Microscopy system and a method of microscopy for analysing a sample

Abstract

A microscopy system (1) is provided for imaging a sample. The microscopy system comprises a light source system (10) configured for providing a first train of optical pulses (T1) at first optical center wavelengths (l1) and a second train of optical pulses (T2) at second optical center wavelengths (l2), both at a first repetition frequency (f1). A repetition rate multiplier (30) is configured to receive the first train of optical pulses (T1) and to output a third train of optical pulses (T3) at a second repetition frequency (f2) different from the first repetition frequency (f1), focusing optics (40) configured to direct the second train of optical pulses (T2) and the third train of optical pulses (T3) to a sample in a focal volume (5). The microscopy system comprises an optical detector (60) configured to detect a transmitted or reflected third train of optical pulses (T31) after interaction in the sample in the focal volume, and a processor (80) configured to process signal components at the first repetition frequency (f1) from the detected transmitted or reflected third train of optical pulses (T31) and to output a signal or a pixel representative of the interaction in the sample.

Description

Title: Microscopy system and a method of microscopy for analysing a sampleDescription

[0001] The present invention claims priority to the Luxembourg patent application LU508061 filed 22 August 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 microscopy system and a method of 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 and 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 for SRS 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] A modulator is introduced to allow extracting the SRS signal from background noise while suppressing laser, thermal, and shot noises. One of the excitation beams (typically the Stokes beam) is modulated by an acousto-optic modulator (AOM) or an electro-optic modulator (EOM) with high frequency before entering the microscope. The intensity of the other excitation beam, typically the pump beam, is detected through one or more filters to filter out the modulated light. The SRS signals can be demodulated using a lock-in amplifier based on the modulation function.

[0009] However optic modulator devices are costly and require high-power radio frequency drivers for operation that make integration into imaging systems in environments with high Electromagnetic compatibility (EMC) safety requirements difficult and cost intensive.

[0010] It is one object of the present invention to propose a microscopy system which is robust and can be simple to integrate.SUMMARY OF THE INVENTION

[0011] To this aim, the present invention proposes a microscopy system for analysing a sample according to claim 1. The microscopy system comprises a 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, both at a first repetition frequency, a repetition rate multiplier configured to receive the first train ofoptical pulses and to output a third train of optical pulses at a second repetition frequency different from the first repetition frequency, focusing optics configured to direct the second train of optical pulses and the third train of optical pulses to a sample in a focal volume, a balanced detector configured to detect a transmitted or reflected third train of optical pulses after interaction in the sample in the focal volume, and a processor configured to process signal components at the first repetition frequency from the detected transmitted or reflected third train of optical pulses and to output a signal or a pixel representative of the interaction in the sample. The light source system includes a fiber-based light source system, and can include a mode-locked fiber laser and a fiber optical parametric oscillator (FOPO). The repetition rate multiplier is a fiber-based rate multiplier.

[0012] Fiber based components are robust and easy to handle, and therefore good candidates for microscopy systems on mass production. However, fiber based light source systems are inherently noisy components, due to amplified spontaneous emission especially prominent in fiber based amplifiers. To overcome the noise due to the fiber laser source, the microscopy system combines a fiber-integrated light source with a stable fiberbased repetition rate multiplier and a balanced detector. In other words, instead of using an active modulation system introducing a new frequency component, the present invention proposes removing a frequency component by passive ways and detect the interaction in the sample at the removed frequency.

[0013] The present invention therefore eliminates the need for cumbersome optical modulators whilst still allowing a high sensitive detection.

[0014] In an aspect, the microscopy system is a stimulated Raman 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.

[0015] The output can be the Raman spectroscopy spectrum for the sample or a pixel of an image representative of the sample.

[0016] Usually, the Stokes wavelength is in the range of 1020-1060 nm, and the pump wavelength in the range of 780-980 nm.

[0017] In an aspect, the optical detector is a balanced detector configured to detect the transmitted or reflected the third train of optical pulses and to receive simultaneously areference train of optical pulses, the reference train of optical pulses being a portion of the third train of pulses not transmitted or reflected through the common focal volume.

[0018] A balanced detection allows sensitive SRS signal detection in the presence of imperfect suppression of side-modes. This allows achieving frequency-encoded SRS signal detection, yet without active modulation, and without external synchronization to clean sources. Preferably, the balanced detector has a high extension ratio, i.e. an extinction ratio of at least 40 dB.

[0019] The microscopy system can further comprise a blocking module between the focal volume and the optical detector, the blocking module being configured to block a transmitted or reflected second train of optical pulses, said transmitted or reflected second train of optical pulses being transmitted or reflected through the common focal volume. This allows ensuring that the only frequency components at the first frequency detected by the optical detector are frequency components comprised in the transmitted or reflected third train of optical pulses. Detected frequency components at the first frequency, i.e. the removed frequency, can only result from interaction in the sample of the second train of optical pulses and of the third train of optical pulses.

