An optical imaging system

Abstract

An optical imaging system (100) for multiplex stimulated Raman scattering (SRS) microscopy. The system (100) comprises: a passively synchronized multi-output amplified fiber laser source (110) configured to generate a plurality of synchronised beams having different wavelength ranges; a laser scanning microscope (120) comprising an objective configured to receive and direct the plurality of beams towards a sample plane; and a detection system (130) configured to receive a plurality of optical signals from the sample plane for multiplex SRS, the detection system (130) comprising a multi-channel lock-in amplifier comprising a channel for each optical signal, each channel comprising at least one demodulator unit and a fixed time constant low-pass filter.

Description

[0001] AN OPTICAL IMAGING SYSTEM

[0002] FIELD OF INVENTION

[0003] The present invention relates to optical imaging systems for histopathology and, in particular, optical imaging systems and methods for imaging a sample volume using Raman spectroscopy.

[0004] BACKGROUND

[0005] Raman spectroscopy enables label-free chemical signatures of tissues and cells. It is based on the Raman scattering effect of molecules with the use of a single continuous wave laser. Such spontaneous Raman scattering is weak, and therefore Raman spectroscopy is typically slow. Coherent Raman spectroscopy (CRS), including coherent anti-Stokes Raman scattering (CARS), and stimulated Raman scattering (SRS) relies on nonlinear excitation of molecules, and can enhance the Raman intensity by orders of magnitude. In theory, such increase in Raman intensity allows measurement to be made at video-rate imaging speeds, which, in theory, means that CRS could be used in many applications in many different fields.

[0006] CRS can be implemented in a narrowband or in a broadband fashion, where narrowband means that a single vibrational frequency is excited at a time and broadband means that multiple vibrational modes are excited simultaneously. The narrowband approach is typically named “hyperspectral CRS” and it is based on narrowband tunable laser sources which permit to reconstruct the vibrational spectrum by serially acquiring the response of the system at different Raman modes. The broadband approach is generally defined as “multiplex CRS” and it relies on the combination of a broadband optical pulse and a narrowband one, thus leading to the parallelization of the Raman modes that can be excited and detected simultaneously. Multiplex CRS has the great advantage of single-shot multiplexed spectral acquisition, which enables fast and chemically-selective imaging at once. Multiplex SRS imaging is a particularly beneficial technique due to increased signal-to-noise ratio enabled by the use of a femtosecond pulse.

[0007] CRS requires the use of synchronised ultra-fast at least pico-second lasers from two laser sources, where pump and Stokes pulses matching the Raman frequency and bandwidths are used for setting up and detecting a vibrational coherence within a sample. Currently, solid-state lasers pumping optical parametric oscillators have been widely used as the laser source for CRS, as these laser sources allow access to the full Raman spectrum (0-4000cm-1). Such solid-state laser sources comprise a bulk piece of doped crystal or glass as the gain medium and require the use of bulky optics. They are, therefore, not only susceptible to misalignment and prone to instability, but their use also incurs a high capital cost. Furthermore, their relatively large footprints prevent them from being deployed effectively in clinical environments, for example, they cannot be easily moved around different wards in a hospital, nor they can be handled conveniently.

[0008] SRS has been widely implemented in microscopy in a narrowband (or hyperspectral) mode, which consists of the use of two synchronized narrowband picosecond pulses (known as the pump and the Stokes) where their frequency difference matches exactly an active Raman vibration. The frequency difference between pump and Stokes can be sequentially tuned, either by changing the central wavelength of the pump or Stokes beam or both, and an image can be collected at each tuning step. At the end of the tuning procedure, N images have been collected sequentially, each one representing the response of the system to a specific Raman vibration.

[0009] The multiplex (or broadband) SRS approach utilizes two synchronized pulse trains, one narrowband and the other broadband. This enables simultaneously excitation of a broadband Raman active region, covered by the bandwidth of the broadband pulse, with a spectral resolution dictated by the narrowband pulse. The are only few academic-research-level multiplex (or broadband) SRS system implementations and all of them suffer from: complex system architecture based on custom solutions which are difficult to replicate, low average power and high noise on the broadband pulse, or the use of solid-state alignment-dependent bulky solutions. Moreover, from the detection point of view, there are not available user- friendly and optimized multi-channel detectors for multiplex SRS microscopy applications. Because of this, narrowband tunable (hyperspectral) SRS systems are still preferred over multiplex SRS systems due to the extended coverage (typically more than 2000 cm'1) and off-the-shelf detection electronic. However, this comes at the cost of the need of a sequential tuning of the exploited narrow laser central wavelengths for accessing the whole Raman spectrum for each pixel.

[0010] It would be desirable to provide an optical imaging system and method overcoming the drawbacks of known optical imaging systems.

[0011] SUMMARY OF INVENTION The invention in its various aspects is defined in the independent claims below to which reference should now be made. Optional features are set forth in the dependent claims.

[0012] In a first aspect, the disclosure provides an optical imaging system for multiplex stimulated Raman scattering (SRS) microscopy. The system comprises: a passively synchronized multioutput amplified fiber laser source configured to generate a plurality of synchronised beams having different wavelength ranges; a laser scanning microscope comprising an objective configured to receive and direct the plurality of beams towards a sample plane; and a detection system configured to receive a plurality of optical signals from the sample plane for multiplex SRS microscopy. The detection system comprises a multi-channel lock-in amplifier comprising a channel for each optical signal, each channel comprising at least one demodulator unit and a fixed time constant low-pass filter.

[0013] Advantageously, the disclosed optical imaging system is able to multiplex together multiple distinct, distant and wide Raman active regions in a single shot with a high signal-to-noise ratio. The disclosed optical imaging solution simplifies the implementation and enhances the performance of multiplex SRS systems due to the use a passively-synchronized multi-output amplified fiber laser source, which can be tailored to generate picosecond / femtosecond combined beams with improved noise performance. The use of an optimized multichannel lock-in amplifier based on a scalable-modular architecture provides enhanced adaptability to a wide variety of experimental conditions.

[0014] In an example, the passively synchronized multi-output amplified fiber laser source may be configured to generate a first broadband optical output and a first narrowband optical output. In an example, the passively synchronized multi-output amplified fiber laser source may be configured to generate a second narrowband optical output, the second narrowband output comprising a beam having a different wavelength range to the first narrowband optical output. In an example, the optical imaging system is configured to cover both the CH-stretching region and the fingerprint region of the Raman spectrum.

[0015] In an example, the passively synchronized multi-output amplified fiber laser source may be configured to emit three synchronized outputs centered at the following wavelength ranges: 750-850 nm, 910-950 nm, and 1000-1 100 nm.

[0016] In an example, the synchronized multi-output amplified fiber laser source may comprise at least one optical output which is amplitude modulated at a frequency lower than the native repetition rate of the oscillators. In an example, the laser scanning microscope may comprise a beam scanning unit and collection optics. In an example, the beam scanning unit may comprise a single axis or dual axis galvo mirror and an optical relay system positioned between the galvo mirror and an objective back focal plane of the laser scanning microscope. In an example, the laser scanning microscope may comprise one or more of: an acousto optic deflector (AOD), a dual-axis galvo mirror, a fast steering mirror, a polygon mirror scanner, or a fixed mirror.

[0017] In an example, the laser scanning microscope does not comprise an active electro-optical descanning system situated between the sample plane and the detection system. In one example, the laser scanning microscope may be coupled to the detection system via an optical fibre coupling.

[0018] In an example, optical imaging system may comprise two optical relay systems separated by a transmission grating between the laser scanning microscope and the multi-channel lock-in amplifier.

[0019] In an example, the laser scanning microscope may comprise a descanning unit. In an example, the descanning unit may comprise a single axis or dual axis galvo mirror and an optical relay system positioned between the collection optics and the galvo mirror. Advantageously, the descanning unit may de-scans the received beams to remove a residual beam pointing error.

[0020] In an example, the optical imaging system may comprise an SHG and TPEF detection system. In an example, the optical imaging system may comprise multiple detectors. The multiple detectors may comprise one or more multichannel lock-in amplifiers, and one or more photo-multipliers tubes (PMTs). In an example, the PMTs may be located upstream from the sample plane and configured for SHG and TPEF detection in epi. SHG and TPEF detection increases the amount of data that can be gathered from the sample. In some examples, SHG and TPEF data is collected using the passively synchronized multi-output amplified fiber laser source.

[0021] In an example, the detection system may be located downstream from the sample plane. In an alternative example, the detection system may be located upstream from the sample plane in an Epi configuration to receive signals propagating upstream from the sample. In an example, the detection system may comprise a filter for selecting a spectrum of interest. For example, the filter may comprise one or more of a short-pass filter, a band-pass filter, or a long-pass filter. In an example, the detection system may comprise a half-wave plate for rotating the polarization of the spectrum of interest onto a desired axis. This may advantageously improve grating diffraction efficiency. In an example, the detection system may comprise a transmission grating for spatially dispersing the spectrum of interest.

