

WO2011099938A1 - Method for supplying light beams for integrated cars and multiphoton microscopy

Info

Publication number: WO2011099938A1
Application number: PCT/SG2011/000051
Authority: WO
Inventors: Zhiwei Huang
Original assignee: National University Of Singapore
Priority date: 2010-02-12
Filing date: 2011-02-02
Publication date: 2011-08-18

Abstract

A method and apparatus for supplying light beams for integrated CARS and multiphoton microscopy, and an integrated CARS and multiphoton microscopy system and method. The method for supplying light beams for integrated CARS and multiphoton microscopy comprises the steps of generating a femtosecond pump beam; optionally generating a femtosecond Stokes beam using a portion of the pump beam; spectrally filtering the Stokes beam, the pump beam, or both such that the filtered beams are in a picosecond range or a femtosecond range depending on a desired microscopy operation mode; and supplying the filtered Stokes beam, pump beam, or both to a scanning microscope.

Description

METHOD FOR SUPPLYING LIGHT BEAMS FOR INTEGRATED CARS AND MULTIPHOTON MICROSCOPY

FIELD OF INVENTION

The present invention relates broadly to a method and apparatus for supplying light beams for integrated CARS and multiphoton microscopy, and to an integrated CARS and multiphoton microscopy system and method.

BACKGROUND

Femtosecond (fs) pulsed lasers have been widely used for multiphoton microscopy imaging, including two-photon excitation fluorescence (TPEF), second- harmonfe generation (SHG), third-harmonic generation (THG), and sum-frequency generation (SFG), etc., due to the high peak power of fs lasers for efficient generation of nonlinear signal radiation. Meanwhile, in the past decade, coherent anti-Stokes Raman scattering (CARS) microscopy has been developed for label-free bio-imaging owing to its outstanding capability of real-time, non-invasive chemical mapping of live cells and tissues based on molecular vibrations. It is well known that picosecond (ps) pulsed lasers are ideal for CARS imaging because their spectral bandwidths match well with the Raman linewidth (about 10 cm^"1) of biomoiecules for optimizing the CARS excitation with improved spectral resolution and minimized nonresonant background. Thus, the early multiphoton microscopy imaging systems were unsuitable for CARS imaging, and vice versa.

Very recently, multimodal nonlinear optical microscopy, which integrates different nonlinear optical imaging techniques (e.g. CARS, TPEF, SHG, THG, SFG) together, has emerged as a powerful tool for label-free bio-molecular imaging with intrinsic biochemical sensitivity and specificity. However, the conventional multimodal imaging technique usually involves using both fs and ps laser sources which make the technique very costly and inconvenient for operation, especially in biological laboratories. To facilitate wide application of multimodal nonlinear optical microscopy in biological and biomedical systems, it is highly desirable to simplify the technique by only employing e.g. one fs laser source while still being able to access the different nonlinear optical microscopy imaging modalities.

A need therefore exists to provide a method and apparatus for supplying light beams for integrated CARS and multiphoton microscopy that seek to address at least one of the above problems.

SUMMARY

In accordance with a first aspect of the present invention, there is provided a method of supplying light beams for integrated CARS and multiphoton microscopy, the method comprising the steps of:

generating a femtosecond pump beam;

optionally generating a femtosecond Stokes beam using a portion of the pump beam;

spectrally filtering the Stokes beam, the pump beam, or both such that the filtered beams are in a picosecond range or a femtosecond range depending on a desired microscopy operation mode; and

supplying the filtered Stokes beam, pump beam, or both to a scanning microscope. Generating the femtosecond Stokes beam may comprise coupling the portion of the pump beam into an optical parametric oscillator.

Spectrally filtering the Stokes or pump beam may comprise directing said beam to a respective pair of gratings.

The method may further comprise aligning an orientation of a second grating in counter-direction to that of a first grating. The method may further comprise disposing, in a sequence, a first achromatic focusing lens, a slit and a second achromatic focusing lens between the pair of gratings. The first and second achromatic focusing lenses may have the same focal length.

The method may further comprise configuring a distance between adjacent elements to be equal to the focal iength.

