FIELD

The present invention relates to a three-photon light sheet imaging system and method for use, in particular though not exclusively, for three-photon light sheet microscopy.

BACKGROUND

Light sheet fluorescence microscopy (LSFM), also known as selective plane illumination microscopy (SPIM), is becoming increasingly important in biological research for monitoring the development of larger three-dimensional samples, especially for monitoring biological samples in vivo. In LSFM, only a thin layer of the sample is illuminated at a time and the images are captured along a detection axis which is generally perpendicular to the illuminated plane. Such optical sectioning ability enables relatively high-contrast, high resolution imaging, whilst minimizing sample exposure and photo-toxicity.

The resolution of LSFM methods along the detection axis is determined by a combination of the numerical aperture of the detection objective and the extent or thickness of the light sheet along the detection axis. For Gaussian-beam illumination, a large field of view requires a relatively thick light sheet, thus compromising the resolution along the detection axis and exposing the sample unnecessarily to irradiation. Known LSFM methods may not provide sufficient image contrast or sufficient signal-to-background ratio (SBR) for some imaging applications. SUMMARY

It should be understood that one or more of the features of any of the following aspects of the present invention may apply alone or in any combination in relation to any of the other aspects of the present invention.

According to an aspect of the present invention there is provided a light sheet imaging system comprising:

an illumination arrangement for generating a light sheet for three-photon excitation of a fluorescent sample; and

a fluorescence collection arrangement for collecting fluorescence generated in the sample as a result of three-photon excitation by the light sheet. It has been found that the average intensity levels required for three-photon light sheet excitation are surprisingly low relative to the photo-toxic average intensity levels which can destroy the viability of a biological sample such as a cell. The use of three-photon excitation for light sheet imaging of a fluorescent sample allows the use of an excitation wavelength which is three times longer than the excitation wavelength required for single photon excitation. This may reduce scattering of the light sheet within the sample. For example, it is well known that Rayleigh scattering is dependent upon 1/λ ⁴ . Consequently, the use of three-photon excitation reduces Rayleigh scattering by a factor of 81 when compared with the use of single-photon excitation. The reduction in scattering of the light sheet within the sample increases the penetration of the light sheet into the sample, permitting light sheet imaging at greater depths compared with the use of single-photon or two-photon excitation for light sheet imaging. The reduction in scattering also enhances the signal-to-background (SBR) ratio for enhanced fluorescence image contrast compared with the fluorescence image contrast obtainable when using single-photon or two-photon excitation for light sheet imaging. In addition, the use of three-photon excitation for light sheet imaging leads to an effective reduction in the beam waist compared with the use of single-photon or two- photon excitation in a direction transverse or generally perpendicular to the light sheet. This may lead to an enhancement in axial resolution along a fluorescence collection or imaging axis.

The light sheet may comprise one or more wavelengths in the range 700 nm to 1800 nm.

The light sheet may be configured for three-photon excitation of endogenous fluorophores such as NADH and/or flavins.

The light sheet may comprise one or more wavelengths which are between

1030 nm and 1050 nm, for example at or around 040 nm.

The light sheet may comprise one or more wavelengths which are between 1540 nm and 1560 nm, for example at or around 1550 nm.

The light sheet may be configured for three-photon excitation of exogenous fluorophores such as green fluorescent protein (GFP) or red fluorescent protein (RFP).

The light sheet may comprise one or more wavelengths which are between 1665 nm and 1685 nm, for example at or around 1675 nm.

The illumination arrangement may comprise an optical source. The optical source may be coherent. The optical source may be tuneable. The optical source may comprise a laser and/or an optical parametric oscillator (OPO). The optical source may be configured to generate a stream of optical pulses. The stream of optical pulses may have an average power of at least 1 mW, at least 5 mW, at least 10 mW, at least 50 mW or at least 100 mW.

Each optical pulse may have a duration of 1 ps or less, 500 fs or less, or 100 fs or less.

Each optical pulse may have an energy of at least 10 nJ, at least 50 nJ, at least 100 nJ or at least 500 nJ.