[0022] In an example, the detection system may comprise a lens to image the dispersed spectrum onto the multi-channel lock-in amplifier with the correct dimension.

[0023] In an example, each channel of the multi-channel lock-in amplifier may comprise two demodulator units operating in phase quadrature.

[0024] In an example, the detection system may comprise a photodiode array. The photodiode array may comprise a number of photodiodes suitable for the desired spectral resolution.

[0025] In an example, the detection system may comprise an electronic board configured to perform pixel triggered averaging of the optical beams received in each channel. In an example, the pixel triggered averaging takes place outside the multi-channel lock-in amplifier.

[0026] In an example, the laser scanning microscope may be an inverted laser scanning microscope. In alternative examples, the laser scanning microscope may be an upright laser scanning microscope.

[0027] In an example, the optical imaging system may be configured for stimulated Raman scattering (SRS) microscopy. In an example, the system may also be configured for second- harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) microscopy.

[0028] In an example, the passively synchronized multi-output amplified fiber laser source may comprise an optical circuit. The optical circuit may comprise a first polarization-maintaining optical cavity and a second polarization-maintaining optical cavity. The first polarizationmaintaining optical cavity may comprise a first gain medium excitable by a first pump light source to generate light at a first range of wavelengths, and a first saturable absorber configured to carry out passive mode locking of the light pulses in the first polarizationmaintaining optical cavity. The second polarization-maintaining optical cavity may comprise a second gain medium different to the first gain medium excitable by a second pump light source to generate light at a second range of wavelengths, and a second saturable absorber configured to carry out passive mode locking of the light pulses in the second polarization- maintaining optical cavity. The first polarization-maintaining optical cavity and the second polarization-maintaining optical cavity may share a common branch, wherein the common branch does not include a saturable absorber.

[0029] Saturable absorbers can be used to initiate and promote strong intra-cavity pulsing through intensity dependent loss, i.e. a pulse (probe) sees a loss reduction caused by a higher energy pulse (pump). Unlike shared saturable absorber laser cavity configurations, the disclosed common branch optical circuit arrangements utilize independent saturable absorbers in each optical cavity. As a result, the operating life of the disclosed optical circuit arrangements may be extended due to the reduced stresses on the saturable absorbers resulting from excessive non-linear saturable absorption / heating induced by simultaneous pulses from multiple optical cavities, as in the known shared saturable absorber laser cavity configurations. Additionally, the inventors of the present disclosure have found that the properties of a saturable absorber may differ depending on the number of wavelengths transmitted through it. This cross-talk effect is unpredictable and typically detrimental for the mode-locking and / or synching mechanism of each individual wavelength in the common branch shared by the two optical cavities. Therefore, the provision of two independent saturable absorbers, one for each optical cavity, may allow each saturable absorber to be optimized for the wavelength associated with its respective optical cavity and avoid undesirable cross-talk effects.

[0030] Like the shared saturable absorber laser cavity configurations, the disclosed common branch optical circuit arrangements utilize passive optical synchronization resulting from crossphase modulation (XPM) interactions in the common branch. The strength of the XPM interaction in the common branch is proportional to the peak intensity of the interacting pulses and the non-linearity of the medium. The interaction length of the common branch influences the XPM-induced synchronization range. Generally, the longer the common branch, the better the XPM interaction, however, an upper limit is imposed on the permissible length of the common branch due to the group-velocity mismatch (GVM) phenomenon. It has been found that the disclosed optical circuits comprising a relatively short common branch may be as effective as a complete cavity injection with regards to passive synchronization. The disclosed common branch optical circuits may exhibit improved passive synchronization over cavity injection arrangements due to their slave-slave optical structure, in which the XPM induced repetition rate change feedback effects the pulses in both optical cavities, increasing the permissible cavity length mismatch in the optical circuit. Furthermore, performing the XPM in a common branch between two optical cavities ensures enough XPM interactions for synchronisation without the need for external amplification, leading to a more efficient optical circuit with a lower lasing threshold.

[0031] A lasing threshold is the lowest excitation level at which a laser's output is dominated by stimulated emission rather than by spontaneous emission. Below the threshold, the laser's output power rises slowly with increasing excitation. Above the lasing threshold, the slope of power vs excitation is orders of magnitude greater. The linewidth of the laser's emission also becomes orders of magnitude smaller above the lasing threshold. Above the lasing threshold, the laser is said to be lasing.

[0032] The configuration of the first and second optical cavities in the disclosed optical circuits may take on several different forms.

[0033] In a first example, the first polarization-maintaining optical cavity may have a ring configuration and the second polarization-maintaining optical cavity may have a ring configuration. In a ring configuration, optical cavities follow a path which forms a complete / continuous optical loop. In a ring-ring configuration, a portion of the two optical cavities is joined by the common branch. Compared with linear cavity configurations, ringring cavity configurations experience lower losses per round trip of the optical cavities, when employing transmissive saturable absorbers (CNTs, Graphene, transmissive semiconductor based SA), since the pulses in each optical cavity only pass through a saturable absorber once. There are no restrictions on the possible location of the common branch within each of the optical cavities except between the pump WDM and the active fiber, since the presence of the common branch WDMs will block pump light from reaching the active fiber due the presence of optical filters.

[0034] In a second example, the first polarization-maintaining optical cavity may have a linear configuration and the second polarization-maintaining optical cavity may have a ring configuration. In a linear configuration, optical cavities follow a linear path, the ends of which do not meet. Compared with a ring-ring configuration, a linear-ring configuration has the advantage of being more compact, since the total fiber length is half with respect to a ring cavity counterpart. Moreover, for dispersion compensated cavities which employ FBGs and SESAMs, the linear cavity arrangement is always less lossy with respect to the ring counterpart, leading to lower lasing thresholds.

[0035] In a third example, the first polarization-maintaining optical cavity may have a linear configuration and the second polarization-maintaining optical cavity may have a linear configuration. Advantageously, a linear-linear optical cavity configuration effectively doubles the XPM interaction length, thereby increasing the acceptable cavity mismatch in the optical circuit. This arises since, in a linear-linear configuration, cavity pulses pass through the common branch twice per round trip of each cavity.

[0036] Optionally, the first saturable absorber is different from the second saturable absorber.

[0037] Optionally, at least one of the first saturable absorber or the second saturable absorber comprises at least one of a Graphene / carbon allotrope, single-walled carbon nanotube (SWCNT), semiconductor saturable absorber mirror (SESAM), or transmissive semiconductor based saturable absorber. Advantageously, these saturable absorber (SA) types may reduce the footprint of the optical cavity arrangement since they can provide effective mode-locking functions with shorter fiber lengths compared to non-linear amplifying loop mirror and non-linear polarization rotation / evolution SA types. This enables a greater proportion of fiber to be used in the common branch leading to a larger cavity mismatch tolerance.

[0038] Any of the SA types described may be used in any of the described cavity configurations. Transmissive SAs, such as Graphene / carbon allotrope, SWCNTs and transmissive semiconductor based SAs are particularly suited to a ring cavity, since the linear losses are experienced once per cavity round-trip, leading to a lower lasing threshold. Reflective SAs however, such as SESAMs, are particularly suited to a linear cavity. Where a transmissive SA is used in a linear cavity arrangement, a fiber coupled mirror must be provided on the output of the SA. Particularly advantageous SA combinations with reduced lasing thresholds include a first transmissive SA and a second transmissive SA for a ring-ring cavity configurations, a first transmissive SA and a second SESAM SA for ring-linear cavity configurations, and a first SESAM SA and a second SESAM SA for linear-linear cavity configurations.

[0039] Optionally, at least one of the first saturable absorber and / or the second saturable absorber is mounted on a temperature controlled system. This ensures that the first and / or second saturable absorber operates in a consistent and predictable manner.

[0040] Optionally, the common branch includes a high non-linearity device or material. The high non-linearity device may comprise one or more of a photonic crystal fiber (PCF), a highly non-linear fiber (HNLF), a small mode area fiber, or a tapered fiber. The high non-linearity material may comprise two-dimensional materials (TDMs), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), and / or cellulose acetate (CA). The non-linear index of a standard single-mode fiber at 1030nm is around 2.7-2.8 x 10-7cm2 / GW. The materials listed above have a higher non-linear index or effective non-linear coefficient with respect to a standard fiber and thus higher non-linearity. The high non-linearity devices typically have the non-linear index the same as a standard fiber, but achieve higher non-linearity due to smaller mode areas. The high non-linearity device or the material may be spliced between two single mode polarization-maintaining fibers.

[0041] Optionally, the common branch comprises a single mode polarization-maintaining fiber.

[0042] Optionally, the common branch comprises a single mode fiber.

[0043] Optionally, at least one of the first polarization maintaining optical cavity or the second polarization-maintaining optical cavity comprises a polarizing isolator or dispersion compensating device or circulator with dispersion compensating device.

[0044] Optionally, at least one of the first gain medium or the second gain medium comprises a ytterbium, erbium, neodymium or thulium doped fiber.