The method may further comprise adjusting a lateral position of the slit for controlling a central wavelength of the filtered beam.

The method may further comprise adjusting the slit width for controlling a bandwidth of the filtered beam such that the filtered beam is in the picosecond range or the femtosecond range depending on the desired microscopy operation mode.

Supplying the filtered Stokes beam, pump beam, or both to the scanning microscope may comprise delaying the filtered pump beam to substantially cancel out optical path differences.

The light source may comprise a Ti:sapphire laser source.

In accordance with a second aspect of the present invention, there is provided an integrated CARS and multiphoton microscopy method, the method comprising the steps of:

supplying light beams to a scanning microscope as defined in the first aspect; focusing the light beams onto a sample;

collecting nonlinear optical signal radiation from the sample; and

obtaining measurements from the radiation.

Obtaining measurements from the radiation may comprise using at least one of a photomultipiier tube and a spectrometer. The sample may comprise a biological or biomedical sample.

In accordance with a third aspect of the present invention, there is provided an , apparatus for supplying light beams for integrated CARS and multiphoton microscopy, comprising:

means for generating a femtosecond pump beam;

means for optionally generating a femtosecond Stokes beam using a portion of the pump beam;

means for spectrally filtering the Stokes beam, the pump beam, or both such that the filtered beams are in a picosecond range or a femtosecond range depending on a desired microscopy operation mode; and

means for supplying the filtered Stokes beam, pump beam, or both to a scanning microscope. The means for generating the femtosecond Stokes beam may comprise an optical parametric oscillator configured for having the portion of the pump beam coupled*thereinto.

The means for spectrally filtering the Stokes or pump beam may comprise a respective pair of gratings.

An orientation of a second grating may be aligned in counter-direction to that of a first grating.

The apparatus may further comprise, in a sequence, a first achromatic focusing lens, a slit and a second achromatic focusing lens between the pair of gratings.

The first and second achromatic focusing lenses may have the same focal length.

A distance between adjacent elements may be equal to the focal length. A lateral position of the slit may be adjustable for controlling a central wavelength of the filtered beam.

The slit width may be adjustable for controlling a bandwidth of the filtered beam such that the -filtered beam may be in the picosecond range or the femtosecond range depending on the desired microscopy operation mode.

The means for supplying the filtered Stokes beam, pump beam, or both to the scanning microscope may comprise means for delaying the filtered pump beam to substantially cancel out optical path differences.

The light source may comprise a Ti:sapphire laser source. in accordance with a fourth aspect of the present invention, there is provided an integrated CARS and multiphoton microscopy system, comprising:

an apparatus for supplying light beams as defined in the third aspect;

means for focusing the light beams onto a sample;

means for collecting nonlinear optical signal radiation from the sample; and means for obtaining measurements from the radiation.

The means for obtaining measurements from the radiation may comprise at least one of a photomultiplier tube and a spectrometer.

The sample may comprise a biological or biomedical sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will be better understood and readily apparent to one of ordinary skill in the art from the following written description, by way of example only, and in conjunction with the drawings, in which:

Figure 1 shows a schematic diagram of an integrated multimodal microscopy system according to an example embodiment. Figure 2 shows a graph of the signal to background ratio using the system of Figure 1 according to an example embodiment. Figure 3(a) shows graphs comparing fs-CARS and ps-CARS spectra using the system of Figure 1 , as well as the spontaneous Raman spectrum, of 465 nm polystyrene beads immersed in water.

Figure 3(b) shows a fs-CARS image of 465 nm polystyrene beads in water using the system of Figure 1.

Figure 3(c) shows a ps-CARS image of 465 nm polystyrene beads in water using the system of Figure 1. Figure 4(a) shows a fs-CARS image of lipid droplets in a 50 pm sectioned fibrotic liver tissue using the system of Figure 1.

Figure 4(a) shows a ps-CARS image of lipid droplets in a 50 pm sectioned fibrotic iiver tissue using the system of Figure 1.

Figure 4(c) shows respective intensity profiles along the lines in Figures 4(a) and 4(b).

