Photothermal microscopy system and a method of photothermal microscopy for analysing a sample

Abstract

The present invention proposes a photothermal microscopy system (1) for analysing a sample, comprising a pulse light source system (10) configured for providing a first train of optical pulses (B1) at first optical center wavelengths (λ1) and a second train of optical pulses (B2) at second optical center wavelengths (λ2) in a burst mode, wherein the microscopy system is a stimulated Raman photothermal microscopy system with a Stokes beam and a pump beam. A probe light source (30) provides a probe beam (P) at a third wavelength (λ3), the third wavelength different from the first and second wavelengths. The system comprises focusing optics (40) configured to direct the first train of optical pulses (B1), the second train of optical pulses (B2) and the probe beam (P) to a sample in a focal volume (5), an optical detector (60) configured to detect a transmitted or reflected probe beam (P1) after interaction in the sample in the focal volume, and a processor (80) configured to process signal components from the detected transmitted or reflected probe beam (P1) and to output a signal or a pixel representative of the interaction in the sample.

Description

Title: Photothermal microscopy system and a method of photothermal microscopy for analysing a sampleDescription

[0001] The present invention claims priority of the Luxembourg patent application LU509071 filed 26 November 2024, the entire content being incorporated therein by reference.TECHNICAL FIELD

[0002] The present invention relates to imaging systems. In particular, the present invention relates to a photothermal microscopy system and a method of photothermal microscopy for analysing a sample.PRIOR ART

[0003] Over the last decades, microscopy imaging systems and improved analytical methods have enabled more and more extensive characterization of samples of all kinds and have contributed to a deep understanding of e.g. the structures and reaction mechanisms of biological systems. Among the most commonly used analytical methods are spectroscopic methods, which are, at least in principle, capable of resolving even larger structures and reaction processes in samples in real time with a spatial resolution in the sub-micrometer range.

[0004] Raman spectroscopy is based on Raman scattering, which results from energy exchange between an incident photon and the molecular vibrational or rotational level. Since every molecule has unique energy levels, the frequency of scattered light from a molecule can describe the structural characteristics of its chemical bonds.

[0005] Laser-based methods such as nonlinear coherent Raman spectroscopy (CRS) can image behaviors of molecules in their chemical environment without additional staining.

[0006] Stimulated Raman spectroscopy (SRS) has been proposed to enhance the sensitivity of Raman spectroscopy and dynamic processes of biological samples. Existing setups forSRS microscopy rely on the active modulation of a property of one of the illumination beams by a modulation system.

[0007] For example, a Stimulated Raman spectroscopy system has been described in the patent publication EP 2 157 415 Al. Two synchronized incoming laser beams, respectively a pump laser beam at a pump frequency and a Stoke laser beam at a Stokes frequency excite a sample. When the difference frequency of the two laser beams matches the vibration frequency of the molecule of the sample, the molecule population is transferred from the ground state through a virtual state to the vibrationally excited state. Due to the coherent excitation of molecular vibration, a pump photon is absorbed by the sample, and a Stokes photon is generated, which results in a loss (SRL) and gain (SRG), resulting in intensity changes in the pump and Stokes beams. As a result of stimulated excitation of the molecular transition, the molecular transition frequency is enhanced in contrast to that in spontaneous Raman.

[0008] Stimulated Raman photothermal microscopy has recently been proposed as a more sensitive sensing technique with a simpler setup than classical SRS microscopy. By introducing a third, so-called probe beam to sense local heating induced by Raman scattering of the material under study, similar to optical photothermal infrared (O-PTIR) microscopy, the need for high-numerical apertures condensers and low-noise pulsed excitation lasers can be mitigated.

[0009] In other words, Stimulated Raman photothermal microscopy is thermal lensing microscopy in which the refractive index modulation is induced by stimulated Raman scattering.

[0010] While stimulated Raman photothermal microscopy allows for a simpler setup than traditional stimulated Raman Scattering microscopy, the best signals are achieved with bulky tabletop laser systems like free-space optical parametric oscillator or low-repetition rate optical parametric amplifiers. For clinical adaptation, such as virtual H&E imaging for pathology or biomarker qualification, a microscope is needed that combines ease of setup, strong signal strength, and robust, mobile fiber laser systems.

[0011] Stimulated Raman spectroscopy intensity is proportional to the product of pump and Stokes peak power. Higher peak power leads to increased energy deposition and local heating, detectable by the probe beam. However, the average power is typically limited to a few tens of milliwatts for biological samples in SRS.

[0012] Tunable fiber lasers are widely used in SRS microscopy due to their costeffectiveness and compactness.

[0013] US patent application US 20240255429A1 discloses a Stimulated Raman photothermal microscopy system in which a classical 80 MHz SRS laser with >1W average power was used. In this system, it has been proposed a reduction in duty cycle of the laser to 5-10% to produce higher peak power at the same average power. This reduction in duty cycle results in a pulse train with a reduced number of optical pulses while maintaining an average power in the tens of milliwatt range, increasing peak power at typical single pulse energies of 50-100 nanojoule.

[0014] It is one object of the present invention to propose a Stimulated Raman photothermal microscopy system which is robust and can be simple to integrate.SUMMARY OF THE INVENTION

[0015] To this aim, the present invention proposes a photothermal microscopy system for analysing a sample according to claim 1, comprising a pulse light source system configured for providing a first train of optical pulses at first optical center wavelengths and a second train of optical pulses at second optical center wavelengths in a burst mode, and a light source for providing a probe beam at a third wavelength, the third wavelength different from the first and second wavelengths. Focusing optics is configured to direct the first train of optical pulses, the second train of optical pulses and the probe beam to a sample in a focal volume. An optical detector is configured to detect a transmitted or reflected probe beam after interaction in the sample in the focal volume, and a processor configured to process signal components from the detected transmitted or reflected probe beam and to output a signal or a pixel representative of the interaction in the sample.

[0016] The microscopy system is a stimulated Raman photothermal microscopy system with a Stokes beam and a pump beam, wherein one of the first train of optical pulses and the second train of optical pulses is the Stokes beam and the other one of the first train of optical pulses and the second train of optical pulses is the pump beam.