[0045] Optionally, at least one of the first polarization maintaining optical cavity or the second polarization-maintaining optical cavity comprises a polarizing fiber coupler.

[0046] Optionally, at least one of the first polarization-maintaining optical cavity or the second polarization-maintaining optical cavity comprises an optical delay line for matching the lengths of the first polarization-maintaining optical cavity and the second polarizationmaintaining optical cavity. Optionally, the optical delay line comprises a fiber-pigtailed optical delay line. The use of delay line in one or more of the optical cavities allows the pairing of non-identical optical cavities by equalizing their lengths.

[0047] Passive synchronization of multiple mode-locked fiber oscillators addresses many technical challenges, including precise timing distribution between different wavelength regions and multi-wavelength synchronized mode-locked laser sources for microscopy. A key advantage of the passive XPM based, shared common-branch synchronization mechanism is the modularity, which enables the scale up to multiple optical cavities by adding new common branches shared with adjacent cavities.

[0048] In a fourth example, the optical circuit may further comprises a third polarization-maintaining optical cavity. The third polarization-maintaining optical cavity comprising: a third gain medium excitable by a third pump light source to generate light at a third range of wavelengths; and a third saturable absorber configured to carry out passive mode locking of the light pulses in the third polarization-maintaining optical cavity. In such embodiments, the second polarization-maintaining optical cavity and the third polarization-maintaining optical cavity may share a common branch, wherein the common branch does not include a saturable absorber.

[0049] The modularity of the disclosed optical circuit facilitates the transfer synchronization from the first and second optical cavities to a third optical cavity adjacent to the second optical cavity through XPM. This geometry enables adoption of the lowest accessible order XPM (3rdorder non linearity) thanks to the two pulse interaction in the common branches, ensuring the strongest synchronization effect. The resulting synchronization range of the neighboring optical cavities remains unaffected by the presence of the other neighboring optical cavities.

[0050] Synchronising multiple oscillators together provides advantages in broadband coherent Raman microscopy by enabling the generation of multiple pump / Stokes couples at the same time covering multiple Raman regions all at once. One implementation for broadband SRS microscopy may use the combination of two narrowband pump / Stokes beams coupled to a broadband Stokes / pump beam: one pump / Stokes will be probing one Raman active region (i.e. CH stretching) through the broadband Stokes / pump and the other pump / Stokes, centred at a different wavelength probing another Raman active region (i.e. fingerprint) through the same broadband Stokes / pump.

[0051] Generating multiple pump / Stokes couples directly from the oscillators, enable each optical cavity to be optimized for the desired wavelength range independent of other optical cavities in the optical circuit. Additionally, the proposed solution may provide increased power spectral density at the detector for broadband SRS, leading to a higher signal-to-noise ratio at constant total average power at the detector and Raman spectral coverage. Furthermore, the proposed solution may enable improved handling of non-ultra-broadband pulses inside microscopes since the use of multiple narrowband pump / Stokes beams requires a less broad Stokes / pump pulse, thus experiencing less dispersion.

[0052] Optionally, the first range of wavelengths and the second range of wavelengths may be nonoverlapping, and wherein the second range of wavelengths and the third range of wavelengths are non-overlapping. Optionally, the first gain medium comprises a Erbium doped fiber, the second gain medium comprises a Ytterbium doped fiber, and the third gain medium comprises a Neodymium doped fiber.

[0053] Optionally, the first gain medium comprises a Erbium doped fiber, the second gain medium comprises a Ytterbium doped fiber, and the third gain medium comprises a Thulium doped fiber.

[0054] Optionally, each common branch in the optical circuit is enclosed between two wavelength division multiplexers. In some examples, each common branch in the optical circuit is enclosed between two micro-optic filter couplers or micro-optic filter splitters.

[0055] In one example, each of the first polarization-maintaining optical cavity, the second polarization-maintaining optical cavity, and the third polarization-maintaining optical cavity have a ring configuration.

[0056] In one example, the first polarization-maintaining optical cavity and the second polarizationmaintaining optical cavity have a ring configuration, and wherein the third polarizationmaintaining optical cavity has a linear configuration.

[0057] In one example, the first polarization-maintaining optical cavity and the third polarizationmaintaining optical cavity have a linear configuration, and wherein the second polarizationmaintaining optical cavity has a ring configuration.

[0058] In an alternative example, the optical circuit may further comprises a third polarizationmaintaining optical cavity. The third polarization-maintaining optical cavity comprising: a third gain medium excitable by a third pump light source to generate light at a third range of wavelengths; and a third saturable absorber configured to carry out passive mode locking of the light pulses in the third polarization-maintaining optical cavity. The first polarizationmaintaining optical cavity, the second polarization-maintaining optical cavity and the third polarization-maintaining optical cavity may share the same common branch, wherein the common branch does not include a saturable absorber.

[0059] In addition to any of the above described optical circuits, the passively synchronized multioutput amplified fiber laser source may also comprise a first optical filter and a second optical filter, wherein the first optical filter and the second optical filter are configured to filter the light from the first polarization-maintaining optical cavity and the second optical polarizationmaintaining optical cavity respectively in order to output first filtered light pulses at a first predetermined range of wavelengths and second filtered light pulses at a second predetermined range of wavelengths.

[0060] The disclosed passively synchronized multi-output amplified fiber laser source enables CRS at fast enough speeds for non-invasive imaging. That is to say, to obtain objective and quantitative information of a tissue, by measuring its detailed molecular composition through its vibrational response detected by CRS. Examples of the passively synchronized multioutput amplified fiber laser source also provide a convenient tool for pump-probe experiments, and provide a suitable pump source for parametric mixing and frequency up / down conversion.

[0061] Broadly, in the passively synchronized multi-output amplified fiber laser source described, each optical cavity may comprise a gain element and single-mode polarization maintaining fibers. The optical cavity lengths may be matched using a fiber-pigtailed optical delay line inserted in one half. Following the synchronized oscillators, fiber amplifiers may be provided to increase the average power of the two branches to hundreds of mW level required for the application. In other words, two independent laser media are mode-locked and synchronised to provide pump and Stokes pulses for CRS. Two independent mode-locked optical cavities are locked in synchronism (i.e. pulses have the same repetition rate and constant optical delay between the two optical pulse trains) through the shared XPM interactions via a common branch between the two optical cavities. Frequency detuning is achieved either in the broadband configuration by employing a narrowband (broadband) pump and a broadband (narrowband) Stokes or in narrowband configuration by a tunable filter stage located either within or outside the cavities.

[0062] In contrast to the known implementation of CRS, where one of the two required independent pulses of different frequencies is generated through parametric amplification, in the passively synchronized multi-output amplified fiber laser source described herein, different laser media emitting at different frequencies are passively synchronized, thus greatly simplifying the generation of broadband or multi-colour (multi-frequency) pulse sequences required for CRS.

[0063] In the examples described, two independent mode-locked oscillators or optical cavities are provided that are synchronized through XPM interactions in a shared cavity segment.

[0064] The laser source described herein passively synchronizes fiber lasers, providing a very simple and low cost laser source for CRS. Fiber lasers enable robust and stable sources, owing to their simple, compact, and cost-effective designs, and an alignment-free operation that does not require bulky optical setups.

[0065] As explained below, examples of the laser source described herein, have been applied to Coherent anti-Stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS), thus proving the concept. Due to their compactness and all-optical synchronization, the examples described are a good source for CRS in the high-wavenumber region and also in the fingerprint region.

[0066] Arrangements are described in more detail below and take the form of a passively synchronized multi-output amplified fiber laser source for outputting filtered light pulses for inducing coherent Raman scattering in a sample.

[0067] Optionally, both of the first optical filter and second optical filter may comprise fiber Bragg grating (FBG) configured to output light pulses at a first predetermined range of wavelengths and at a second predetermined range of wavelengths. A fiber Bragg grating is a short segment of fiber that reflects particular wavelengths of light and transmits all the others. This effect is obtained by creating a periodic variation in the refractive index of the fiber core, which generates a wavelength-selective mirror. FBGs can be as well designed as chirped mirrors, thus introducing a predetermined dispersion on the reflected wavelengths of light.

[0068] Optionally, both of the first optical filter and the second optical filter may comprise a fixed wavelength optical filter configured to set the first predetermined range of wavelengths and the second predetermined range of wavelengths respectively.

[0069] Optionally, both of the first optical filter and second optical filter may comprise a tunable optical filter and configured to vary the first predetermined range of wavelengths and the second predetermined range of wavelengths respectively. Tunable optical filters allow the ranges of wavelengths to be specified by the user so that the range of wavelengths of the pump and Stokes light pulses can be varied with respect to the sample being measured.