The illumination arrangement may comprise a pulse compression arrangement for compressing the optical pulses.

The pulse compression arrangement may comprise one or more elements or components for providing anomalous dispersion. For example, the pulse compression arrangement may comprise one or more chirped mirrors and/or a hollow-core photonic bandgap fibre.

The pulse compression arrangement may comprise one or more elements or components for providing normal dispersion. For example, the pulse compression arrangement may comprise one or more elements or components comprising normally dispersive glass and/or a normally dispersive optical fibre.

The illumination arrangement may comprise a wavelength conversion element or component for converting or adjusting a wavelength of light generated by the optical source. The wavelength conversion element or component may comprise a photonic crystal rod for soliton self-frequency shifting.

The illumination arrangement may comprise a light sheet generating element. The light sheet generating element may be configured for use in converting an optical beam, such as a circularly symmetric optical beam, into the light sheet. The light sheet generating element may comprise a cylindrical lens.

The light sheet generating element may comprise a cylindrical reflector or a cylindrical mirror.

The light sheet generating element may comprise a scanner, for example an acousto-optical scanner. Such a scanner may be used to scan a beam such as a circularly symmetric beam generated by the optical source across a plane of the sample to progressively generate the light sheet. Scanning a beam progressively in this way may allow light sheet imaging to be performed without unduly reducing the intensity of the light sheet.

The light sheet may be formed from and/or may comprise a superposition or an array of beams. The use of a superposition or an array of beams may allow a light sheet to be more easily or more rapidly formed compared with the use of a single beam because the use of a superposition or an array of beams means that the light sheet can be formed by scanning or dithering each beam over a smaller distance in a direction transverse to the direction of propagation.

The light sheet may be a non-diffractive, propagation-invariant light sheet.

One of ordinary skill in the art will understand that a perfectly non-diffractive, perfectly propagation-invariant optical beam such as a propagation-invariant light sheet only exists in theory and that, in practice, a non-diffractive, propagation-invariant optical beam may only be considered to be approximately non-diffractive and propagation- invariant over a finite distance. In particular, a non-diffractive, propagation-invariant optical beam has a transverse intensity profile which varies more slowly than a diffractive, propagation-variant optical beam such as a Gaussian beam of a comparable beam waist.

As such, a propagation invariant beam such as a propagation-invariant light sheet is used herein to refer to a beam that maintains its transverse profile or shape on propagation over a finite distance. Specifically, this can mean that a propagation invariant beam maintains its transverse profile or shape in a transverse plane (x,y) on propagation over a finite distance along a z direction (assuming x,y,z are orthogonal Cartesian co-ordinates). Ideally this means these beams would satisfy a relation such that l(x,y,z=0)=l(x,y,z>0) where I denotes the transverse spatial profile. In practice, however, such beams cannot be propagation invariant or diffraction-free indefinitely and such beams can only be considered to be propagation invariant or diffraction-free over a finite distance.

One of ordinary skill in the art will understand that for a propagation invariant beam which has a structured pattern in the transverse plane (x,y), the spacing between features in the transverse (x,y) plane remains the same after propagation over a finite distance. That is, there is no radial scaling after propagation over a finite distance. For such propagation invariant beams, it should be understood that although the relative intensities of the different features of the transverse spatial profile may vary after propagation over a finite distance, there is no change in spacing between the different features of the transverse spatial profile i.e. there is no radial scaling. For example, a propagation-invariant Bessel beam includes a plurality of concentric rings in the transverse plane (x,y), wherein the spacing between the concentric rings remains the same after propagation over a finite distance, even if the relative intensities of the concentric rings actually change slightly after propagation over the finite distance. Similarly, an Airy beam includes a plurality of discrete lobes or features in the transverse plane (x,y), wherein the spacing between the discrete lobes or features remains the same after propagation over a finite distance, even if the relative intensities of the discrete lobes or features actually change slightly after propagation over the finite distance.