[0017] The first train of optical pulses comprises bursts of pulses with burst parameters, the burst parameters including a first burst duration taul, a first burst separation T1 and a first number of pulses in a burst Nl, wherein the second train of optical pulses comprisesbursts of pulses with burst parameters, the burst parameters including a second burst duration tau2, a second burst separation T2 and a second number of pulses in a burst N2. A first and second temporal pulse spacing dtl, dt2 less than 10ns. The first and second number of pulses in a burst Nl, N2 is greater than 100, and / or the first and second burst duration shorter than 2ps

[0018] In other words, the present invention proposes a thermal lensing microscopy system in which the refractive index modulation is induced by stimulated Raman scattering.

[0019] In typical tunable fiber laser systems, single pulse energy is limited by nonlinear effects to around 5 nJ. Thus, reducing the duty cycle and therewith the number of pulses while maintaining high average power is challenging for creating a strong heating effect. In the prior art such as in US 2024 / 255429 cited above, it is proposed to use a 80 MHz SRS laser with an average power less than 1 W together with AOM-gated pulses to reduce the duty cycle to 5-10 %, with the aim of increasing the peak power at the same average power.

[0020] Fiber lasers lack the average power required to maintain tens of milliwatts when reducing the duty cycle to 5-10%.

[0021] The present invention proposes a counterintuitive approach of increasing the repetition rate in burst mode, instead of reducing the number of pulses. This approach proposes stacking more pulses at lower peak power in a shorter time to obtain high peak power for useful photothermal signal generation. The total available average power of the laser source can be utilized for heating the sample before thermal diffusion counteracts the process, typically within Ips. The thermal capacity of the sample acts as a signal reservoir that can be filled with multiple low energy pulses thus allowing to harness high repetition rate short pulse bursts for efficient signal generation.

[0022] The burst mode can be defined as a type of train of pulses with pulse bursts. A burst has a burst duration tau and a number of pulses N in the burst leading to a time separation dt of dt = tau / (N-l) between pulses in the burst. Two bursts are separated by a burst separation time T. Usually the burst separation time T is much greater than the burst duration tau, eg. T is more than 5*tau.

[0023] With a single pulse energy E, a train of pulses in burst mode has an average power P of P=N*E*T.

[0024] To achieve better Signal-to-Noise Ratio (SNR), it is therefore proposed to define a new, thermally confined regime with the burst parameters of temporal pulse spacing dtl, dt2 less than 10ns, and a number of pulses in a burst Nl, N2 is greater than 100, and / or the first and second burst duration shorter than 2ps. The burst parameters lead to a concentration of excitation power into a single heating event per pixel, rather than to a distribution of the same power across multiple events as done in the prior art. This results in a stronger, more detectable signal, efficiently processed using matched filtering. With the burst parameters, the effective repetition rate is increased in burst mode, so that many low-energy pulses are stacked in a short burst, with dt < 10 ns, N > 100, T < 2 ps. Energy is deposited before thermal diffusion cancels the heating.

[0025] The first and second temporal pulse spacings dtl, dt2 can be less than 6.25ns

[0026] In known Stimulated Raman Spectroscopy systems, it is not possible to increase the repetition rate in widely tunable fiber laser systems for SRS to above 100 MHz. It is therefore not possible to extract bursts of pulses from available fiber lasers with dt<10ns or N>200 in a burst having a duration tau of 2ps. At repetition frequencies greater than 100 MHz, very short cavity lengths are required, which typically cannot accommodate the necessary components for widely tunable fiber lasers. While active mode-locking at harmonics of the repetition frequency has been shown, these systems suffer from higher noise and long pulses that are not optimized for SRS.

[0027] The present document therefore also proposes a light source system for providing the pump beam and the Stokes beam in a burst mode. The light source system allows reconfiguring burst parameters. Burst parameters include burst length or duration tau, burst separation T, and number of pulses N in a burst, also called pulse range.

[0028] The light source system can be a fibre based light source, allowing to realise a thermally confined burst of many low-energy pulses using fiber lasers. Conventional light source system such as conventional dual-output free-space laser cannot simply be driven to such effective repetition rates.

[0029] In one aspect, the bursts parameters are reconfigurable using a modulator. The modulator can be an acousto-optic modulator or an electro-optic modulator. In another aspect, the bursts parameters are further reconfigurable using a repetition rate multiplier, in addition to the modulator.

[0030] Using an oscillator at a lower repetition frequency and employing external repetition frequency multiplication can circumvent the above issues of higher noise and long pulses. In addition, by utilizing the same pulse train as the source for both the pump and Stokes beams, the proposed light source system avoids signal degradation from imperfect repetition rate multiplication and timing mismatches.

[0031] In an aspect, the light source system comprises a light source followed by a modulator followed by a repetition rate multiplier, leading to a first arm and a second arm, wherein the output repetition rate multiplier is a seed burst Ts, which can be passed into the first arm and the second arm to generate the first and second trains of optical pulses with reconfigurable burst parameters.

[0032] In another aspect, the light source system can utilize a light source with a limited tuning range covering only part of the Raman spectrum (e.g. 2800 / cm to 3200 / cm) but with a higher repetition rate followed by a modulator. In this aspect a repetition rate multiplier is not necessary. This leads to a less complex light source system.

[0033] The burst length and burst separation can be adjusted via the Acousto-Optic Modulator for each pixel dwell time. Depending on the sample type and signal strength, the required pixel dwell time can range from 5 ps to 20 ps, leading to a pulse range between 100 and 400 pulses per burst.

[0034] If the burst duration tau exceeds the thermal confinement time (depending on the sample, for biological samples this can be e.g., 2 ps), the signal strength decreases.

[0035] A lower limit for the repetition rate multiplier ratio RR can be set to maintain thermal confinement, using the following relationship:Tconf > (Nmax-1) * dt, with dt = RR / f_oscillator with Tconf being the thermal confinement time, Nmax the number of optical pulses per burst, f oscillator being the oscillator repetition rate, dt being the time separation of two pulses in the burst.

[0036] Hence, the repetition rate multiplier ratio RR fulfills the following requirements:RR > (Nmax -1) / f_oscillator*Tconf

[0037] For example, with a 40 MHz oscillator frequency and a pulse range of 100 and 400 pulses per burst, the repetition rate multiplier ratio is set higher than five. A repetition rate multiplier having three cascaded fiber interferometers can have a repetition rate of 8.