[0070] Optionally, the tunable or fixed wavelength optical filter may comprises an etalon based fiber optic tunable or fixed wavelength filter. An etalon is a dielectric material where its specific thickness and refraction index dictates the bandwidth of each transmission peak, and only one wavelength is transmitted with maximum transmission. An etalon based fiber optic tunable or fixed wavelength filter works by selecting the refraction index of the medium of the material to select a specific resonant wavelength. The wavelength in resonance with the optical length of the cavity is transmitted, whereas the other wavelengths are reflected.

[0071] Optionally, the first optical filter and the second optical filter are positioned within the first polarization-maintaining optical cavity and second polarization-maintaining optical cavity respectively, and wherein the first polarization-maintaining optical cavity and the second polarization-maintaining optical cavity respectively outputs the filtered light pulse at a first optical outlet and a second optical outlet. Having the optical filters fitted inside the optical cavities ensures light pluses with undesired ranges of wavelengths are promptly filtered after their generation.

[0072] Optionally, the first optical filter and the second optical filter are positioned externally to the first polarization-maintaining optical cavity and second polarization-maintaining optical cavity respectively, and wherein the first polarization-maintaining optical cavity and the second polarization-maintaining optical cavity respectively output the light pulses at a first optical outlet and a second optical outlet. Having the optical filters placed externally to the optical cavities eliminates the need to filter the recirculating filtered light pulses repeatedly, as well as permitting the construction of simple and compact optical cavities.

[0073] The light pulses may be filtered such that only light pulses within a defined range of wavelengths are output to coherent Raman spectroscopy, which yields a more accurate measurement. Furthermore, the use of two synchronized and mode-locked laser sources greatly reduces the impact of optical filters on the optical power of pump and Stokes pulses, making it a versatile choice for CRS.

[0074] Optionally, the laser source further comprises a first fiber amplifier doped with the first gain medium at the first optical outlet and a second fiber amplifier doped with the second gain medium at the second optical outlet for amplifying the light pulses or the filtered light pulses. This ensures the amplified light pulses are amplified in the correct wavelength range. The use of amplifiers mitigates the reduction in optical power when optical filters are in place.

[0075] Optionally, the laser source further comprises a second harmonic generation crystal operably coupled to an optical outlet of at least one of the first fiber amplifier or the second fiber amplifier. The second harmonic generation crystal may be formed from one or more of periodically poled lithium niobate PPLN or periodically poled potassium titanyl phosphate PPKTP. Optionally, the laser source further comprises an acousto-optic modulator or an electrooptic modulator which is operably coupled to at least one of the first amplifier or the second amplifier.

[0076] Optionally, the laser source further comprises an acousto-optic modulator or an electrooptic modulator which is operably coupled to the outlet of at least one of the first polarization-maintaining optical cavity or the second polarization-maintaining optical cavity.

[0077] Optionally, the spectrum of both the light pulses exiting the first polarization maintaining cavity or the second polarization maintaining cavity can be broadened in the fiber amplifier section due to self-phase modulation in order to increase the spectral bandwidth and reduce the compressed pulse duration.

[0078] Optionally, the laser source is a fiber laser. Optionally, the laser source is an all-fiber laser. Optionally, the first optical cavity and second optical cavity comprises an isotropic optical fiber. Optionally, each of the first optical cavity and second optical cavity comprises a singlemode optical fiber.

[0079] Optionally, the laser gain media comprises ytterbium or erbium, where optionally the predetermined range of wavelengths generated by said laser gain media corresponds to full Raman spectrum of 0-4000crrr1.

[0080] Optionally, the predetermined range of wavelengths comprises the range of 1000nm to 110Onm and / or 1535nm to 1600nm and / or 91 Onm to 950nm and / or 1800nm to 1900nm.

[0081] Optionally, frequency conversion of the amplified light pulses can be implemented using second-harmonic generation crystals (i.e. periodically poled lithium niobate - PPLN -, periodically poled potassium titanyl phosphate -PPKTP-).

[0082] Optionally, an acousto-optic modulator (AOM) or an electro-optic modulator (EOM) can be placed outside of both the first polarization maintaining cavity and the second polarizationmaintaining cavity.

[0083] In an example, the passively synchronized multi-output amplified fiber laser source may comprise two collimators configured to collimate the filtered light pulses. This limits the divergence of filtered light pulses. Optionally, one of the collimators comprises a delay stage configured to achieve an overlap on the measured sample. Optionally, the laser source further comprises a dichroic mirror configured to combine the collimated light pulses from both of the two collimators.

[0084] Optionally, the laser source comprises a bandpass or short-pass filter for removing the pair of filtered light pulses prior to CARS detection.

[0085] Optionally, the laser source comprises a bandpass or long-pass filter for removing the pump light pulses prior to SRG detection.

[0086] Optionally, the laser source comprises a bandpass or short-pass filter for removing the Stokes light pulses prior to SRL detection.

[0087] In an example, the optical circuit of the passively synchronized multi-output amplified fiber laser source may comprise N polarization-maintaining optical cavities. Each of the N polarization-maintaining optical cavities comprising: a gain medium excitable by a pump light source to generate light at a range of wavelengths; and a saturable configured to carry out passive mode locking of the light pulses in the polarization-maintaining optical cavity. Each of the N polarization-maintaining optical cavities share a common branch with at least one other polarization-maintaining optical cavity of the N polarization-maintaining optical cavities. Wherein the common branches between the N polarization-maintaining optical cavities do not comprise a saturable absorber.

[0088] Optionally, the gain medium of each polarization-maintaining optical cavity is excitable by a pump light source to generate light at a range wavelengths which does not overlap with a range of wavelengths of light generated by exciting the gain medium of adjacent polarizationmaintaining optical cavities.

[0089] Optionally, at least N-1 of the polarization-maintaining optical cavities comprise an optical delay line for matching the length of the cavities for synchronization purposes.

[0090] Optionally, the optical delay line comprises a fiber-pigtailed optical delay line.

[0091] Optionally, each of the N polarization-maintaining optical cavities have a ring configuration.

[0092] Optionally, 1 < M < 2 cavities of the N polarization-maintaining optical cavities have a linear configuration and N-M cavities of the N polarization-maintaining optical cavities have a ring configuration. It will be appreciated that the features described in relation to each of the above passively synchronized multi-output amplified fiber laser source examples of the disclosure may also be applied to the other examples except where stated otherwise.

[0093] In an example, the optical imaging system according to any of the above described examples may be used for histopathology. In an example, the optical imaging system according to any of the above described examples may be used for one or more of: multiplex SRS, SRS, CRS, or CARS.

[0094] In a second aspect, the disclosure provides a method of analysing a sample using multiplex stimulated Raman scattering (SRS). The method comprises: generating a plurality of passively synchronised beams having different wavelength ranges; directing the plurality of beams through a sample plane in a laser scanning microscope; simultaneously receiving, using a multi-channel lock-in amplifier, a plurality of optical signals from the sample plane; and analysing the received plurality of optical signals in parallel for multiplex SRS.

[0095] In an example, generating a plurality of passively synchronised beams having different wavelength ranges may comprise: generating light at respective different ranges of wavelengths with a first polarization-maintaining optical cavity comprising a first gain medium and a second polarization-maintaining optical cavity comprising a second gain medium different to the first gain medium, wherein the first gain medium and the second gain medium are each excitable by a pump light source. Mode-locking may be performed via a first and second saturable absorber optically coupled to the first polarization-maintaining optical cavity and the second polarization-maintaining optical cavity respectively. The method may further comprise synchronising, via a common branch between the first polarization-maintaining optical cavity and the second polarization-maintaining optical cavity, the light from the first polarization-maintaining optical cavity and the second polarization-maintaining optical cavity, wherein the common branch does not include a saturable absorber.

[0096] In an example, generating a plurality of passively synchronised beams may comprise: generating light at respective different ranges of wavelengths using one of the above described passively synchronized multi-output amplified fiber laser sources.

[0097] BRIEF DESCRIPTION OF THE DRAWINGS

[0098] The invention will be described in more detail, by way of example, with reference to the accompanying drawings, in which: Figure 1 is a schematic of an optical imaging system according to an example of the disclosure;

[0099] Figure 2 is a schematic of a dual output laser optical imaging system according to an example of the disclosure;

[0100] Figure 3 is a schematic of an Epi-detected optical imaging system according to an example of the disclosure;

[0101] Figure 4 is a schematic of a non-descanned optical imaging system according to an example of the disclosure;

[0102] Figure 5 is a schematic of a non-descanned optical imaging system according to an example of the disclosure;

[0103] Figure 6 is a schematic of a triple output laser optical imaging system according to an example of the disclosure;

[0104] Figure 7 is a schematic of an individual channel in a multi-channel lock-in amplifier according to an example of the disclosure.

[0105] Figure 8 is a schematic of an optical circuit having a ring-ring configuration according to an example of the disclosure;

[0106] Figure 9 is a schematic of an optical circuit having a linear-ring configuration according to an example of the disclosure;

[0107] Figure 10 is a schematic of an optical circuit having a linear-linear configuration according to an example of the disclosure;

[0108] Figure 11 is a schematic of an optical circuit having a linear-ring-linear configuration according to an example of the disclosure;

[0109] Figure 12 is a schematic of an optical circuit having a ring-ring-linear configuration according to an example of the disclosure;

[0110] Figure 13 is a schematic of an optical circuit having a ring-ring-ring configuration according to an example of the disclosure; and Figure 14 is a schematic of an optical device according to an example of the disclosure.