Figure 4(d) shows a SMG image of lipid droplets in a 50 pm sectioned fibrotic liver tissue using the system of Figure 1.

Figure 4(e) shows a TPEF image of iipid droplets in a 50 pm sectioned fibrotic Iiver tissue using the system of Figure 1. Figure 4(f) shows a merged image based on the images of Figures 4(b), 4(d) and 4(e).

Figure 5 shows a flow chart illustrating a method for supplying light beams for integrated CARS and multiphoton microscopy according to an example embodiment. DETAILED DESCRIPTION The example embodiments describe spectral filtering a femtosecond (fs) laser source to provide both high-contrast coherent anti-Stokes Raman scattering (CARS) microscopy and high-quality multiphoton microscopy on the same platform for label-free biomolecular imaging. Figure 1 shows a schematic diagram of an integrated multimodal (e.g. CARS and multiphoton) microscopy system 100, e.g. for use in bio-imaging, according to an example embodiment. The system 100 comprises a fs laser source 102 (e.g. a Titanium (Ti):sapphire laser source) and an optical parametric oscillator (OPO) system 104 for providing both pump and Stokes excitation light beams. Details of such T sapphire laser source 102 and OPO system 104 can be found in [Fake Lu, Wei Zheng, and Zhiwei Huang, Heterodyne polarization coherent anti-Stokes Raman scattering microscopy, AppJ. Phys. Lett. 92 (12), 123901 (2008)] and [Fake Lu, Wei Zheng, Colin Sheppard, Zhiwei Huang, Interferometric polarization coherent anti-Stokes Raman scattering microscopy, Optics Letters, 33(6), 602-604 (2008)], the contents of which are hereby incorporated by cross-reference.

In the example embodiment, the pump beam 103 and Stokes beam 107 are delivered into two separate spectral filtering systems 110, 120 (to be described in detail below) respectively for pulse shaping via e.g. a beam splitter BS and a mirror 106, as shown in Figure 1. The spectrally shaped pump and Stokes beams that emerge from the spectral filtering systems 1 10, 120 respectively are coliinearly combined through a dichroic mirror DM (e.g. Model No. SWP-45-RU1080-TU830, manufactured by CVI Melles Griot), and delivered into a customized confocai laser scanning microscope 140 (e.g. Model No. FV300, manufactured by Olympus), and then focused onto a sample 150 by a microscope objective MO (e.g. Model No. UPlanSApo 40x, numerical aperture (N.A.) 0.9, manufactured by Olympus) for multimodal nonlinear optical imaging. The customized confocai laser scanning microscope 140 in the example embodiment comprises a high near infra-red (NIR) reflection mirrors design for scanning and a high NIR transmission tube lenses design.

The nonlinear optical signal radiation from the sample 150 is collected in the example embodiment by a condenser 156 (e.g. Model No. U-TLD, N.A. 0.9, manufactured by Olympus), and detected by a detector in the form of a photomultiplier tube PMT (e.g. Model No. R3896, manufactured by Hamamatsu) through spectral filters F. In addition, a flip mirror FM is disposed optically before the photomultiplier tube PMT to allow forward CARS spectral measurements by a spectrometer 160 (e.g. Model No. HR4000, manufactured by Ocean Optics Inc., Dunedin, Florida).

Additionally, the system 100 comprises a mirror 108 and a delay 130 optically coupled to the output beam from the spectral filtering system 1 0 for controlling the path of this output beam before striking the dichroic mirror DM. For example, the position of the delay 130 is adjustable such as to substantially cancel out the optical path differences between the output beam from the spectral filtering system 1 10 and the output beam from the spectral filtering system 120. The path length difference for the Stokes beam in the example embodiment is largely due to the OPO system 104. The system 100 further comprises optical elements such as mirrors 142, 152, lens 154, 158 appropriately positioned for directing and focusing respectively, as will be appreciated by a person skilled in the art.