[0038] In one aspect, the light source is adapted to create a source pulse train at a repetition rate frep, passed to the modulator, wherein the modulator is adapted to transform the source pulse train in an initial burst of optical pulses, and wherein the repetition rate multiplier is provided to receive the initial burst of optical pulses, and to output a seed burst of seed pulses Bs with N pulses per burst and a burst duration tau = 1 / frep * N.

[0039] The initial burst B0 can comprise an initial number of pulses N AOM = N / RR with a burst separation and with RR being the the repetition rate multiplier ratio, i.e. the rate of multiplication of the repetition rate multiplier, and N being the number of pulses in the first and second trains of optical pulses.

[0040] It is possible to reconfigure the repetition rate multiplication using optional interferometer elements, which are integrated or removed via fiber connectors within the repetition rate multiplier.

[0041] In an aspect, the first arm of the light source comprises a first amplifier followed by a fiber optical parametric oscillator, wherein the seed burst is amplified in said first amplifier and coupled as a pump source into the fiber optical parametric oscillator to generate the first train of optical pulses.

[0042] The fiber optical parametric oscillator has a length L set to be a multiple k of the inverse of the first burst separation Tl, hence L=k / Tl.

[0043] The repetition rate multiplier can be a passive component, in particular an extra cavity repetition rate multiplier such as a chain of interferometers in cascade, preferably a fiber Mach Zehnder interferometer.

[0044] With the present light source system, bursts parameters can be adjusted by changing the settings of the acousto-optic modulator and / or of the repetition rate multiplier.

[0045] The light source system can comprise on a fiber laser based e of: a mode-locked ytterbium pump laser and a fiber based optical parametric oscillator, an erbium doped fiber laser, a nonlinear fiber and an ytterbium doped amplifier system, and a synchronized erbium doped fiber laser and an ytterbium doped fiber laser with a shared element in one or both of their optical cavities.

[0046] A tunable fiber based laser light source system provides the thermally confined burst regime in a robust implementation.

[0047] In one aspect, the optical detector is a detector configured to detect the transmitted or reflected probe beam and to block the transmitted or reflected first and second train of optical pulses. This improves signal detection of the probe beam only.

[0048] A blocking module can be provided between the focal volume and the optical detector, the blocking module being configured to block transmitted or reflected first and second trains of optical pulses, said transmitted or reflected first and second train of optical pulses being transmitted or reflected through the common focal volume.

[0049] The present document also proposes a method of microscopy analysing of a sample, comprising generating a first train of optical pulses at first optical center wavelengths and a second train of optical pulses at second optical center wavelengths, generating a probe beam at third optical center wavelength, directing the first train of optical pulses and second train of optical pulses and the third optical center wavelength to a sample in a focal volume, collecting a signal transmitted or reflected through the focal volume after interaction with the sample in the focal volume, comprising a transmitted or reflected first and second train of optical pulses and a transmitted or reflected probe beam, optically detecting the transmitted or reflected probe beam, and processing the transmitted or reflected probe beam to output a signal or a pixel representative of the interaction in the sample.

[0050] The first train of optical pulses comprises bursts of pulses with burst parameters, the burst parameters including a first burst duration taul, a first burst separation T1 and a first number of pulses in a burst Nl, wherein the second train of optical pulses comprises bursts of pulses with burst parameters, the burst parameters including a second burst duration tau2, a second burst separation T2 and a second number of pulses in a burst N2. A first and second temporal pulse spacing dtl, dt2 less than 10ns. The first and second number of pulses in a burst Nl, N2 is greater than 100, and / or the first and second burst duration shorter than 2ps.

[0051] The present method defines a new, thermally confined regime with the burst parameters of temporal pulse spacing dtl, dt2 less than 10ns, and a number of pulses in a burst Nl, N2 is greater than 100, and / or the first and second burst duration shorter than 2ps.

[0052] The proposed stimulated Raman photothermal microscopy system is therefore simple, low-maintenance and robust, which makes it suitable for mass production.DESCRIPTION OF THE DRAWINGS

[0052] Other characteristics and advantages of the invention will be more clearly evident upon reading the description of several currently preferred embodiments, provided as examples only, with reference to the attached drawings, wherein:

[0053] Figure 1 shows a microscopy system according to the present disclosure,

[0054] Figure 2 shows an embodiment of the microscopy system according to the present disclosure,

[0055] Figures 3 to 5 shows a light source system according to different aspects of the present disclosure,

[0056] Figure 6 shows a method according to the present disclosure,

[0057] Figure 7 shows a view of a spectrum of a first and second trains of optical pulses,

[0058] Figure 8 show a view of a spectrum of a first and second trains of optical pulses according to an aspect of the present disclosure,

[0059] Figure 9 show according to an aspect of the present disclosure,

[0060] Figure 10 shows a light source system according to the present disclosure,

[0061] Figure 11 shows a pulse repetition rate multiplier usable with the light source system of Figure 10 according to an aspect of the present disclosure,

[0062] Figure 12 shows a generation of bursts using the light source system of Figure 10,

[0063] Figure 13 shows how bursts parameters can be adjusted in the light source system of Figure 10 in an aspect,

[0064] Figure 14 shows how bursts parameters can be adjusted in the light source system of Figure 10 in an aspect,

[0065] Figure 15 shows the output from the light source system of Figure 10

[0066] Fig. 16 shows optical spectra of the tunable light source system of Figure 3 in burst mode.DETAILED DESCRIPTION

[0067] In the figures, identical parts are identified using the same reference numbers.

[0068] Figure 1 shows a microscopy system 1 according to this disclosure. The microscopy system 1 is provided for analysing a sample placed in a common focal volume 5, and Figure 2 shows an embodiment of the microscopy system.

[0069] The microscopy system 1 comprises a light source system 10, a probe light source 30, focusing optics 40, an optical detector 60, and a processor 80. The different components of the microscopy system and their arrangement will now be explained in detail in the following.

[0070] The light source system 10 comprises a light source 15 configured for providing a first train of optical pulses Bl at first optical centre wavelengths XI and a second train B2 of optical pulses at second optical centre wavelengths X2.