[0020] In an aspect, the repetition rate multiplier is a passive component and / or wherein the second repetition frequency is a multiple of the first repetition frequency, preferably the double of the first repetition frequency.

[0021] The repetition rate multiplier can be an extra cavity repetition rate multiplier, such as a chain of interferometers in cascade, preferably a fiber Mach Zehnder interferometer.

[0022] A passive repetition-frequency multiplication, in particular fiber based extra-cavity repetition-frequency multiplication is particularly suitable and allows adapting easily the systems depending on the sample and requirements.

[0023] A fiber optic amplification stage configured to amplify the third train of pulses outputted from the frequency remover can be provided

[0024] In an aspect, the processor comprises a lock-in amplifier.

[0025] In an aspect, the 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, or a synchronized erbium doped fiber laser and an ytterbium doped fiber laser with a shared element in one or both of their optical cavities

[0026] In yet another aspect, the light source system integrates both the light source and the repetition rate multiplier.

[0027] The present invention also proposes a method of microscopy analysing of a sample, comprising generating, by an optical source system, a first train of optical pulses at first optical center wavelengths and a second train of optical pulses at second optical center wavelengths, both at a first repetition frequency, manipulating the first train of optical pulses to output a third train of optical pulses at a second repetition frequency different from the first repetition frequency, directing the second train of optical pulses and the third train of optical pulses to a sample in a focal volume, collecting a signal transmitted or reflected through the common focal volume after interaction with the sample in the focal volume, comprising a transmitted or reflected second train of optical pulses and a transmitted or reflected third train of optical pulses, optically detecting the transmitted or reflected third train of optical pulses, processing the transmitted or reflected third train of optical pulses at the frequency first repetition frequency to output a signal or a pixel representative of the interaction in the sample.

[0028] In an aspect, the method comprises blocking the transmitted or reflected second optical train before or simultaneously to optically detecting the transmitted or reflected third train of optical pulses.

[0029] The step of optically detecting can be is performed by a balanced detector receiving a reference signal of the third train of pulses, for sensitive detection of weak SRS signal, in the presence of imperfect suppression of the first frequency in the third train of optical pulses, i.e. imperfect side modes suppression.

[0030] In an aspect, the step of manipulating the first train of optical pulses or the second train of optical pulses comprises passively removing the first frequency from the first train of optical pulses or the second train of optical pulses, preferably using a repetition rate multiplier.

[0031] The proposed microscopy system is therefore simple, low-maintenance and robust, which makes it suitable for mass production.DESCRIPTION OF THE DRAWINGS

[0032] 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:

[0033] Figure 1 shows a microscopy system according to the present disclosure,

[0034] Figure 2 shows an embodiment of the microscopy system according to the present disclosure,

[0035] Figures 3 to 5 shows a light source according to different aspects of the present disclosure,

[0036] Figure 6 shows a pulse repetition rate multiplier according to an aspect of the present disclosure,

[0037] Figure 7 shows a light source integrated with a pulse repetition rate multiplier according to the present disclosure,

[0038] Figure 8 shows a method according to the present disclosure,

[0039] Figure 9 shows a view of a spectrum of a third train of optical pulses,

[0040] Figure 10 show a view of a spectrum of a third train of optical pulses according to an aspect of the present disclosure,

[0041] Figure 11 show a view of a spectrum of a third train of optical pulses according to an aspect of the present disclosure,

[0042] Figure 12 shows an example of measured stimulated Raman scattering signal according to an aspect of the present disclosure.

[0043] DETAILED DESCRIPTION

[0044] In the figures, identical parts are identified using the same reference numbers.

[0045] 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.

[0046] The microscopy system 1 comprises a light source system 10, a repetition rate multiplier 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.

[0047] The light source system 10 comprises a light source 15 configured for providing a first train of optical pulses T1 at first optical centre wavelengths 11 and a second train T2 of optical pulses at second optical centre wavelengths 12.

[0048] The microscopy system 1 is preferably 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 w lies in Raman spectroscopy bands of the sample. Hence, one of the first optical centre wavelengths 11 or second optical centre wavelengths 12 corresponds to the Stokes frequency, and the other one of the first optical centre wavelengths 11 or second optical centre wavelengths 12 corresponds to the pump frequency.

[0049] Usually, the Stokes wavelength is in the range of 1020-1060 nm, and the pump wavelength in the range of 780-980 nm.

[0050] The first train of optical pulses T1 and the second train of optical pulses T2 are provided at a first repetition frequency fl, also called first pulse rate.

[0051] Either the first optical centre wavelengths 11 or the second optical centre wavelengths 12 or both are tuneable, with their energy difference w being tuneable, preferably over the Raman spectroscopy band.

[0052] The light source can be a dual frequency laser system, with an oscillator and a preamplifier, allowing the synchronisation of the first and second trains of optical pulses.