The use of a non-diffractive, propagation-invariant light sheet for three-photon light sheet imaging may provide superior axial resolution across a given field of view when compared with the use of a light sheet formed from and/or comprising a Gaussian beam for three-photon light sheet imaging across the same field of view. Alternatively, the use of a non-diffractive, propagation-invariant light sheet for three- photon light sheet imaging may provide the same axial resolution across an extended field of view when compared with the use of a light sheet formed from and/or comprising a Gaussian beam for three-photon light sheet imaging.

The illumination arrangement may comprise an optical component for use in generating the non-diffractive, propagation-invariant light sheet.

The optical component may be configured to convert the optical beam generated by the optical source into a non-diffractive, propagation-invariant optical beam and the light sheet generating element may be configured to convert the non- diffractive, propagation-invariant optical beam into the non-diffractive, propagation- invariant light sheet.

The light sheet generator may be configured to convert the optical beam generated by the optical source into an intermediate light sheet and the optical component may be configured to convert the intermediate light sheet into the non- diffractive, propagation-invariant light sheet.

The light sheet may have a symmetric intensity profile transverse to a direction of propagation.

The light sheet may be formed from and/or comprise a Bessel beam. Studies have suggested that the use of a light sheet formed from and/or comprising a Bessel beam for three-photon light sheet imaging may provide superior axial resolution across a given field of view defined by the light sheet within a sample when compared with the use of a light sheet formed from and/or comprising a Gaussian beam for three-photon light sheet imaging. Alternatively, the use of a light sheet formed from and/or comprising a Bessel beam for three-photon light sheet imaging may provide the same axial resolution across an extended field of view defined by the light sheet within a sample when compared with the use of a light sheet formed from and/or comprising a Gaussian beam for three-photon light sheet imaging.

The light sheet may be formed from and/or may comprise an Airy beam.

The light sheet may be formed from and/or may comprise a parabolic beam. The optical component may comprise a fixed, static and/or passive optical component.

The optical component may comprise a phase mask.

The optical component may comprise an axicon. An axicon may be used to generate a Bessel beam.

The optical component may comprise a cylindrical lens which is tilted relative to a direction of propagation. Such a tilted cylindrical lens may be used to generate an Airy beam.

The optical component may be dynamic and/or re-configurable. The optical component may comprise a diffractive optical element. The optical component may comprise a spatial light modulator. The optical component may comprise a digital micro-mirror.

The light sheet may be formed from and/or may comprise at least one of a superposition or an array of Bessel beams, a superposition or an array of Airy beams, and a superposition or an array of parabolic beams.

The illumination arrangement may comprise an input lens for coupling the light sheet into the sample. The fluorescence collection arrangement may comprise an output lens for collecting fluorescence emitted from the sample. One or both of the input and output lenses may comprise a microscope objective. The input and output lenses may be arranged along orthogonal axes.

The illumination arrangement may comprise an input optical fibre for coupling light to the input lens. The fluorescence collection arrangement may comprise an output optical fibre for receiving light from the output lens. The input and output fibres may be arranged side-by-side.

One or both of the input and output lenses may comprise a gradient-index (GRIN) lens. The input and output lenses may be arranged with their optical axes parallel to one another.

The illumination arrangement may comprise a reflector, mirror, deflector or the like, for example a micro-prism deflector, for deflecting light received from the input lens. The use of input and output GRIN lenses arranged with their respective optical axes parallel to one another together with the deflector may provide a compact three- photon fluorescence light sheet imaging system for use in in vivo imaging. The use of input and output optical fibres arranged side-by-side may further render the three- photon fluorescence light sheet imaging system suitable for use in in vivo imaging.

The fluorescence collection arrangement may comprise one or more further lenses and/or an image sensor such as a CMOS or CCD image sensor or a camera for imaging the collected fluorescence.

The light sheet imaging system may comprise or form part of, a light sheet microscope.

According to an aspect of the present invention there is provided a light sheet microscope comprising:

an illumination arrangement for generating a light sheet for three-photon excitation of a fluorescent sample; and

a fluorescence collection arrangement for collecting fluorescence generated in the sample as a result of three-photon excitation by the light sheet.