[0071] The microscopy system 1 is a Raman spectroscopy imaging system with a pump laser beam at a pump frequency and a Stoke laser beam at a Stokes frequency to excite a sample when their energy difference lies in Raman spectroscopy bands of the sample. Hence, one of the first optical centre wavelengths XI or second optical centre wavelengths X2 corresponds to the Stokes frequency, and the other one of the first optical centre wavelengths XI or second optical centre wavelengths X2 corresponds to the pump frequency.

[0072] Usually, the Stokes wavelength is in the range of 1020-1060 nm, and the pump wavelength in the range of 780-980 nm.

[0073] The first train of optical pulses Bl comprises bursts of pulses with a first burst duration taul and a first burst separation of Tl, and a first number of pulses in the burst of Nl. The second train of optical pulses B2 comprises bursts of pulses with a second burst duration tau2 and a second burst separation of T2, and a second number of pulses in the burst of N2.

[0074] The first train of optical pulses Bl and the second train of optical pulses B2 are provided respectively with a first burst duration taul and a first number of pulses in the burst Nl and a second burst duration tau2 and a second number of pulses in the burst N2

[0075] Preferably, the first and second burst durations taul, tau 2 are equal, the first and second burst separations Tl, T2 are equal to achieve synchronisation.

[0076] As will be explained later, the temporal pulse spacing dtl = taul / (Nl-l) and dt2 = tau2 / (N2-l) is preferably less than 10ns or less than 6.25ns, , with a number of pulses in a burst of Nl, N2 greater than 100 and / or a burst duration shorter than 2ps.

[0077] Either the first optical centre wavelengths XI or the second optical centre wavelengths X2 or both are tuneable, with their energy difference o being tuneable, preferably over the Raman spectroscopy band.

[0078] The light source can comprise a dual frequency laser system, with an oscillator and a preamplifier, allowing the synchronisation of the first and second trains of optical pulses.

[0079] An example of light source system 10 is illustrated on Figure 3, showing a fiberbased laser system. The light source 10 may comprise a mode-locked ytterbium pump laser 17, a modulator 416 and a fiber based optical parametric oscillator 16. A fiber based optical parametric oscillator laser provides a light source tuneable over the entire Raman spectroscopy band, with high peak power pulses.

[0080] In another example shown on Figure 4, the light source system 10 comprises an erbium doped fiber laser 216, a modulator 416, a nonlinear fiber 217 and an ytterbium doped amplifier system 218. In this example, the light source system is tuneable over a limited part of the Raman spectroscopy band the CH_2 and CH_3 stretch vibration bands. On the other hand, the light source system is less complex.

[0081] In yet another example shown on Figure 5, the light source system 10 can comprise a synchronized erbium doped fiber laser 316 and an ytterbium doped fiber laser 318 with a shared element 317 in their optical cavities allowing for the synchronisation of the first and second trains of pulses Bl, B2. As illustrated in Fig. 5, the shared element 317 can be either be present in both optical cavities or only in one optical cavity leading to a master and slave laser configuration. Preferably the shared element is either a saturable absorber element or a fiber optical element with an intensity dependent refractive index for synchronization via cross-phase modulation. In this example, the light source is tuneable over a limited part of the Raman spectroscopy spectrum, providing a straightforward and easy to implement setup.

[0082] In the light source systems of Figs 3 to 5, the burst parameters, including the number of pulses in the bursts, the burst length and burst separation are set by the oscillation and amplification parameters of the fiber based oscillators as well as by the modulator 416. The modulator 416 can be used to pick the bursts from a common train of optical pulses and can be reconfigured via gate time and separation. In other words, the modulator 16 can act as a burst adjustment module. The modulator 416 can preferably be an acousto-optic modulator but could also be an electro-optic modulator.

[0083] As will be explained later in reference to Figures 10 to 14, the burst parameters can be adjusted by the modulator 416. It is also possible to have a light source system integrating a repetition rate multiplier as well for the generation of the first and second train of optical pulses, in particular for additional adjusting of burst parameters during operation.

[0084] Referring back to the microscopy system 1, in addition to the light source 15, the probe light source 30 is provided to provide a probe beam P at a third wavelength 3, also called probe wavelength. The probe wavelength is different from the first and second wavelengths.

[0085] The probe beam P is provided to sense changes of a local refractive index in the sample resulting from a temperature rise induced by the molecules vibrations due to Stimulated Raman Scattering. The temperature rise in the sample depends, among others, on the power of the first and second train of pulses, i.e. Stokes and pump beams, repetition rate of the first and second train of pulses.

[0086] It should be noted that the changes of the local refractive index propagate to surroundings around the focal center, i.e. at the point hit by the first and second pulse trains in the sample. With other words, a thermal lens is formed in the sample being a thermo- optical material as a result of its inhomogeneous heating.

[0087] The probe beam P is a continuous wave (cw) beam. The probe light source 30 may be for example a single frequency, cw diode pumped laser at 532nm or 660nm.

[0088] As known in thermal lensing, a tunable telescope 32, as shown in Fig. 2, is added to the probe beam to detune the focal position of the probe beam P in front or beyond of the common focus of the pump and Stokes beams Bl, B2.

[0089] The telescope 32 can be for a example a Galilean beam expander in a sliding lens design to minimize walk-off during adjustment.

[0090] Turning back to the microscopy system 1, the focusing optics 40 is provided for directing and focusing the first and second trains of optical pulses Bl, B2 as well as the probe beam P onto the focal volume 5 where the sample can be placed.

[0091] The focusing optics 40 can be adapted to the microscopy requirements and comprise dichroic mirror 41, a galvanometer scanner 42 allowing scanning of the sample in the focal volume, and a set of lenses 43, as well as an objective lens 44, as exemplified in Figure 2.

[0092] An aperture 50 is provided for collecting the signal transmitted or reflected through the common focal volume T4 and to transmit said signal to the detector 60.

[0093] It is noted that the microscopy system is a Burst-pulsed stimulated Raman photothermal microscope, and not a Stimulated Raman microscope. The useful signal is therefore the transmitted or reflected probe beam and not the Raman scattering signal. Therefore, there is no need for a high numerical aperture to collect the light after the sample.