[0111] DETAILED DESCRIPTION OF THE INVENTION

[0112] Optical imaging systems for stimulated Raman scattering (SRS) according to examples of the present invention are described below with reference to Figures 1 to 6.

[0113] Figure 1 is a schematic of an optical imaging system 100 according to an example of the disclosure. The imaging system 100 comprises a synchronised multi-output amplified fiber laser source 110 coupled to a detection system 130 via a laser scanning microscope 120. The laser scanning microscope 120 is configured to receive a sample volume within the sample plane for histopathological examination.

[0114] The laser scanning microscope 120 includes a beam scanning unit 122 for directing received optical signals to a microscope objective 124. The laser scanning microscope 120 further comprises a condenser 126 for receiving optical signals from a sample plane, which are directed to a descanning unit 128 before being received by the detection system 130. The detection system 130 comprises a multi-channel lock-in amplifier.

[0115] Figure 2 is a schematic of an optical imaging system 200 according to an example of the disclosure. The system 200 comprises a passively synchronised dual output amplified fiber laser source 210 configured to generate two synchronised beams having different wavelength ranges. The laser source 210 generates a first broadband Stokes beam centred at 1030nm and a first narrowband pump beam centred at 792nm. The passively synchronised dual output amplified fiber laser source 210 is coupled to a beam scanning unit 222.

[0116] The beams are directed into the beam scanning unit 222 to a first optical relay system 234 via a first single axis galvo mirror 232 which facilitates strip mosaicking laser scanning onto the sample. To conjugate the galvo pivot point with an objective back focal plane of the laser scanning microscope, the first optical relay system 234 has a certain magnification Mi. This magnification relates input beam diameter (D) and angle (0) entering the first optical relay system 234 with the ones exiting with the very well-known formula:

[0117] Then, the desired Field-of-View (FoV) at the objective focal plane is:

[0118] In some embodiments, the first single axis galvo mirror 232 may be replaced with one of: an acousto optic deflector (AOD), a dual-axis galvo mirror, a fast steering mirror, a polygon mirror scanner, or a fixed mirror.

[0119] From first optical relay system 234 the beams are directed through a sample plane 240 via a microscope objective 224. Situated between the first optical relay system 234 and the microscope objective 224 is a dichroic mirror 252 configured to reflect epi generated second harmonic generated (SHG) and two-photon excitation fluorescence (TPEF) signals by the sample through a lens 254 to concentrate them onto two different photo multiplier tubes (PMTs) 256, 258. The separation of SHG signals from TPEF signals is achieved by an extra dichroic mirror 262 positioned between the lens and each of the PMTs 256, 258.

[0120] The beams are directed through the sample volume positioned in the sample plane 240 and collected by a condenser 226. In some embodiments, the condenser 226 can be replaced with one of: a single lens, for example a metalens, grin lens, or Fresnel lens, a multi-lens, or other collection optics system.

[0121] The condenser 226 is configured to direct the collected beams through a descanning unit 228 comprising a second optical relay system 242 having a magnification M2and a second single axis galvo mirror 246. The descanning unit 228 is aligned on the same axis as the first single axis galvo mirror 232 of the beam scanning unit 222, and fully de-scans the received beams to remove the residual beam pointing error. The relationship between the instantaneous angle of the first and second single axis galvo mirror 232, 246 is:

[0122] The two angles are equal only in the situation where the ratio between the condenser 226 effective focal length and the magnification of the descanning optical relay system 242 is the same as the ratio between the objective effective focal length and the magnification of the scanning unit optical relay system 234. In this embodiment, the descanning optical relay system 242 is an afocal relay system.

[0123] In some embodiments, the second single axis galvo mirror 246 may be replaced with one of: an acousto optic deflector (AOD), a dual-axis galvo mirror, a fast steering mirror, a polygon mirror scanner, or a fixed mirror.

[0124] The optical imaging system 200 of Figure 2 is shown with a descanning plane of the galvo mirror oriented parallel to the plane of incidence of the grating. In a preferred embodiment, descanning plane is orthogonal to the plane of incidence of the grating in order to minimize the effect of the residual pointing error of the beam on the grating diffraction efficiency.

[0125] Coupled to the descanning unit 228 is the detection system 230. The detection system 230 comprises a series coupling of a long-pass filter 263, a half-wave plate 264, a transmission grating 266, a lens 268 and a single phase multi-channel lock-in amplifier 270. The detection system 230 is configured to extract optical signals from the received beams. In this specific embodiment, the broadband Stokes beam centered at 1030 nm is dispersed and imaged on a 38-channel photodiode array, resulting in a spectral coverage of 2800-3100 cm-1with a spectral resolution lower than 8 cm-1on average.

[0126] Figure 3 is a schematic of an Epi-detected optical imaging system 300 according to an example of the disclosure. The system 300 is similar to the system 200 shown in Figure 2, however the system 300 does not comprise either a condenser (or collection optics) or a separate descanning unit. In the embodiment of Figure 3, the detection system 300 is coupled between the passively synchronised dual output amplified fiber laser 310 and the beam scanning unit 322 via a polarizing beam splitter 372 and a quarter waveplate 374. In this embodiment, the polarizing beam splitter 372 separates the reflected and / or back scattered Stokes and pump beams from the optical path between the laser source 310 and the beam scanning unit 322, and directs the beam to the detection system 330. Such embodiments are particularly suited to analysing sample volumes which are opaque or too thick for light beams to effectively penetrate.

[0127] Figures 4 & 5 illustrate schematics of non-descanned optical imaging systems 400, 500 according to examples of the disclosure. Non-descanned optical imaging systems 400, 500 can be advantageous over systems as they avoid the need for complex electronics control and synchronisation, however can be more susceptible to misalignment. The system 400 of Figure 4 is similar to the system 200 of Figure 2, however does not include an active electro-optical descanning system. Rather than a second single axis galvo mirror, or equivalent descanning component, the system 400 in Figure 4 utilises an optical fibre 480 coupling between the second optical relay system 442 and the detection system 430. In this embodiment, the back focal plane of the condenser 426 is imaged through the second optical relay system 442 onto the core of the optical fiber 480. The light coupled in the fiber 480 is then collimated at the output and sent to the detection system 430, which acts as a standalone system.

[0128] The system 500 of Figure 5 provides an alternative non-scanned solution to the system 400 of Figure 4. The system 500 of Figure 5 comprises a third optical relay system 580 having a magnification M3positioned in the detection beam path. The third optical relay system 580 is configured to conjugate the spot on the transmission grating 566 with the single phase multichannel lock-in amplifier 570 of the detection system. The two conjugated pivot points in this arrangement guarantee that the signal is collected at all the galvo mirror scanning angles. In this embodiment, the dispersion plane (the plane where the wavelengths of the detected beam are dispersed by the grating) must be perpendicular to the scanning plane (the plane where the beam is scanned by the galvo). In the described system 500, the magnification of the second optical relay system 542 is set depending on specification of the transmission grating 566 and the magnification of the third optical relay system 580 is set depending on specification of the detection system 530.

[0129] This configuration ensures two important conditions: firstly, to ensure that all the beams emerging with different angles from the galvo are imaged onto the same point at the detection system; and secondly, so that the grating diffraction efficiency is set by the beam angle of incidence on an orthogonal plane with respect to the galvo scanning plane, such that the wavelength dispersion efficiency is constant and not varying.

[0130] Figure 6 is a schematic of an optical imaging system 600 according to an example of the disclosure. The system 600 in Figure 6 is similar to the system 200 of Figure 2, however comprises a passively synchronised triple output amplified fiber laser source 610 configured to generate three synchronised beams having different wavelength ranges. In this embodiment, the laser generates a first broadband Stokes beam centred at 1030nm, a first narrowband pump beam centred at 792nm and a second narrowband pump beam centred at 920nm. In this embodiment, a dual phase multi-channel lock-in amplifier is required in the detection system. In this embodiment, imaging may cover both the CH-stretching region and the fingerprint region. In such embodiments, each channel of the multi-channel lock-in amplifier 670 comprises two demodulators working in phase quadrature, thus ensuring a simultaneous coverage of the -2770-3370 cm-1and -1000-1610 cm-1wavenumber regions. In the embodiment of Figure 6, the multi-channel lock-in amplifier 670 comprises 76 channels.

[0131] The first and second narrow pump beams can be amplitude modulated at a different frequency and / or with a different phase (for instance, modulated in quadrature -with a phase difference of TT / 2- with respect to each other) and then sent collinearly with the broadband Stokes beam through the sample volume.