According to an aspect of the present invention there is provided a method of light sheet imaging comprising:

using a light sheet for three-photon excitation of a fluorescent sample; and collecting fluorescence generated in the sample as a result of three-photon excitation of the sample by the light sheet.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is described herein by way of non-limiting example only with reference to the drawings of which:

Figure 1 is a schematic of a three-photon light sheet fluorescence microscopy system; Figure 2A is a two-dimensional transverse intensity profile of a Bessel beam of the microscopy system of Figure 1 ;

Figure 2B is a one-dimensional transverse intensity profile of the Bessel beam of the microscopy system of Figure 1 ; Figure 2C is a schematic side view showing the arrangement of the detection objective above the light sheet formed using the Bessel beam of the microscopy system of Figure 1 ; Figure 3A is a simulated two-dimensional single-photon transverse intensity profile of a Bessel beam at the focal plane;

Figure 3B is a simulated two-dimensional three-photon transverse intensity profile of a Bessel beam at the focal plane;

Figure 3C shows simulated one-dimensional single- and three-photon transverse intensity profiles corresponding to the two-dimensional Bessel beam profiles of Figures 3A and 3B at the focal plane; Figure 3D is a simulated two-dimensional single-photon transverse intensity profile of a Gaussian beam at the focal plane;

Figure 3E is a simulated two-dimensional three-photon transverse intensity profile of a Gaussian beam at the focal plane;

Figure 3F shows simulated one-dimensional single- and three-photon transverse intensity profiles corresponding to the two-dimensional Gaussian beam profiles of Figures 3D and 3E at the focal plane; Figure 4A is a simulated two-dimensional single-photon transverse intensity profile of a Bessel beam at a distance of 50 μηη from the focal plane;

Figure 4B is a simulated two-dimensional three-photon transverse intensity profile of a Bessel beam at a distance of 50 μΓη from the focal plane;

Figure 4C shows simulated one-dimensional single- and three-photon transverse intensity profiles corresponding to the two-dimensional Bessel beam profiles of Figures 4A and 4B at a distance of 50 μηπ from the focal plane; is a simulated two-dimensional single-photon transverse intensity profile of a Gaussian beam at a distance of 50 μηη from the focal plane; is a simulated two-dimensional three-photon transverse intensity profile of a Gaussian beam at a distance of 50 μιη from the focal plane; shows simulated one-dimensional single- and three-photon transverse intensity profiles corresponding to the two-dimensional Gaussian beam profiles of Figures 4D and 4E at a distance of 50 μηι from the focal plane; is a plot of the simulated modulation transfer function against normalised spatial frequency for single- and three-photon Bessel and Gaussian beams at the focal plane; is a plot of the simulated modulation transfer function against normalised spatial frequency for single- and three-photon Bessel and Gaussian beams at a distance of 50 μνη from the focal plane; is a schematic of a first alternative illumination and detection arrangement for use with the three-photon light sheet fluorescence microscopy system of Figure 1 ; is a schematic of a second alternative illumination and detection arrangement for use with the three-photon light sheet fluorescence microscopy system of Figure 1 ; is a schematic of a third alternative illumination and detection arrangement for use with the three-photon light sheet fluorescence microscopy system of Figure 1 ; is a schematic of an alternative sample chamber arrangement for use with the three-photon light sheet fluorescence microscopy system of Figure 1 ; Figure 8B is a detailed schematic of the alternative sample chamber arrangement of Figure 7A; and

Figure 9 is a schematic of a three-photon light sheet fluorescence microscopy system for use in vivo.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring initially to Figure 1 there is provided a three-photon light sheet fluorescence microscopy system for imaging a sample such as a tissue sample, one or more particles, cells, colloids, organisms, organelles or the like. The sample may have been treated or otherwise exposed to an exogenous fluorescent substance such as an exogenous fluorophore or the like for three-photon excitation by a light sheet generated by the microscopy system. Alternatively, the sample may include an endogenous fluorescent substance such as an endogenous fluorophore such as NADH and/or one or more flavins for three-photon excitation by a light sheet generated by the microscopy system.