[0094] The aperture can be a condenser 50 with a low numerical aperture, e.g. 0.5 to act as an aperture, but this is an example only. Other apertures can be an iris or a multiwell plate.

[0095] The detector 60 is an optical detector configured to detect the transmitted or reflected probe beam Pl. For example, the optical balanced detector 60 comprises a photodetector, 61 to detect the transmitted or reflected probe beam at the wavelength 3.

[0096] It should be noted that the transmitted or reflected first train of pulses Bl l at the wavelength XI and the transmitted or reflected second train of pulses B21 at the wavelength X2 are not detected by the detector 60.

[0097] It is also possible to provide a blocking filter 55 between the condenser 50 and the detector 60 to filter out the transmitted or reflected first and second trains of optical pulses Ti l, T21.

[0098] The detector 60 can be an optical detector, such as a photodetector optimized for high photocurrents e.g. to detect >10mW laser light.

[0099] Finally, a processor 80 is provided to process said the transmitted or reflected probe beam at the wavelength 13. The processor 80 can detect the modulation transferred to the probe beam at a frequency inversely proportional to the burst separation T1 = T2 e.g. by box-filtering, matched filtering or lock-in detection. The processor 70 can comprise a lock- in amplifier.

[0100] The processor can output a signal or image representative of the heating due to the Stimulated Raman excitation of the sample or a pixel of an image representative of the sample.

[0101] A method of photothermal microscopy imaging will now be described with reference to figure 6, performed using the microscopy system of Fig. 1.

[0102] In a first step SI, a first train of optical pulses Bl at first wavelength XI and a second train of optical pulses B2 at second wavelength X2 are generated. The first and second wavelengths are the Stokes and pump wavelengths.

[0103] In a second step S2, a probe beam P is generated at a third wavelength X3. The probe beam is a continuous beam.

[0104] The first train of optical pulses Bl and the second train of optical pulses B2, as well as the probe beam P are directed and focused by the focusing optics 40 onto the sample in the common focal volume 5 (step S3). The first train of optical pulses T1 and the second train of optical pulses T2 interact in the sample to trigger Raman scattering when the difference of the wavelengths of the two laser beams matches the vibration frequency of the molecule of the sample.

[0105] The working principle of the photothermal microscope 1 is the following: initial pulse pairs of the first and second train of optical pulses Bl, B2 interact in the sample and excite a molecular vibration of the Stimulated Raman scattering. Subsequent pulse pairs of the first and second train of optical pulses Bl, B2 heat the sample, resulting in a thermal lens.

[0106] In addition, the probe beam P transmitted or reflected through the common focal volume to form respectively a transmitted or reflected probe beam Pl to detect a change of optical transmission induced by the thermal lens.

[0107] In the next step S4, the signal transmitted or reflected through the common focal volume is collected by a condenser 50, and then detected by the detector 60. The detector 60 is an optical detector configured to detect the transmitted or reflected probe beam, but not signals at the first or second wavelengths. The detector 60 measures the optical intensity of the transmitted or reflected probe beam Pl.

[0108] It is also possible to filter out the transmitted or reflected first and second train of optical pulses are filtered out with a passband filter 55 provided between the condenser 50 and the detector 60.

[0109] Finally, a processor 80 is provided to process transmitted or reflected probe beam Pl and output an image or a signal representative of a pixel or sample. The processor detects the modulation transferred to the probe beam P at a frequency inversely proportional to the burst separation T1 = T2 e.g. by box-filtering, matched filtering or lock- in detection.

[0110] The photothermal microscope system is constrained by two key limits: the Sample Destruction Threshold and the Laser Pulse Energy for the heating induced by the Stimulated Raman excitation and thermal diffusion.

[0111] The Sample Destruction Threshold (P max) is a limit imposed by the maximum power supported by a biological sample, typically P max around 50mW.

[0112] The Laser Pulse Energy is determined by the light source, in particular fiber laser, used for the generation of the Stokes and pump beams. State of the art lasers have an energy per pulse of typically E_max=2.5 nJ.

[0113] Figure 7 illustrates a state-of-the-art stimulated Raman photothermal (SRP) microscope signal generation. In the top graph, the SRP signal generated due to the sample heating is depicted for a duty cycle of a high-power free space laser system, such as an optical parametric oscillator, of 50% (dashed line) and 25% (solid line) at the same average power on the sample. The bottom graph shows the respective trains of optical pulses Bl, B2. The average power on the sample is limited to e.g. 40mW per beam.

[0114] Typical high-power laser systems are, therefore, attenuated before being sent to the sample. As for a fiber laser the pulse energy cannot be scaled to the higher peak power shown in the 25% duty cycle case, the present invention proposes to utilize short bursts of low pulse energy to deposit energy before thermal relaxation can counteract the process.

[0115] Fig. 8 illustrate a stimulated Raman photothermal microscope signal generation according to the invention. The top graph of Figure 8 shows a generated SRP signal in a first excitation scheme SI with rapid burst pulses according to the invention, compared to the signals generated respectively by a 50% and 25% duty cycle SRP microscope as described in the state of the art in prior art excitation schemes S2 and S3, respectively. The bottom graph depicts the first and second trains of optical pulses Bl, B2 used for excitation for the three excitation schemes SI to S3. All three excitation schemes utilize the same average power on the sample which can be matched to a destruction threshold of same sample.

[0116] The present invention is based on the recognition that concentrating excitation power into a single heating event per pixel, rather than distributing the same power across multiple events, results in a stronger, more detectable signal, with other words to a better Signal-to-Noise Ratio (SNR).

[0117] The first train of optical pulses Bl and second train of optical pulses B2 are therefore provided with burst parameters including with a first burst duration taul, and a first burst separation T1 and a first number of pulses in the burst Nl, for the first train of optical pulses Bl, and a second burst duration tau2 and a second burst separation T2 and a second number of pulses in the burst N2, for the second train of optical pulses B2.

[0118] The temporal pulse spacing dtl = taul / (Nl-l) and dt2 = tau2 / (N2-l) is preferably less than 10ns or less than 6.25ns, with a number of pulses in a burst of Nl, N2 > 100 and / or a burst duration shorter than 2ps.