Each spectral filtering system 110, 120 comprises a first grating 1 , 121 ; a first achromatic focusing lens 1 12, 122; a slit 1 13, 123; a second achromatic focusing lenses 114, 124; and a second grating 115, 125. The achromatic focusing lenses 1 12, 122, 1 14, 124 have the same focal iength f. Also, the separation between consecutive elements is equal to one focal Iength f of the achromatic focusing lenses 112, 122, 114, 124, which will be referred to herein as a 4-f configuration, in addition, each spectral filtering system 110, 120 comprises a mirror 116, 126 for directing the input beam to the first grating 1 11 , 121 , and a mirror 117, 127 for directing the output beam from the second grating 1 5, 125. In the example embodiment, when e.g. 100-fs pump 103 and Stokes 107 light beams are directed into the 4-f configured paired-gratings spectral filtering systems 110, 120 respectively, different spectral or wavelength components of the incident light are angularly dispersed into different directions by the first grating 111, 121 and then focused into a iine oh the focal plane of the first achromatic focusing lens 112, 122 (e.g. focal length f = 100 mm). Both spatial (x) and temporal (t) profiles of the input puise (E_m) are assumed to be Gaussian, that is:

£,„(x,t) = £₀exp(-x²/w²)exp(-a₀/²) (1) where E₀ is the amplitude; w is the beam radius at the e^"1 level; and ₀ = τ^'7 , τ being the temporal FWHM of the input pulse.

For example, if the width of the slit 113, 123 positioned on the spectral plane of the first focusing lens 112, 122 is D (Figure 1) the temporal property of the output pulse (E_out) through the 4-f paired-gratings filtering system 110, 120 can be expressed as: exp[-(&¾:/ -t)^~ 14p]\ H(y/)sxp(-s /^~ -iry/)dy/dx (2)

Hence, the spectral bandwidth (E_out(w)) of the tailored (i.e. shaped) pulses can be derived by performing Fourier transform of Equation (2):

E_oul{co)= E_oul{t)eKp{-icot)dt (3) where

r = (ax + ₀kpM>²t)/( ² +c₀k² β²Μ>²) (3.2) p = \I _Q +k² w² /4a² where A is a constant accounting for the overall reflection and absorption losses as well as slowly varying factors; Η(ψ) is the amplitude transmittance function for a single silt; a=cos(Q₀)/cos(y₀) and β=2ποη/[ω₀ ²άοο8(γ₀) c is the velocity of iight in vacuum; k=ix>₀ /c is the wave number at central frequency (ω₀); m is the diffraction order {m = 1); d is the groove spacing of the grating; θ₀, and y¾ are the incident and diffraction angles of the input pulse at the central frequency respectively. in the dual 4-f paired-gratings spectral filtering systems 110, 120 of the example embodiment, the slit 113, 123 disposed on the spectral plane of the respective first lens 112, 122 for spectral shaping or tailoring comprises a motorized tunable slit. According to Equations (1)-(3), the lateral position of the slit 113, 123 determines the central wavelength, while the slit width (D) controls the bandwidth of the output filtered laser pulses. Since different spectral components dispersed travel through different optical paths in this process, pulse group velocity dispersion (GVD) may result. In the example embodiment/ the respective second achromatic focusing lens 114, 124 and second grating 115, 125 (same as respective first ones), are positioned in the 4-f optics configuration, and advantageously substantially cancel out the optical path differences to minimize the overall GVD. Such arrangement helps to maintain the high pulse peak power of the spectrally filtered output laser pulses for effective nonlinear optical signal generation in the example embodiment. Also, in the dual 4-f configured paired-gratings filtering design of the example embodiment, the orientations of the respective paired gratings (11 and 115; 121 and 125) are preferably aligned in counter-directions such that the spectrally filtered components are recombined at the second grating 115, 125, and their angular dispersions are substantially compensated.