The illumination propagates along the x-direction, the beam is expanded along the y-direction to form the light sheet, and fluorescence is collected along the z- direction. The three-photon light sheet fluorescence microscopy system of Figure 1 permits imaging of fluid-immersed samples on a horizontal surface such as a standard microscope slide or dish. This is enabled by positioning illumination and detection objectives at 45 degrees to the horizontal sample. By translating the sample and the detection objective along the z-direction as indicated by the "scan dir." arrow, different "planes" of the sample are illuminated sequentially by the light sheet and the fluorescence generated in each plane is collected and imaged onto an image sensor such as a camera (CAM^ so that a three dimensional volumetric image can be recorded.

The three-photon light sheet fluorescence microscopy system of Figure 1 includes an illumination arrangement including a laser, an optical component MKi and a light sheet generating element (LSG). The optical component MK may be a static optical component such as an axicon or a mask for the generation of a Bessel beam. Alternatively, the optical component MK-i may be a dynamic optical component configured so as to generate a Bessel beam. For example, the optical component may be a spatial light modulator or a digital micro-mirror configured so as to generate a Bessel beam by reflection therefrom. As shown in Figures 2A and 2B, a Bessel beam has a bright central spot surrounded by a series of concentric rings which decrease in intensity with distance from the central spot. The LSG can be a cylindrical lens or a scanning device such as an acousto-optical deflector (AOD) for progressively generating a light sheet.

The beam-shaping arrangement includes a series of lenses L1 to L6 for coupling light from the laser towards the sample via the optical component MK-, and the LSG. The beam-shaping arrangement further includes irises and \ ₂ , a quarter waveplate λ/4, mirrors M1 and M2 and an illumination objective OBJ

The three-photon light sheet fluorescence microscopy system of Figure 1 includes a detection objective OBJ ₂ , a third mirror M ₃ , a fourth mirror M ₄ , a filter F for blocking light at the one or more excitation wavelengths and an image sensor or camera CAM-].

In use, light from the laser is expanded by lenses L and L ₂ to match the optical component MK Having passed through the optical component MK^ the Bessel beam is then passed through lens L ₃ , where is it focussed onto the iris I-, and through another lens l where it is shaped to the diameter of the LSG. The light sheet that is formed by the LSG is then passed through the lens L ₅ , where it is focussed onto the iris \ ₂ and through another lens L _6| which collimates the light sheet onto the quarter wave plate λ/4, which provides polarization control. From there, the light sheet is reflected from the first mirror IV^ onto the second mirror M ₂ and from there onto the illumination objective OBJi, which is positioned at 45 degrees to the horizontal sample stage. Figure 2C shows a side view of the detection objective positioned adjacent the light sheet generated using the Bessel beam corresponding to the profiles of Figures 2A and 2B. Fluorescence generated in the sample passes through the detection objective OBJ ₂ onto the third mirror M ₃ and from there onto the fourth mirror M ₄ , where it is directed to the image sensor or camera CAMi via the filter F.

The mirrors to M ₄ can be used to facilitate the vertical alignment of the objectives with the sample. In one example, mirrors M ₂ and M ₃ can be fixed with respect to the axis of the objectives OBJ _! and OBJ ₂ , respectively. Appropriate alignment of mirrors M and M ₄ allows a vertical translation of both objectives OB^ and OBJ ₂ and mirrors M ₂ and M ₃ with respect to the sample stage without altering the alignment. Axial translation stages on the objectives may facilitate fine-tuning the alignment.

Various optional elements are included to control the polarization and to filter the light. Optionally, the irises, ^ and l ₂ may be included as shown in Figure 1 to select the first diffraction orders to allow more flexibility and prevent unwanted illumination of the sample. Lenses L to L ₆ form telescopes to relay the light between the various components. The quarter waveplate λ/4 can be introduced to control the polarization of the light, e.g. by converting linear polarized light to circularly polarized light that produces a more uniform excitation of fluorescence. Light filters, F, e.g. to block excitation light and select fluorescence emission, can be introduced between OBJ ₂ and the camera, CAMi . If necessary or convenient, elements of the illumination path and the detection path can be brought closer than the mirrors to OB ^ and OBJ ₂ , respectively.