[0132] Broadband multiplexed stimulated Raman gain (SRG) / stimulated Raman loss (SRL) is detected by the multichannel lock-in amplifier 670, which can demodulate the signals at the two different frequencies and / or phases. In this embodiment, it is possible to reconstruct simultaneously the broadband SRG / SRL spectra generated by the first and second narrow pump beams on the broadband Stokes beam. The output of detection system 630 comprises two slices of the whole Raman spectrum, whose centers and widths can be tuned by varying the central wavelength of the two narrow pump beams and the bandwidth of the broadband Stokes beam, respectively. Advantageously, the system 600 of Figure 6 can be easily tuned and optimized to cover the desired Raman active regions with independent spectral resolution. The amount of Raman active regions to be covered may be set by the number of narrowband beams generated by the laser source 610, which can be easily modulated separately. Furthermore, the use of narrowband beam multiplexing enables amount of Raman active regions covered simultaneously to be increased without also increasing the number of channels required in the multi-channel lock-in amplifier 670.

[0133] It will be appreciated that the imaging systems of figures 3 - 5 may also be configured for both CH-stretching and fingerprint imaging with the use of a triple output amplified fiber laser source 610 and a dual phase multi-channel lock-in amplifier 670, as described in Figure 6.

[0134] Figure 7 is a schematic an individual channel in a multi-channel lock-in amplifier according to an example of the disclosure. As illustrated in Figure 7, each channel comprises a photodiode, an amplifier with both DC and AC gain control, one or more demodulators with low-pass filters and an analog-to-digital converter (ADC). The output of each channel is fed to a processing unit. The signal generated by each photodiode of a photodiode array of the multi-channel lock-in amplifier is amplified by a dedicated chain of amplifiers with firmware (FW) based controllable gain in order to maximize the signal-to-noise ratio. Moreover, the amplifier in each channel is able to divide the DC component of the signal from the AC component and amplify the two components independently with different gain values. The DC component is sent directly to the ADC and it’s used for two reasons: firstly, to reconstruct the linear transmission image of the sample as a function of the detected wavelength; and secondly, to normalize the AC component, in order to reconstruct the correct relative spectral shape.

[0135] The amplified AC component is sent to one or more demodulators (mixers) at the same time, each one receiving a reference signal with a certain frequency and phase. Multiple configurations of the demodulators are possible, including common phase with different frequencies, common frequency with different phases or different phase different frequency.

[0136] At the output of all the demodulators there is a dedicated fixed-time-constant low pass filter with a built in amplifier having a bandwidth designed to guarantee a rise time compatible with the shortest pixel dwell time chosen. After the signal has been digitized at the output stage of the lock-in amplifier, there is an external processing unit which performs averaging with an average window which is set by two consecutive pixel triggers. This solution facilitates an almost-all analog fixed bandwidth lock-in amplifier for each channel which can generate, through an external board, an effective time constant which is pixel triggered and therefore dynamically adapted, in an autonomous way, to the imaging speed.

[0137] Optical circuits for use in laser sources of the optical imaging systems of Figures 1 -6 are described below with reference to Figures 8 to 14.

[0138] Figure 8 illustrates an optical circuit 1000 according to a first embodiment of the present invention. Optical circuit 1000 comprises two independent mode-locked optical cavities, oscillators or resonators for generating two sets of light pulses at order of picosecond durations at different ranges of wavelengths suitable for CRS. The two optical cavities 1 100a, 1100b are joined together at a common branch 1200.

[0139] In more detail, continuing to refer to optical circuit 1000 of Figure 8, each of the two optical cavities 1100a, 1100b of optical circuit 1000 are arranged in the form of a loop. The ends of optical fibres making up optical cavities 1100a, 1100b are optically connected using any suitable couplers, in order to circulate the light pulses in the loops until their discharge from the optical cavities at their respective optical outlets. Furthermore, each of the optical outlets comprises fibre couplers to provide approximately 20-30% output for their respective cavities. The mode-locked optical cavities 1100a, 1100b each include a pump light source 1300a, 1300b to each of the optical cavities 1 100a, 1 100b to excite gain elements 1400a, 1400b that are located or deposited inside the optical cavities 1 100a, 1100b. The gain elements 1400a, 1400b, in this example, are optical fibres doped with rare earth gain elements. Laser sources using such gain elements are commonly referred to as fiber lasers.

[0140] The choice of pump light sources 1300a, 1300b and the gain elements 1400a, 1400b depend on the light spectra required by the CRS. The example shown in Figure 8 uses two different optical fibers as the gain elements. One optical fiber is doped with a rare earth gain element in the form of ytterbium (Yb) 1400a. The other optical fiber is doped with a rare earth gain element in the form of erbium (Er) 1400b. In this example, the pump light sources to excite the gain elements are a 976nm wavelength pump light source 1300a to excite the Yb doped fiber; and a 976nm wavelength pump light source 1300b to excite the Er doped fiber. The light pulses generated from the Yb and Er gain media are in the range of desirable pump and Stokes wavelengths.

[0141] An optical isolator 1500a, 1500b is optically coupled in each of the optical cavities 1100a, 1100b in order to force the same direction of circulation inside the common branch 1200. This ensures that the light pulses generated by the gain media 1400a, 1400b in the optical cavities travel in a single direction in the loops forming the optical cavities 1100a, 1100b. That is, light pulses generated from the gain media 1400a, 1400b are directed towards the optical outlets. In this example, the optical isolators 1500a, 1500b are fiber based Faraday isolators. In other examples, optical isolators 1500a, 1500b may comprise polarizing circulators with dispersion compensating devices and output couplers.

[0142] The pair of light pulses generated in each of the optical cavities 1 100a, 1 100b are passively synchronized via XPM interactions in common branch 1200, shared by both loops forming the optical cavities 1100a, 1100b. In this example, common branch 1200 comprises a high non-linearity device or material 1600 to enhance XPM interaction strength for synchronization.

[0143] Optical cavities 1100a, 1 100b each comprise their own saturable absorber 1700a, 1700b outside of common branch 1200. The function of a saturable absorber is described in the summary of invention section above. A saturable absorber is a light absorber whose degree of absorption is reduced at high optical intensities. In optical circuit 1000, this allows passive mode-locked pulses to circulate in each of optical cavities 1 100a, 1 100b. That is, passive mode-locking allows the generation of femtosecond light pulses. Saturable absorber 1700a, 1700b possesses a sufficiently short recovery time so that fast loss modulation is achieved.

[0144] The saturable absorber 1700a, 1700b in Figure 8 may be a graphene based polymer- composite saturable absorber, which has ultrafast recovery time and broadband operation. A graphene saturable absorber may be prepared by exfoliating bulk graphite by mild ultrasonication, wherein a dispersion first enriched with obtained single layer graphene and few layer graphene is mixed with an aqueous solution of polyvinyl alcohol, resulting in a polymer composite. Other saturable absorbers may alternatively be used for carrying out passive mode locking of the light pulses, for example saturable absorbers comprising singlewall carbon nanotubes (CNT), however any of the saturable absorbers described in the summary of invention section above may be utilized.

[0145] The pair of optical cavities 1 100a, 1 100b do not need to be identical. The difference in cavity lengths between the two optical cavities 1100a, 1100b is compensated for by the addition of an optical delay line 1800a, 1800b to either one or both of the optical cavities. In this example, an optical delay line 1800a, 1800b is located in both optical cavities 1 100a, 1100b after isolator 1500a, before isolator 1500b. In this example, optical delay line 1800a, 1800b comprises a fiber-pigtail delay line. The fiber-pigtail delay line is optically coupled to the outlet of the isolator 1500a, to the inlet of 1500b in each optical cavities 1100a, 1 100b. In other examples, the optical delay line 1800a, 1800b may comprise an output coupler in series.

[0146] The ranges of wavelengths of the light pulses generated at each of the optical cavities 1 100a, 1100b are dictated by the type of gain media being excited in the respective optical cavity.

[0147] Each of the optical cavities 1 100a, 1100b have an outlet to together output first filtered light pulses at a first predetermined range of wavelengths and second filtered light pulses at a second predetermined range of wavelengths from the optical circuit 1000. In an example, the outlets may be located in 1500a, 1500b if they are circulators with dispersion compensating device and output couplers. The optical outlets may each be coupled to a different fiber amplifier. The relevant fiber amplifier may be doped with a gain element corresponding to the gain medium 1400a, 1400b that is responsible for light pulse generation. In an example, Yb- and Er- doped fibre amplifiers are respectively provided for optical cavities 1100a, 1 100b, in order to amplify the light pulses at the Yb and Er wavelengths to 100mW average power.

[0148] Figure 9 illustrates an optical circuit 2000 according to a second embodiment of the present invention. Similarly to the optical circuit 1000 shown in Figure 8, optical circuit 2000 comprises two independent mode-locked optical cavities, oscillators or resonators for generating two sets of light pulses at order of picosecond durations at different ranges of wavelengths suitable for CRS. The two optical cavities 2100a, 2100b are joined together at a common branch 2200.