Each of the paired gratings 111 and 115 (e.g. density of 1200 gr/mm, blazed at 750 nm) used for spectral filtering of the 100-fs pump beam 103 provides an approximately 75% diffraction efficiency in the 700-1600 nm range in the example embodiment. Each of the other paired-gratings 121 and 125 (e.g. density of 1200 gr/mm, blazed at 1000 nm) for the fs Stokes beam 107 filtering also has about 75% diffraction efficiency in the 880-1600 nm range in the example embodiment. The 4-f configured paired-gratings spectral filtering systems 110, 120 of the example embodiment offer a large tunability of Raman shifts covering from e.g. 100 to 8000 cm^"1 for CARS imaging. The overall diffraction efficiency measured from each of the 4-f paired-gratings systems 1 10, 120 of the example embodiment (including paired gratings, 2 lenses, slit, mirrors) is about 6% for the pump beam 103, and about 8% for the Stokes beam 107. CARS experiments have proved that the power level with about 6 to 8% diffraction efficiency is sufficient for high contrast ps-CARS imaging of thick tissues.

In the example embodiment, by adjusting the slit widths, for instance, down to about 150 m and 200 m, respectively, for the input pump and Stokes light beams (the beams have different wavelengths) with pulse widths of about 100 fs (FWHM of about 120 cm^"1), output pump and Stokes laser pulses with puise widths of about 1 ps (FWHM of about 11 cm^"1) can be achieved from the 4-f paired-gratings filtering systems 1 0, 120 for ps-CARS imaging, which match well with calculation results based on Equations (1)-(3). That is, the pulse width and FWHM of each central wavelength are controlled by the slit in the example embodiment.

The FWHM of the spectrally filtered pump and Stokes light beams using the system of the example embodiment is 10-fold narrower than the fs laser beams. This indicates that the ps-CARS imaging technique of the example embodiment can provide at least 10-fold improvements in spectral resolution, as compared to e.g. fs- CARS. Thus, simply by changing the slit widths in the 4-f paired-gratings filtering systems 110, 120, switching between the 1 ps and 100 fs laser pulses is achieved in the example embodiment for CARS imaging (ps pulses) and multiphoton imaging (fs pulses) on the same platform, e.g. for biomedical applications.

Figure 2 shows a graph 200 of the signal to background ratios (SBR) using the system of Figure 1 according to an example embodiment. Here, the SBR are measured based on CARS images of 465 nm polystyrene beads immersed in water using different pulse bandwidths of the pump and Stokes beams. As can be seen in Figure 2, the smaller the spectral FWHM of the excitation pulses, the higher the SBR of CARS imaging obtained. Figure 3(a) shows graphs comparing fs-CARS and ps-CARS spectra using the system of Figure 1 , as well as the spontaneous Raman spectrum, of 465 nm polystyrene beads immersed in water. Figure 3(b) shows a fs-CARS image of 465 nm polystyrene beads in water using the system of Figure 1. Figure 3(c) shows a ps- CARS image of 465 nm polystyrene beads in water using the system of Figure 1.

Here, the spontaneous Raman spectrum is recorded using a micro-Raman spectrometer system (e.g. inVia series, manufactured by Renishaw, UK), and CARS spectra are obtained by scanning the wavelengths of the Stokes beam, e.g. using the OPO system 104 (Figure 1 ), from 1099 nm to 1130 nm while fixing the wavelength of the pump beam at 830 nm. The puise widths of the Stokes beam may change slightly, but can be controlled by the slit, as described above. The average powers of the pump and Stokes beams are about 4 and 2 milliwatts (mW), respectively, for both fs-CARS and ps-CARS imaging.

As can be seen in Figure 3(a), compared to the fs-CARS spectrum (line 302), the ps-GARS spectrum (line 304) shows much iess spectral iineshape distortion and smaller frequency shift (relative to the spontaneous Raman spectrum (line 306)). This indicates that the nonresonant background in ps-CARS is relatively smaller than that in fs-CARS. The fs-CARS and ps-CARS imaging (e.g. based on aromatic C-H stretching vibration at 3054 cm^"1) of 465 nm polystyrene beads in water confirms a 2-fold improvement in the suppression of nonresonant background using ps-CARS imaging over fs-CARS imaging. For example, the graph inserted in the fs- CARS image of Figure 3(b) (for the intensity distribution along iine 312) shows a vibrational contrast (a ratio of peak intensity to background intensity) of about 1.5:1 , while the corresponding graph inserted in the ps-CARS image of Figure 3(c) (for the intensity distribution along line 314) shows a vibrational contrast of about 3:1.