[0119] This allows excitation allows to deposit the energy on the sample, preferably on a pixel, before thermal relaxation can counteract the process.

[0120] The competing effects of the thermal warming and thermal relaxation are illustrated on Fig. 9 shows a thermal warming by stimulated Raman excitation and competing diffusion effects for an excitation scheme SI as described in this disclosure and for a standard excitation scheme S2 of 50% duty cycle SRP microscope (Temperature T in C vs time t in ps). To illustrate these effects, numerical simulations of the thermal diffusion under optical heating conditions by stimulated Raman excitation were conducted using Finite Difference Time Domain (FDTD) simulations under realistic experimental conditions In the case of the excitation scheme SI, the light source system generates the Stokes and pump beams in a burst mode. In this example, a fiber laser based on a FOPO pumped by a repetition rate multiplied ytterbium oscillator is used with 400 pulses per burst and a burst duration tau of 1.35ps also with 5nJ pulse energy. In the standard excitation scheme S2, the light source is a fiber laser with a repetition rate of 40 MHz and a pulse energy of 5 nJ and 400 pulses in the 50% duty cycle. Both experiments were, therefore, conducted with the same average power and pulse peak power.

[0121] According to the present disclosure, the burst parameters can be set so that a pixel dwell time (pdT) is determined based on a required SNR at each pixel, influencing a goal number of pulses required.

[0122] The number of pulses per burst N is given by the Sample Destruction Threshold P max, the laser pulse energy E max :N=(P_max-pdT) ZE max.

[0123] Depending on the sample type and signal strength, the required pixel dwell time pdT can range from 5 ps to 20 ps, leading to a target number of pulses in a burst lying in the pulse range between 100 and 400 pulses per burst.

[0124] Figure 10 illustrates a fiber-based light source system 410 with reconfigurable burst parameters for the first and second train of optical pulses Bl, B2.

[0125] The light source system 410 comprises a light source 415, such as a dual frequency laser system, with a modulator 416, followed by a repetition rate multiplier 417 connected to a first and a second arms. The output of the repetition rate multiplier 417 is a seed burst Bs, which is then passed into the first and second arms to generate the first and second trains of optical pulses Bl, B2 with reconfigurable burst parameters.

[0126] In this example, the light source 415 is a mode-locked ytterbium laser, adapted to create a source pulse train at a repetition rate frep., passed to the modulator 416.

[0127] The modulator 16 is an acousto-optic modulator , and is provided to transform the source pulse train in an initial burst B0 of optical pulses by picking a first number of pulses N AOM = N / RR with a burst separation and with RR being the rate of multiplication of the repetition rate multiplier and N the number of pulses of the seed burst Bs.

[0128] The initial burst B0 of optical pulses can be configured by adjusting a gate length and gate separation of the acousto-optic modulator 416.

[0129] The initial burst B0 is passed to the repetition rate multiplier 417. After repetition rate multiplication, a seed burst of seed pulses Bs with N pulses and a burst duration tau = 1 / frep * N is created.

[0130] The seed burst Bs is then passed into the first and the second arm of the light source system to generate the first and second trains of optical pulses Bl, B2.

[0131] The first arm comprises a first amplifier 420 followed by a fiber optical parametric oscillator 422. The seed burst Bs is amplified in said first amplifier 420 and coupled as a pump source into the fiber optical parametric oscillator 422 to generate tunable SRP-Pump pulses, here the first train of optical pulses Bl.

[0132] The length of the fiber optical parametric oscillator 422 needs to be a multiple k of the inverse of the burst separation T, hence L=k / T, so that each burst of oscillating SRP-Pump pulses in the fiber optical parametric oscillator is synchronously pumped by a pump burst.

[0133] In the second arm, the seed burst Bs is amplified in an amplifier 430 to create the second train of optical pulses B2, here the Stokes beam. The amplifier 430 can be an Ytterbium doped amplifier.

[0134] The generated first train of optical pulses Bl and the second train of optical pulses B2 are characterized by bursts parameters which can be adjusted using the repetition rate multiplier 417 and the acousto-optic modulator 416. The burst parameters include the first and second burst duration taul, tau2, the first and second burst separation Tl, T2, and the first and second number of pulses in the burst Nl, N2, respectively.

[0135] Preferably, the first burst duration taul and the second burst duration tau2 are equal, the first burst separation Tl is equal to the second burst separation T2.

[0136] As mentioned before, the number of pulses in a burst Nl, N2 is greater than 100 and / or a burst duration shorter than 2ps.

[0137] If the burst duration exceeds the thermal confinement time depending on the sample, for biological samples this can be e.g., 2 ps, the signal strength decreases.

[0138] Given the pixel dwell time and the maximum number of pulses N max, a lower bound for the repetition rate multiplier ratio RR to maintain thermal confinement can be set based on the oscillator repetition rate f oscillator and the time separation between two pulses in the burst dt.

[0139] The lower limit for the repetition rate multiplier ratio RR can be set to maintain thermal confinement, using the following relationship:Tconf > (Nmax-1) * dt, with dt = RR / f_oscillator

[0140] with Tconf being the thermal confinement time, Nmax the number of optical pulses per burst, f oscillator being the oscillator repetition rate, dt being the time separation of two pulses in the burst

[0141] The lower limit for the repetition rate multiplier ratio RR is therefore set :RR > (Nmax -1) / f_oscillator*Tconf

[0142] For example, with a 40 MHz oscillator frequency and a number of pulses comprised between 100 and 400, the repetition rate multiplier ratio should be set greater than 5.

[0143] Using an oscillator at a lower repetition frequency and employing external repetition frequency multiplication allows providing optical train with high repetition rate, whilst ensuring synchronisation by utilizing the same pulse train as the source for both the pump and Stokes beams.

[0144] An example of repetition rate multiplier 417 is shown in figure 11. The repetition rate multiplier 417 has 3 cascaded fiber interferometers. The repetition ratemultiplier 417 can achieve a repetition rate multiplier ratio of 8. The repetition rate multiplier can be reconfigured using optional interferometer elements, which are integrated or removed via fiber connectors within the repetition rate multiplier, to change the repetition rate multiplier ratio.