[0149] Unlike optical circuit 1000, in optical circuit 2000, only one of the two optical cavities 2100b of optical circuit 2000 is arranged in the form of a loop, while the other optical cavity 2100a is arranged in a linear configuration. As in optical circuit 1000, the mode-locked optical cavities 2100a, 2100b of optical circuit 2000 each include a pump light source 2300a, 2300b to each of the optical cavities 2100a, 2100b to excite gain elements 2400a, 2400b that are located or deposited inside the optical cavities 2100a, 2100b. The gain elements 2400a, 2400b, in this example, are optical fibres doped with rare earth gain elements.

[0150] An optical isolator 2500 is optically coupled within optical cavity 2100b. This is in order to ensure that the light pulses generated by the gain media 2400b travel in a single or in one and only one direction in the loop forming optical cavity 2100b. That is, light pulses generated from the gain media 2400b are directed towards the optical outlet. In this example, the optical isolator 2500 may include a fiber based Faraday isolator. In other examples, optical isolator 2500 may comprise polarizing circulators with dispersion compensating devices and output couplers.

[0151] As described in relation to optical circuit 1000, the pair of light pulses generated in each of the optical cavities 2100a, 2100b are passively synchronized via XPM interactions in common branch 2200, shared by both optical cavities 2100a, 2100b. In this example, common branch 2200 comprises a high non-linearity device or material 2600 to enhance XPM interaction strength for synchronization.

[0152] Optical cavities 2100a, 2100b each comprise their own saturable absorber 2700a, 2700b outside of common branch 2200. As described in relation to optical circuit 1000, in optical circuit 2000, this allows passive mode-locked pulses to circulate in each of optical cavities 2100a, 2100b. Saturable absorber 2700a, 2700b possesses a sufficiently short recovery time so that fast loss modulation is achieved. Any of the saturable absorbers mentioned in this disclosure may be utilised in optical circuit 2000. Where a transmissive saturable absorber is provided in a linear optical cavity, a fiber-coupled mirror must be provided within the optical cavity after the saturable absorber. The difference in cavity lengths between the two optical cavities 2100a, 2100b is compensated for by the addition of an optical delay line 2800a, 2800b to either one of the optical cavities. In this example, an optical delay line 2800a, is located in optical cavity 2100a after gain medium 2400a, while optical delay line 2800b, is located in optical cavity 2100b after isolator 2500. In this example, optical delay lines 2800a, 2800b comprises a fiber-pigtail delay line. The fiber-pigtail delay line is optically coupled to the outlet of the isolator 2500 in optical cavity 2100b. In other examples, the optical delay line 2800a, 2800b may comprise an output coupler in series.

[0153] Optical cavity 2100a has a dispersion compensator and output coupler (chirped fiber Bragg grating in this example) 1900 to output first filtered light pulses at a first predetermined range of wavelengths from optical circuit 2000. Optical cavity 2100b has a corresponding outlet to output second filtered light pulses at a second predetermined range of wavelengths from the optical circuit 2000. In an example, the outlets may be located in 1900 and in 2800b. The optical outlets may each be coupled to a different fiber amplifier. The relevant fiber amplifier may be doped with a gain element corresponding to the gain medium 2400a, 2400b that is responsible for light pulse generation. In an example, Yb- and Er- doped fibre amplifiers are respectively provided for optical cavities 2100a, 2100b, in order to amplify the light pulses at the Yb and Er wavelengths to 10OmW average power.

[0154] Figure 10 illustrates an optical circuit 3000 according to a third embodiment of the present invention. Similarly to optical circuits 1000 and 2000, optical circuit 3000 comprises two independent mode-locked optical cavities, oscillators or resonators for generating two sets of light pulses at order of picosecond durations at different ranges of wavelengths suitable for CRS. The two optical cavities 3100a, 3100b are joined together at a common branch 3200.

[0155] Unlike optical circuits 1000 and 2000, in optical circuit 3000, both of the two optical cavities 3100a, 3100b of optical circuit 3000 are arranged in a linear configuration. As in optical circuits 1000 and 2000, the mode-locked optical cavities 3100a, 3100b of optical circuit 3000 each include a pump light source 3300a, 3300b to each of the optical cavities 3100a, 3100b to excite gain elements 3400a, 3400b that are located or deposited inside the optical cavities 3100a, 3100b. The gain elements 3400a, 3400b, in this example, are optical fibres doped with rare earth gain elements. Optical circuit 3000 may comprise a polarizing coupler 3500a, 3500b optically coupled to the outlet of gain medium 3400a, 3400b in optical cavity 3100a, 3100b respectively. This is used to select the polarization state on the slow axis and can act as the optical outlet.

[0156] As described in relation to optical circuits 1000 and 2000, the pair of light pulses generated in each of the optical cavities 3100a, 3100b are passively synchronized via XPM interactions in common branch 3200, shared by both optical cavities 3100a, 3100b. In this example, common branch 3200 comprises a high non-linearity device or material 3600 to enhance XPM interaction strength for synchronization. In this linear-linear optical cavity arrangement, the effective XPM interaction length is increased since each generated light pulse in each optical cavity 3100a, 3100b passes common branch 3200 twice. This improves the permissible cavity mismatch in the optical circuit 3000.

[0157] Optical cavities 3100a, 3100b each comprise their own saturable absorber 3700a, 3700b outside of common branch 3200. As described in relation to optical circuits 1000 and 2000, in optical circuit 3000, this allows passive mode-locked pulses to circulate in each of optical cavities 3100a, 3100b. Saturable absorber 3700a, 3700b possesses a sufficiently short recovery time so that fast loss modulation is achieved. Any of the saturable absorbers mentioned in this disclosure may be utilised in optical circuit 3000.

[0158] The difference in cavity lengths between the two optical cavities 3100a, 3100b is compensated for by the addition of an optical delay line 3800a, 3800b to either one of the optical cavities. In this example, an optical delay line 3800a, is located in optical cavity 3100a after gain medium 3400a, while optical delay line 3800b, is located in optical cavity 3100b after gain medium 3400b. In this example, optical delay lines 3800a, 3800b may comprise a fiber-pigtail delay line.

[0159] Optical cavities 3100a, 3100b have dispersion compensators and output couplers 3900a, 3900b to output first filtered light pulses at a first predetermined range of wavelengths and second filtered light pulses at a second predetermined range of wavelengths from the optical circuit 3000 (chirped fiber Bragg grating in this example). The optical outlets may each be coupled to a different fiber amplifier. The relevant fiber amplifier may be doped with a gain element corresponding to the gain medium 3400a, 3400b that is responsible for light pulse generation. In an example, Yb- and Er- doped fibre amplifiers are respectively provided for optical cavities 3100a, 3100b, in order to amplify the light pulses at the Yb and Er wavelengths to 10OmW average power. In other embodiments, dispersion compensators and output couplers 3900a, 3900b may be replaced with high reflectivity mirrors while polarizing couplers 3500a, 3500b are used as the output couplers.

[0160] In each of the disclosed optical circuits 1000, 2000 and 3000, the position of the SAs and / or the common branch with respect to components within the circuit may vary, providing the common branch 3200 and dedicated WDMs are positioned within each cavity after the pump diodes 3300a, 3300b and the active fiber-gain elements 3400a, 3400b. The SAs can be positioned either before or after the common branch.

[0161] Figure 1 1 illustrates an optical circuit 5000 according to an embodiment of the present invention comprising three optical cavities 5100a, 5100b, 5100c, each comprising a pump light source 5300a, 5300b, 5300c to each of the optical cavities 5100a, 5100b, 5100c to excite gain elements 5400a, 5400b, 5400c that are located or deposited inside the optical cavities 5100a, 5100b, 5100c. The gain elements 5400a, 5400b, 5400c in this example, are optical fibres doped with rare earth gain elements.

[0162] The first optical cavity 5100a shares a common branch with the second optical cavity 5100b in a manner equivalent to that in optical circuit 2000 of Figure 9. Unlike optical circuit 2000, second optical cavity 5100b of optical circuit 5000 comprises a second common branch shared with a third optical cavity 5100c.

[0163] An optical isolator 5500 is optically coupled within optical cavity 5100b. This is in order to ensure that the light pulses generated by the gain media 5400b travel in a single or in one and only one direction in the loop forming optical cavity 5100b. That is, light pulses generated from the gain media 5400b are directed towards the optical outlet. In this example, the optical isolator 5500 may include a fiber based Faraday isolator. In other examples, optical isolator 5500 may comprise polarizing circulators with dispersion compensating devices and output couplers.

[0164] Light pulses generated in each of the optical cavities 5100a, 5100b, 5100c are passively synchronized via XPM interactions in common branches, shared between optical cavities 5100a - 5100b, and 5100b - 5100c. In this example, the common branches comprises a high non-linearity device or material 5600a, 5600c to enhance XPM interaction strength for synchronization. Optical cavities 5100a, 5100b, 5100c each comprise their own saturable absorber 5700a, 5700b, 5700c outside of the common branches. As described above, this allows passive mode-locked pulses to circulate in each of optical cavities 5100a, 5100b, 5100c. Saturable absorber 5700a, 5700b, 5700c possesses a sufficiently short recovery time so that fast loss modulation is achieved. Any of the saturable absorbers mentioned in this disclosure may be utilised in optical circuit 5000.