The inventors have applied the integrated CARS and multiphoton microscope system of the example embodiment to biomedical imaging. Figure 4(a)-(f) show results of an example use of the multimodal nonlinear optical imaging system (e.g. CARS, TPEF, and SHG) of the example embodiment, acquired from a sectioned fibrotic liver tissue induced by bile duct ligation (BDL) in a rat model, in this example, the average powers of the pump and Stokes beams are 6 and 3 mW, respectively, for both fs-CARS and ps-CARS imaging, while the average power of the laser beam at 800 nm for SHG and TPEF imaging is about 0 mW.

As shown in Figures 4(a) and 4(b), the hepatic lipid droplets occurring in rat liver tissue are clearly identified in both fs- and ps-CARS images using a CARS-only microscopy mode (e.g. based on symmetric CH₂ stretch vibration of hepatic lipids at 2840 cm^"1). In addition, ps-CARS imaging gives a higher vibrational contrast (about 4:1), compared to that of fs-CARS imaging (about 2:1), as depicted in Figure 4(c), which shows intensity profiles 412, 414 across lines 402, 404 indicated in Figure 4(a) and 4(b), respectively. The lipid droplet distributions and variations inside the hepatic cells can be observed more clearly in ps-CARS imaging due to its improved sensitivity and spectral resolution (Figure 4(c)).

By swapping the 4-f grating filtering from the ps mode to fs mode, e.g. by adjusting the slit width, it is also possible to measure the multiphoton microscopy images of the same tissue in tandem in the example embodiment. The SHG image and TPEF image of fibrotic liver tissue excited by the 100 fs laser light (using only the pump beam) at 800 nm are shown in Figures 4(d) and 4(e), respectively. The dense while aggregated fibrillar collagen are clearly identified by SHG signals of fibrotic liver tissue (Figure 4(d)), while minimal SHG signals of collagen fibers can be detected in normal liver tissue (not shown). The hepatocyte morphology is also observed by TPEF signals arising from nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavins' autofluorescence in liver tissue (Figure 4(e)). The merged image of ps-CARS, SHG and TPEF of liver tissue (Figure 4(f)) reveais that the collagen fibers are more preferentially produced in the areas where the hepatocytes are damaged, whereas the distribution of hepatic lipid droplets does not show the similar trend in fibrotic liver tissue.

Hence, the integrated CARS microscopy and multiphoton microscopy system of the example embodiment can be used for label-free imaging of significant biochemical components and structures, e.g. of liver tissues with potential for monitoring the onset and progression of liver diseases. Advantageously, the system of the example embodiment can provide broad spectral/temporal tenability, compact spectral filtering design and ease of operation for swapping between ps and fs modes. Many simultaneous wavelengths can be provided to precisely match the optimal Raman linewidth of interest, for maximizing their signal-to-noise ratios for high-fidelity, multiband, ratiometric imaging. The technique according to the example embodiment is also cost effective as it only requires one femtosecond laser source and an OPO system.

Figure 5 shows a flow chart 500 illustrating a method of supplying light beams for integrated CARS and multiphoton microscopy. At step 502, a femtosecond pump beam is generated. At step 504, optionally, a femtosecond Stokes beam is generated using a portion of the pump beam. At step 506, the Stokes beam, the pump beam, or both are spectrally filtered such that the filtered beams are in a picosecond range or a femtosecond range depending on a desired microscopy operation mode. At step 508, the filtered Stokes beam, pump beam, or both are supplied to a scanning microscope.

It will be appreciated by a person skilled in the art that numerous variations and/or modifications may be made to the present invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

Claims

1. A method of supplying light beams for integrated CARS and multiphoton microscopy, the method comprising the steps of:

generating a femtosecond pump beam;

supplying the filtered Stokes beam, pump beam, or both to a scanning microscope.

2. The method as claimed in claim 1 , wherein generating the femtosecond Stokes beam comprises coupling the portion of the pump beam into an optical parametric oscillator.