The laser is configured to generate light at one or more wavelengths in the range 1000 nm to 1800 nm for three-photon excitation of the fluorophore with which the sample is treated. For example, the laser may be configured to generate light having wavelengths around 1675 nm for the three-photon excitation of exogenous fluorophores. Alternatively, the laser may be configured to generate light having wavelengths around 1040 nm for the three-photon excitation of endogenous fluorophores such as NADH and/or one or more flavins.

The laser generates a stream of optical pulses with each pulse having a duration of the order of 100 fs or less and an energy of the order of 100 nJ at the light sheet. The illumination arrangement may include one or more elements or components for the compensation of dispersion associated with propagation of light via the various optical components between the laser and the sample. The laser may generate a light sheet with sufficient peak power to generate three-photon excitation of the fluorophore at relatively low average powers of only a few mWs and may provide enhanced image contrast or improved SBR at average intensity levels which are surprisingly low relative to the average intensity levels which can give rise to phototoxicity. Moreover, as described below with reference to Figures 3A to 3F, 4A to 4F, 5A and 5B, theoretical studies have demonstrated that use of a light sheet for three-photon fluorescence light sheet microscopy generated using a Bessel beam has the potential to provide high image contrast and superior image resolution across the light sheet compared with the use of a Gaussian beam.

Figures 3A and 3B show the two-dimensional transverse single-photon and three-photon intensity profiles of a Bessel beam at a focal plane of an ideal lens respectively for a numerical aperture (NA) of 0.2 and the corresponding one- dimensional transverse single-photon and three photon intensity profiles are plotted in Figure 3C. Similarly, Figures 3D and 3E show the two-dimensional transverse single- photon and three-photon intensity profiles of a Gaussian beam at the focal plane of the ideal lens respectively for a NA of 0.2 and the corresponding one-dimensional transverse single-photon and three-photon intensity profiles are plotted in Figure 3F, All units are in μηι.

Comparing Figures 3C and 3F, it may be seen that the three-photon intensity profile of the Bessel beam is surprisingly similar to the three-photon intensity profile of the Gaussian beam at focal plane of the ideal lens. More importantly, as shown in Figures 4A - 4F the three-photon intensity profile of the Bessel beam is essentially propagation-invariant for distances of up to 50 pm from the focal plane of the ideal lens. The profiles of Figures 3A - 4C suggest that a light sheet generated using a Bessel beam may have a three-photon intensity at the focal plane which is comparable to, or only marginally less than, the three-photon intensity at the focal plane of a light sheet generated using a Gaussian beam and three-photon intensities in regions either side of the focal plane which may actually be significantly greater than the three-photon intensities in regions either side of the focal plane of a light sheet generated using a Gaussian beam. In other words, the profiles of Figures 3A - 4C suggest that a light sheet generated using a Bessel beam has the potential to provide a fluorescence image with an enhanced contrast or signal-to-background ratio (SBR) across an extended field of view compared with a Gaussian beam.

Figure 5A is a plot of the modulation transfer function (MTF) against normalised spatial frequency for the one- and three-photon Bessel and Gaussian beams of Figures 3A, 3B, 3C and 3D at the focal plane of the ideal lens for a numerical aperture (NA) of 0.2. Similarly, Figure 5B is a plot of the modulation transfer function against normalised spatial frequency for the one- and three-photon Bessel and Gaussian beams of Figures 4A, 4B, 4C and 4D at a distance of 50 pm from the focal plane of the ideal lens for a NA of 0.2. In each case, the spatial frequency is normalised to the maximum transmitted spatial frequency 2ΝΑ/λ, for a peak excitation wavelength λ of 800nm. In each case, the one- and three-photon Bessel and Gaussian beams were assumed to comprise a stream of optical pulses that would typically have a Gaussian spectrum but no constraint on pulse duration is assumed. As shown in Figures 5A and 5B, the MTF for a three-photon Bessel beam is only marginally lower than the MTF for a three-photon Gaussian beam at the focal plane whereas the MTF for the three- photon Bessel beam is significantly higher than the MTF for the three-photon Gaussian beam at a distance of 50 pm from the focal plane. This demonstrates that the use of a Bessel beam for three-photon light sheet microscopy has the potential to provide fluorescence images across an extended field of view with both an axial and a lateral spatial resolution which are comparable to, or only marginally less than, those provided by a Gaussian beam at the focal plane.