[0165] The difference in cavity lengths between the optical cavities 5100a - 5100b and between optical cavities 5100b -5100c is compensated for by the addition of optical delay lines 5800a, 5800b, 5800b in one or more of the optical cavities. In this example, optical delay lines 5800a and 5800c are located in optical cavities 5100a and 5100c after gain mediums 5400a and 5400c respectively, while optical delay line 5800b, is located in optical cavity 5100b after isolator 5500. In this example, optical delay lines 5800a, 5800b, 5800c comprises a fiberpigtail delay line. The fiber-pigtail delay line is optically coupled to the outlet of the isolator 5500 in optical cavity 5100b. In other examples, the optical delay line 5800a, 5800b, 5800c may comprise an output coupler in series.

[0166] Optical cavities 5100a and 5100c have dispersion compensators and output coupler (chirped fiber Bragg grating in this example) 5900a, 5900c to output first filtered light pulses at a first predetermined range of wavelengths from optical circuit 5000. Optical cavity 5100b has a corresponding outlet to output second filtered light pulses at a second predetermined range of wavelengths from the optical circuit 5000. In an example, the outlets may be located in 5900a, 5900c and in 5800b. The optical outlets may each be coupled to a different fiber amplifier. The relevant fiber amplifier may be doped with a gain element corresponding to the gain medium 5400a, 5400b, 5400c that is responsible for light pulse generation. In an example, Er-, Yb- and Nd- doped fibre amplifiers are respectively provided for optical cavities 5100a, 5100b, 5100c in order to amplify the light pulses at the Er, Yb and Nd wavelengths to 100mW average power.

[0167] Figure 11 illustrates an optical circuit 5000 having three optical cavities 5100a, 5100b, 5100c arranged in a linear-ring-linear configuration. However it will be appreciated that alternative arrangements are also possible. For example, figure 12 illustrates a similar optical circuit 6000 to that shown in figure 1 1 , and has three optical cavities 6100a, 6100b, 6100c. In optical circuit 6000, however, optical cavity 6100a is in a ring configuration, thereby forming a ring- ring-linear configuration optical circuit 6000. Despite the differences in configuration, optical circuit 6000 may function in a similar manner to optical circuit 5000. Figure 13 illustrates having three optical cavities 7100a, 7100b, 7100c arranged in a ring- ring-ring configuration. As discussed in relation to optical circuit 6000, the features described with respect to optical circuit 5000 apply equally to optical circuit 7000.

[0168] It will be appreciated that although not illustrated, optical circuits in accordance with the present invention may comprise more than three optical cavities and in a variety of configurations. In one example, the optical circuit comprising N optical cavities, each optical cavity sharing a common branch with at least one adjacent optical cavity. In some examples, all N optical cavities may be arranged in a ring / loop configuration. In other examples, 1 <M<2 optical cavities of the N optical cavities may be arranged in a linear configuration, while N-M optical cavities are arranged in a ring / loop configuration.

[0169] Figure 14 illustrates an optical device 8000 according to the present invention. Optical device 8000 comprises an optical circuit 8100 along with further optical elements 8200. Optical circuit 8100 may comprise any of the optical circuits described above. Optical elements 8200 direct the light pulses generated by optical circuit 8100 to illuminate a sample 8500 on which coherent Raman scattering is being carried out. The scattering from the sample is filtered by a short-pass, bandpass or long-pass filter 8600 before entering a multichannel dispersive detector 8700 (i.e. spectrometer, multi-channel lock-in amplifier).

[0170] The optical elements 8200 of the arrangement or setup illustrated in Figure 14 includes collimators 8300a, 8300b. The optical circuit 8100 outputs two filtered light pulses each through a collimator. Thus, the two filtered light pulses are collimated in their respective collimators 8300a, 8300b, in order to limit the divergence of the beams of light pulses when they are combined in a subsequent combination step by dichroic mirror 8400a. In some cases, where it is necessary to achieve an overlap on a sample 8500, one of the two collimators 8300a, 8300b may be placed on a delay stage 8300c. Alternatively or additional, a delay stage 8300c may be provided before the collimators 8300a, 8300b.

[0171] The optical elements 8200 of the arrangement or setup illustrated in Figure 14 also includes a dichroic mirror 8400a. A dichroic mirror is a mirror with different reflection and transmission properties at different wavelengths. The two collimated light pulses from the different cavities are combined using the dichroic mirror 8400a. They are then focused into the sample 8500. A short-pass or bandpass or long-pass filter 8600 and then a multichannel dispersive detector 8700 are located downstream of the sample. In CARS detection configuration, the pump and Stokes light pulses from the sample are removed using the short-pass filter 8600. A short-pass filter is a filter with a very sharp transition from transmission to reflection. The resulting CARS spectrum is measured at the spectrometer 8700. In SRS configuration (either SRG or SRL), the pump (SRG) or Stokes (SRL) light pulses are removed after the sample with a long-pass (SRG) or short-pass (SRL) optical filter 8600. The resulting SRG or SRL spectrum is measured at the multichannel lock-in amplifier 8700. Embodiments of the present invention have been described. It will be appreciated that variations and modifications may be made to the described embodiments within the scope of the present invention.

Claims

CLAIMS1 . An optical imaging system for multiplex stimulated Raman scattering (SRS) microscopy, the system comprising: a passively synchronized multi-output amplified fiber laser source configured to generate a plurality of synchronised beams having different wavelength ranges; a laser scanning microscope comprising an objective configured to receive and direct the plurality of beams towards a sample plane; and a detection system configured to receive a plurality of optical signals from the sample plane for multiplex SRS, the detection system comprising a multi-channel lock-in amplifier comprising a channel for each optical signal, each channel comprising at least one demodulator unit and a fixed time constant low-pass filter.

2. The optical imaging system according to any preceding claim, wherein the passively synchronized multi-output amplified fiber laser source comprises a first broadband optical output and a first narrowband optical output.

3. The optical imaging system according to claim 2, wherein the passively synchronized multi-output amplified fiber laser source further comprises a second narrowband optical output, the second narrowband output being configure to emit a beam at a different wavelength range to the first narrowband optical output.

4. The optical imaging system according to any preceding claim, wherein the passively synchronized multi-output amplified fiber laser source emits three synchronized outputs centred at the following wavelength ranges: 750-850 nm, 1000-1100 nm, 910-950 nm and1800-1900nm.

5. The optical imaging system according to any preceding claim, wherein the synchronized multi-output amplified fiber laser source comprises at least one optical output which is amplitude modulated at a frequency lower than the native repetition rate of the oscillators.

6. The optical imaging system according to any preceding claim, wherein the passively synchronized multi-output amplified fiber laser source comprises an optical circuit, the optical circuit comprising: a first polarization-maintaining optical cavity comprising:a first gain medium excitable by a first pump light source to generate light at a first range of wavelengths; and a first saturable absorber configured to carry out passive mode locking of light pulses in the first polarization-maintaining optical cavity; and a second polarization-maintaining optical cavity comprising: a second gain medium different to the first gain medium excitable by a second pump light source to generate light at a second range of wavelengths; and a second saturable absorber configured to carry out passive mode locking of light pulses in the second polarization-maintaining optical cavity, wherein the first polarization-maintaining optical cavity and the second polarizationmaintaining optical cavity share a common branch, and wherein the common branch does not include a saturable absorber.

7. The optical imaging system according to any preceding claim, wherein the laser scanning microscope comprises a beam scanning unit and collection optics.

8. The optical imaging system according to claim 7, wherein the beam scanning unit comprises a single axis or dual axis galvo mirror and an optical relay system between the galvo mirror and an objective back focal plane.

9. The optical imaging system according to any preceding claim, wherein the optical imaging system does not comprise an active electro-optical descanning system situated between the sample plane and the detection system.

10. The optical imaging system according to any one of claims 1 - 8, wherein the optical imaging system comprises a descanning unit.11 . The optical imaging system according to claim 10, wherein the descanning unit comprises a single axis or dual axis galvo mirror and an optical relay system positioned between the collection optics and the galvo mirror.

12. The optical imaging system according to any preceding claim, wherein the multichannel lock-in amplifier is located downstream from the collection optics.

13. The optical imaging system according to any preceding claim, further comprising an SHG and TPEF detection system.

14. The use of the optical imaging system according to any preceding claim for histopathology.

15. A method of analysing a sample using a multi-modal non-linear imaging system, the method comprising: generating a plurality of passively synchronised beams having different wavelength ranges; directing the plurality of beams through a sample plane in a laser scanning microscope; simultaneously receiving, using a multi-channel optical detector, a plurality of optical signals from the sample plane for histopathological analysis.

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