3. The method as claimed in any one of the preceding claims, wherein spectrally filtering the Stokes or pump beam comprises directing said beam to a respective pair of gratings.

4. The method as claimed in claim 3, further comprising aligning an orientation of a second grating in counter-direction to that of a first grating.

5. The method as claimed in claims 3 or 4, further comprising disposing, in a sequence, a first achromatic focusing lens, a slit and a second achromatic focusing lens between the pair of gratings.

6. The method as claimed in claim 5, wherein the first and second achromatic focusing lenses have the same focal length.

7. The method as claimed in claim 6, further comprising configuring a distance between adjacent elements to be equal to the focal length.

8. The method as claimed in any one of claims 5 to 7, further comprising adjusting a lateral position of the slit for controlling a central wavelength of the filtered beam.

9. The method as claimed in any one of claims 5 to 8, further comprising adjusting the slit width for controlling a bandwidth of the filtered beam such that the filtered beam is in the picosecond range or the femtosecond range depending on the desired microscopy operation mode.

10. The method as claimed in any one of the preceding claims, wherein supplying the filtered Stokes beam, pump beam, or both to the scanning microscope comprises delaying the filtered pump beam to substantially cancel out optical path differences.

11. The method as claimed in any one of the preceding claims, wherein the light source comprises a Ti:sapphire laser source.

12. An integrated CARS and multiphoton microscopy method, the method comprising the steps of:

supplying light beams to a scanning microscope as claimed in any one of claims 1 to 11 ;

focusing the light beams onto a sample;

collecting nonlinear optical signal radiation from the sample; and

obtaining measurements from the radiation.

13. The method as claimed in claim 12, wherein obtaining measurements from the radiation comprises using at least one of a photomultipiier tube and a spectrometer.

14. The method as claimed in claims 12 or 13, wherein the sample comprises a biological or biomedical sample.

15. An apparatus for supplying light beams for integrated CARS and multiphoton microscopy, comprising: means for generating a femtosecond pump beam;

means for supplying the filtered Stokes beam, pump beam, or both to a scanning microscope.

16. The apparatus as claimed in claim 15, wherein the means for generating the femtosecond Stokes beam comprises an optical parametric oscillator configured for having the portion of the pump beam coupled thereinto.

17. The apparatus as claimed in claims 15 or 16, wherein the means for spectrally filtering the Stokes or pump beam comprises a respective pair of gratings.

1s8. The apparatus as claimed in claim 17, wherein an orientation of a second grating is aligned in counter-direction to that of a first grating.

19. The apparatus as claimed in claims 17 or 18, further comprises, in a sequence, a first achromatic focusing lens, a slit and a second achromatic focusing lens between the pair of gratings.

20. The apparatus as claimed in claim 19, wherein the first and second achromatic focusing lenses have the same focal length.

21. The apparatus as claimed in claim 20, wherein a distance between adjacent elements is equal to the focal length.

22. The apparatus as claimed in any one of claims 19 to 21 , wherein a lateral position of the slit is adjustable for controlling a central wavelength of the filtered beam.

23. The apparatus as claimed in any one of claims 19 to 22, wherein the slit width is adjustable for controlling a bandwidth of the filtered beam such that the filtered beam is in the picosecond range or the femtosecond range depending on the desired microscopy operation mode.

24. The apparatus as claimed in any one of claims 15 to 23, wherein the means for supplying the filtered Stokes beam, pump beam, or both to the scanning microscope comprises means for delaying the filtered pump beam to substantially cancel out optical path differences.

25. The apparatus as claimed in any one of claims 15 to 24, wherein the light source comprises a TLsapphire laser source.

26. An integrated CARS and multiphoton microscopy system, comprising: an apparatus for supplying light beams as claimed in any one of claims 5 to

25;

means for focusing the light beams onto a sample;

27. The system as claimed in claim 26, wherein the means for obtaining measurements from the radiation comprises at least one of a photomultiplier tube and a spectrometer.

28. The system as claimed in claims 26 or 27, wherein the sample comprises a biological or biomedical sample.