[0145] Figure 12 shows a generation of bursts using the light source of Figure 10, to illustrate how the output from a mode-locked fiber oscillator is transformed into bursts of output pulses using an Acousto-Optic Modulator (AOM) and a Repetition Rate (RR) multiplier.

[0146] In the upper graph, a 40 MHz fiber oscillator output is fed into the AOM 416. The AOM gating window is depicted by the dashed curve. The middle graph shows the resulting output from the AOM, here three bursts, which are then directed into the RR multiplier. The bottom Graph displays the final output from the repetition rate multiplier with a repetition rate multiplier ratio of 4.

[0147] The skilled person understands that the AOM and / or the repetition rate multiplier can be reconfigured to adjust the burst parameters.

[0148] Figure 13 shows how bursts parameters can be adjusted in the light source system of Figure 10. In this aspect, the bursts can be reconfigured using the acousto-optic modulator settings while keeping the repetition rate multiplier unchanged.

[0149] The top graph illustrates the combined output of the acousto-optic modulator 416 and repetition rate multiplier 417, showing the resulting pulse burst alongside the initial AOM gate window.

[0150] The middle graph shows that the burst separation can be adjusted by modifying the gate separation of the acousto-optic modulator 416. Adjusting the burst separation allows control over the timing between bursts.

[0151] The bottom graph shows that the number of output pulses can be increased by increasing the gate length, which simultaneously extends the burst duration.

[0152] In other words, burst parameters can be adjusted by adjusting the gate length and gate separation of the acousto-optic modulator 416.

[0153] Figure 14 shows how bursts parameters can be adjusted in the light source system of Figure 10. In this aspect, the pulse bursts can be reconfigured using the repetition rate multiplier while keeping acousto-optic modulator settings unchanged.

[0154] This figure shows how altering the RR multiplier can change the output pulse bursts without modifying the AOM parameters.

[0155] The top graph illustrates the combined output of the acousto-optic modulator 416 and repetition rate multiplier 417 with RR=4, showing the resulting pulse burst alongside the initial AOM gate window.

[0156] The bottom graph illustrates the bursts when the repetition rate multiplication ratio is changed from RR=4 to RR=2. Decreasing the adjustment repetition rate multiplication ratio decreases the number of pulses while keeping the burst duration the same.

[0157] Therefore, in the light source system of Figures 3 to 5, the modulator acts as a burst adjustment module, whilst in the example of Figure 10, the burst adjustment module 411 comprises both the modulator 416 and the repetition rate multiplier 417.

[0158] Fig. 15 shows a measured output from the light source system of Figure 10 recorded with a photodiode. The measured photocurrent at the FOPO output shows a burst Bl with Nl=120 optical pulses and a time separation between two pulses in the burst dt of 6.2ns.

[0159] Fig. 16 shows an exemplarily recorded optical spectra of the widely tunable light source system in burst mode, such as the light source system of Fig.3. The optical spectra corresponds to the source system’s center wavelengths tuned to 762, 858, 935 and 993nm, respectively, covering the Raman bands from 600-3500 / cm.

[0160] The foregoing description of the preferred embodiments of the disclosure has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention. The embodiment was chosen and described in order to explain the principles of the invention and its practical application to enable one skilled in the art to utilize the invention in various embodiments as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.List of reference numbers:Microscopy system 1light source system 10 light source 15 first train of optical pulses Bl at first optical center wavelengths 11 second train of optical pulses B2 at second optical center wavelengths 12. Probe beam P transmitted or reflected first train of optical pulses Bl 1 transmitted or reflected first second train of optical pulses B21 transmitted or reflected probe beam Pl probe light source 30 coupler 32 amplification stage 38 focusing optics 40, dichroic mirror 41, galvanometer scanner 42, set of lenses 43, objective lens 44, numerical aperture 50, e.g. condenser optical detector 60, processor 80 mode-locked ytterbium pump laser 17, fiber based optical parametric oscillator 16 erbium doped fiber laser 216 nonlinear fiber 217 ytterbium doped amplifier system 218 synchronized erbium doped fiber laser 316 ytterbium doped fiber laser 318 shared element 317 mode-locked ytterbium laser (light source) 415 modulator 416 repetition rate multiplier 417

Claims

-22-Claims1. A photothermal microscopy system (1) for analysing a sample, comprising a pulse light source system (10) configured for providing a first train of optical pulses (Bl) at first optical center wavelengths (XI) and a second train of optical pulses (B2) at second optical center wavelengths (X2) in a burst mode, wherein the microscopy system is a stimulated Raman photothermal microscopy system with a Stokes beam and a pump beam, wherein one of the first train of optical pulses (Bl) and the second train of optical pulses (B2) is the Stokes beam and the other one of the first train of optical pulses (Bl) and the second train of optical pulses (B2) is the pump beam, a probe light source (30) for providing a probe beam (P) at a third wavelength (X3), the third wavelength different from the first and second wavelengths focusing optics (40) configured to direct the first train of optical pulses (Bl), the second train of optical pulses (B2) and the probe beam (P) to a sample in a focal volume (5), an optical detector (60) configured to detect a transmitted or reflected probe beam (Pl) after interaction in the sample in the focal volume, and a processor (80) configured to process signal components from the detected transmitted or reflected probe beam (Pl) and to output a signal or a pixel representative of the interaction in the sample, wherein the first train of optical pulses comprises bursts of pulses with burst parameters, the burst parameters including a first burst duration taul, a first burst separation T1 and a first number of pulses in a burst Nl, wherein the second train of optical pulses comprises bursts of pulses with burst parameters, the burst parameters including a second burst duration tau2, a second burst separation T2 and a second number of pulses in a burst N2, and with a first and second temporal pulse spacing dtl, dt2 less than 10ns, and wherein the first and second number of pulses in a burst Nl, N2 is greater than 100, and / or the first and second burst duration shorter than 2ps.

2. The photothermal microscopy system (1) according to claim 1, wherein the first and second temporal pulse spacing dtl, dt2 is less than 6.25ns.