Figure 6 shows a first alternative illumination and detection arrangement for replacing the mirror Μ·, and all of components in the optical path downstream of the mirror M-i in the three-photon light sheet fluorescence microscopy system of Figure 1. A sample (not shown) is located in a sample chamber SC and remains fixed as the light sheet is sequentially directed along different "planes" of the sample by a scanning mirror SM via a pair of relay lenses RL and an illumination objective 01. The fluorescence emitted from the generally "planar" illuminated region of the sample is imaged onto the image sensor or camera CAM by a detection objective 02, an electrically tuneable lens .ETL, and a tube lens TL. The scanning mirror SM and the electrically tuneable lens ETL are synchronised so that the "plane" of illumination and the plane of detection (i.e. the imaged plane) correspond. This not only allows the sample to be kept stationary whilst image stacks are taken, but also allows fast scanning of the sample. This may be particularly important when monitoring one or more live and/or mobile samples.

Figure 7A is a schematic of a second alternative illumination and detection arrangement for use with the three-photon light sheet fluorescence microscopy system of Figure 1 in which the illumination objective 01 is oriented horizontally along a horizontal illumination axis and the imaging objective 02 is arranged vertically along a vertical imaging axis. Conversely, as shown in Figure 7B, the illumination objective 01 may be oriented vertically along a vertical illumination axis and the imaging objective 02 may be arranged horizontally along a horizontal imaging axis.

Figures 8A and 8B show an alternative sample chamber arrangement SC2 for use with the three-photon light sheet fluorescence microscopy system of Figure 1 in which the illumination and imaging objectives are arranged along orthogonal axes below a sample 14. The alternative sample chamber arrangement SC2 includes a transparent membrane 10 for containing a fluid 12 surrounding the sample 14. Such an alternative sample chamber arrangement SC2 may permit the fluid 12 and the sample 14 to be replaced without re-alignment of objective 01 and 02. The sample 14 may be positioned using optical trapping and/or acoustic trapping.

Figure 9 shows a three-photon light sheet fluorescence microscopy system for imaging a sample (not shown) in vivo. The three-photon light sheet fluorescence microscopy system of Figure 9 includes an illumination arrangement which includes a femtosecond laser, a dispersion compensation element DC, an input fibre coupling lens an input optical fibre OF-i, an illumination GRIN lens arrangement GRII^ and a micro-prism beam deflector BD.

The three-photon light sheet fluorescence microscopy system of Figure 9 further includes a fluorescence collection arrangement which includes detection GRIN lens arrangement GRIN ₂ , an output optical fibre OF-i, an output fibre coupling lens L ₂ , a filter F for blocking light at the excitation wavelength, a tube lens TL and an image sensor or camera CAM.

The illumination GRIN lens arrangement GRINi includes at least one cylindrical GRIN lens for the generation of a light sheet at a position below the detection GRIN lens arrangement GRIN ₂ .

In use, a sample (not shown) is either treated with, or otherwise exposed to, an exogenous fluorescent substance such as a green fluorescent protein or a red fluorescent protein or the like for three-photon excitation by a light sheet generated by the microscopy system. Alternatively, the sample (not shown) may include an endogenous fluorescent substance such as an endogenous fluorophore such as NADH and/or one or more flavins for three-photon excitation by a light sheet generated by the microscopy system.