3. The photothermal microscopy system according to claim 1 to 2, wherein the light source system is a fiber-laser-based light source system with a fiber laser, wherein the fiber-laser-based light source system comprises one of a mode-locked ytterbium pump laser and a fiber based optical parametric oscillator, an erbium doped fiber laser, a nonlinear fiber and an ytterbium doped amplifier system, a synchronized erbium doped fiber laser and an ytterbium doped fiber laser with a shared element in one or both of their optical cavities4. The photothermal microscopy system (1) of any of claims 1 to 3, wherein the optical detector (60) is a detector configured to detect the transmitted or reflected probe beam (Pl) and to block the transmitted or reflected first and second train of optical pulses (Bl 1, B21), in particular wherein a blocking module (55) is provided between the focal volume (5) and the optical detector (60), the blocking module (55) being configured to block transmitted or reflected first and second trains of optical pulses (Bl l, B21), said transmitted or reflected first and second train of optical pulses (Bl l, B21) being transmitted or reflected through the common focal volume.

5. The photothermal microscopy system according to any of claims 1 to 4, wherein an aperture (50) is provided for collecting signal transmitted or reflected through the common focal volume and to transmit said signal to the detector, in particular the aperture (50) being a condenser with a low numerical aperture, e.g. 0.5.

6. The photothermal microscopy system (1) according to any of claims 1 to 5, wherein the first and second trains of optical pulses (Bl, B2) have reconfigurable burst parameters, the light source system comprising a modulator (416) for reconfiguring the burst parameters.

7. The photothermal microscopy system (1) according to claim 6, wherein the light source system further comprises a repetition rate multiplier (417) for further reconfiguring the burst parameters.

8. The photothermal microscopy system (1) according to claim 7, wherein the light source system (400) comprises a light source (415) followed by the modulator (416) followed by the repetition rate multiplier (417), leading to a first arm and a second arm, wherein the output of the repetition rate multiplier (417) is a seed burst (Bs), which can be passed into the first arm and the second arm to generate the first and second trains of optical pulses (Bl, B2) with reconfigurable burst parameters.

9. The photothermal microscopy system (1) according to claim 7 or 8, wherein the light source (415) is adapted to create a source pulse train at a repetition rate frep, passed to the modulator (416), wherein the modulator (416) is adapted to transform the source pulse train in an initial burst (BO) of optical pulses, and wherein the repetition rate multiplier (417) is provided to receive the initial burst of optical pulses (BO), and to output a seed burst of seed pulses (Bs) with N pulses per burst and a burst duration tau = 1 / frep * N.

10. The photothermal microscopy system (1) according to claim 9, wherein the initial burst (BO) comprises an initial number of pulses N AOM = N / RR wherein RR is the the repetition rate multiplier ratio and N the number of pulses in the first and second trains of optical pulses (Bl, B2).

11. The photothermal microscopy system according to any of claims 8 to 10, wherein the first arm of the light source comprises a first amplifier (420) followed by a fiber optical parametric oscillator (422), wherein the seed burst (Bs) is amplified in said first amplifier (420) and coupled as a pump source into the fiber optical parametric oscillator (422) to generate the first train of optical pulses (Bl).

12. The photothermal microscopy system according to claim 11, wherein the fiber optical parametric oscillator (422) has a length L set to be a multiple k of the inverse of the first burst separation Tl, hence L=k / Tl.-25-13. The photothermal microscopy system (1) according to any of claims 7 to 12, wherein the repetition rate multiplier (30) is a passive component, in particular an extra cavity repetition rate multiplier such as a chain of interferometers in cascade, preferably a fiber Mach Zehnder interferometer.

14. The photothermal microscopy system (1) according to any of claims 1 to 13, wherein the processor comprises a lock-in amplifier.

15. A method of microscopy analysing of a sample, comprising generating a first train of optical pulses (Bl) at first optical center wavelengths (XI) and a second train of optical pulses(B2) at second optical center wavelengths (X2), generating a probe beam (P) at third optical center wavelength (X3), wherein the method is a method of microscopy analysing via a stimulated Raman photothermal microscopy with a Stokes beam and a pump beam, wherein one of the first train of optical pulses (Bl) and the second train of optical pulses (B2) is the Stokes beam and the other one of the first train of optical pulses (Bl) and the second train of optical pulses (B2) is the pump beam, configuring burst parameters of the first and second trains of optical pulses (Bl, B2), the burst parameters including a first burst duration taul, a first burst separation T1 and a first number of pulses in a burst N1 of the first train of optical pulses, a second burst duration tau2, a second burst separation T2 and a second number of pulses in a burst N2 of the second train of optical pulses, with a first and second temporal pulse spacing dtl, dt2 less than 10ns, wherein dtl = taul / (Nl-l) and dt2 = tau2 / (N2-l), generating first and second trains of optical pulses (Bl, B2) with a first or second number of pulses in a burst Nl, N2 greater than 100, and / or a first or second burst duration taul, tau 2 shorter than 2ps, directing the first train of optical pulses (Bl), the second train of optical pulses (B2) and the probe beam (P) to a sample in a focal volume, collecting a signal transmitted or reflected through the focal volume after interaction with the sample in the focal volume, comprising a transmitted or-26- reflected first and second train of optical pulses (Bl l, B21) and a transmitted or reflected probe beam (Pl), optically detecting the transmitted or reflected probe beam (Pl), processing the transmitted or reflected probe beam (Pl) to output a signal or a pixel representative of the interaction in the sample16. The method according to claim 15, comprising blocking the transmitted or reflected first and second optical train (Bl l, B21) before or simultaneously to optically detecting the transmitted or reflected probe beam (Pl).

17. The method according to any one of claims 15 to 16, comprising the step of configuring burst parameters of the first and second trains of optical pulses (Bl, B2), with the first and second temporal pulse spacing dtl, dt2 less than 6.25ns, wherein dtl = taul / (Nl-l) and dt2 = tau2 / (N2-l) .

18. The method according to any one of claims 15 to 17, wherein the first and second trains of optical pulses are generated using a fiber-laser-based light source system with a fiber laser, wherein the fiber-laser-based light source system comprises one of a mode-locked ytterbium pump laser and a fiber based optical parametric oscillator, an erbium doped fiber laser, a nonlinear fiber and an ytterbium doped amplifier system, a synchronized erbium doped fiber laser and an ytterbium doped fiber laser with a shared element in one or both of their optical cavities.

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