The laser generates a stream of pulses at a wavelength in the range 1000 to 1800 nm. The dispersion compensation element DC is used to compensate for dispersion in the input fibre coupling lens the input optical fibre OF-i, the cylindrical illumination GRIN lens arrangement GRIN^ the micro-prism beam deflector BD and the sample (not shown) to ensure that the pulses having a duration of the order of a hundred fs or less and a peak power of the order of 100 nJ or less at the light sheet for the three-photon excitation of the fluorophore in the sample (not shown). The input optical fibre OFi may be configured to provide anomalous dispersion and the dispersion compensation element DC may comprise a normally dispersive element to pre-chirp the optical pulses generated by the laser for compression of the pulses at the light sheet. For example, the input optical fibre OF-, may be a hollow-core photonic bandgap fibre. The dispersion compensation element DC may comprise or be formed from a normally dispersive material such as glass, may comprise a normally dispersive optical fibre and/or may comprise one or more chirped mirrors.

The fluorescence generated in the sample is collected by the detection GRIN lens arrangement GRIN ₂ and imaged onto the camera CAM via the output optical fibre OFi, the output fibre coupling lens L ₂ , the filter F and the tube lens TL. One of ordinary skill in the art will understand that the compact arrangement provided by the input optical fibre 0F1 , the illumination GRIN lens arrangement GRIN and the micro-prism beam deflector BD, makes the three-photon light sheet fluorescence microscopy system of Figure 8 particularly suitable for in vivo imaging applications,

One of ordinary skill in the art will understand that in each of the illumination and detection arrangements described with reference to Figures 1 - 9, a sample is illuminated by one or more light sheets for three-photon excitation of the sample along an illumination axis and fluorescence is collected along a detection axis which is generally orthogonal to the illumination axis. Other illumination and detection arrangements are also possible in which the illumination axis and the detection axis are arranged orthogonally. Additionally or alternatively, a sample may be illuminated by a first light sheet travelling in a first direction along an illumination axis and a second light sheet travelling in a second direction along the illumination axis, wherein the second direction is opposite to the first direction.

At least some of optical components of the illumination arrangement of the three-photon light sheet fluorescence microscopy system of Figure 1 may be rearranged. For example, the order in which the optical component MK-t and the light sheet generating element LSG appear in the optical path may be reversed.

The light sheet may be formed from and/or may comprise a superposition or an array of beams such as an array of Bessel beams.

Rather than using a Bessel beam, it may be possible to use a different non- diffractive and/or propagation invariant beam such as an Airy beam and/or a parabolic beam to create the light sheet. One of ordinary skill in the art will appreciate that such non-diffractive and/or propagation invariant beams may be produced by replacing the component MK1 of Figure 1 with a suitably configured spatial light modulator and/or a digital micro mirror. Alternatively, an Airy beam may be produced using a cylindrical lens which is tilted with respect to a direction of propagation of the light from the laser. One of ordinary skill in the art will also appreciate that some non-diffractive and/or propagation invariant beams such as an Airy beam or a parabolic beam may be considered to propagate along a curved path in the sense that such beams may have a central intensity maximum and one or more intensity side-lobes, wherein the intensity maximum follows a curved path which may, for example, be parabolic. For such beams, the resulting light sheet may be curved and references to a "plane" as used in the description above should be understood to encompass such a curved sheet. One of ordinary skill in the art will also understand that linear deconvolution can be used to compensate for artefacts in the image of the fluorescence generated when using a curved light sheet such as a light sheet formed from an Airy beam, thereby effectively "cancelling" the curvature of the Airy beam light sheet.

The light sheet may be formed from and/or may comprise a superposition or an array of Airy beams.

The light sheet may be formed from and/or may comprise a superposition or an array of parabolic beams.

An optical source other than a laser may be used. For example, a different coherent optical source such as an optical parametric oscillator (OPO) may be used.

The optical source may comprise a wavelength conversion element or component for converting or adjusting a wavelength of the generated light. For example, the optical source may comprise a photonic crystal rod for soliton self- frequency shifting. The optical source may be tuneable.