[0053] An example of light source is illustrated on Figure 3, showing a fiber-based laser system. The light source 15 may comprise a mode-locked ytterbium pump laser 16 and a fiber based optical parametric oscillator 17. A fiber based optical parametric oscillator laser provides a light source tuneable over the entire Raman spectroscopy band, with high peak power pulses. In addition, a fiber-based light source system, including a mode-locked fiber laser and a fiber optical parametric oscillator (FOPO), is a robust system easy to handle.

[0054] In another example shown on Figure 4, the light source 15 comprises an erbium doped fiber laser 216, a nonlinear fiber 217 and an ytterbium doped amplifier system 218. In this example, the light source 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 is less complex.

[0055] In yet another example shown on Figure 5, the light source 15 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 Tl, T2. 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.

[0056] Turning back to the microscopy system 1, the repetition rate multiplier 30 is provided to receive the first train of optical pulses Tl and output a third train of optical pulses T3 at a second repetition frequency f2 different from the first repetition frequency fl. With other words, the repetition rate multiplier 30 is designed to remove components at the first repetition frequency fl from the inputted first train of optical pulses Tl.

[0057] In an example, the second repetition frequency f2 is a multiple of the first repetition frequency fl. The second repetition frequency f2 is preferably the double of the first repetition frequency flBut this is a non-limiting example and other multiples, and even fractional multiples, are possible.

[0058] Figure 6 shows an example of the repetition rate multiplier 30. In the example of Figure 6, the repetition rate multiplier 30 is a cascaded fiber interferometer 30.

[0059] The cascaded fiber interferometer 3 is a chain of N length-imbalanced interferometers with an optical path length difference close to c / (2*N*fl) receiving the first or second train of optical pulses Tl, T2 and outputting the third train of optical pulses T3, with c being the speed of light and N between 1 and 3.

[0060] Advantageously, with the cascade fiber interferometer, a fiber based coupler 32 can easily be provided to couple out a reference signal Tref of the third train of optical pulses T3. The reference signal Tref is a portion of the third train of pulses which will be transmitted to the detector. The reference signal Tref can be passed from the repetition rate multiplier 30 via an optical fiber onto an input of the detector 60.

[0061] A fiber-based repetition rate multiplier couples easily with the fibre based light source system described earlier.

[0062] The interferometers are preferably fiber Mach Zehnder interferometers, which are robust.

[0063] In another embodiment, the repetition rate multiplier 30 can be a repetition-rate multiplication of picosecond optical pulse trains based on temporal self-imaging in a dispersive element.

[0064] In addition, a fiber optic amplification stage 38 can be provided to amplify the third train of pulses T3 and compensate the loss introduced by the repetition rate multiplier and the coupler at the output of the repetition rate multiplier 30.

[0065] The output of the fiber optic amplification stage, if present, or the third train of optical pulses T3 at the output of the repetition rate multiplier 30 is passed to the focusing optics 40.

[0066] In an aspect shown figure 7, the light source system 410 integrates both the light source and the repetition rate multiplier. Figure 7 illustrates a fiber-based light source system 410 integrating a light source 415 and a fiber-based repetition rate multiplier 430. The light source 415 is a dual frequency laser system, with an oscillator 416 and a preamplifier 417, allowing the synchronisation of the first and second trains of optical pulses. A fiber based optical parametric oscillator 17 is provided. In this example, the repetition rate multiplier 430 is provided for the Stokes beam.

[0067] Turning back to the microscopy system 1, the focusing optics 40 is provided for directing and focusing the second train of optical pulses T2 and the third train of optical pulses T3 onto the focal volume 5 where the sample can be placed.

[0068] 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.

[0069] A condenser 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.

[0070] In an embodiment, the detector 60 is an optical balanced detector configured to detect the transmitted or reflected third train of optical pulse T31. The optical balanced detector 60 comprises two photodetectors, 61, 62, with a first photodetector 61 configured to detect the transmitted or reflected third train of optical pulse T31, and a secondphotodetector 62 configured to detect a reference signal Tref of the third train of optical pulses passed from the repetition rate multiplier 30.

[0071] In the words, the first photodetector 61 is a microscopy detector for detecting the components of the microscopy signal having the transmitted or reflected third train of optical pulses T31, and the second photodetector is a reference photodiode for detecting the reference signal Tref.

[0072] It should be noted that the transmitted or reflected second train of pulses T21 at the wavelength 12 and at the first frequency fl is therefore blocked and not detected by the detector 60. It is also possible to provide a blocking filter 55 between the condenser 50 and the detector 60.

[0073] When the light source system and the repetition rate multiplier are fiber- based components, the balanced detector has preferably an extinction ratio of at least 40 dB, to a enable SRS signal detection, without having to add active modulation or external synchronisation to clean sources.

[0074] Finally, a processor 80 is provided to detect a signal component at the first repetition frequency fl in the transmitted or reflected third train of optical pulse T31 and process said signal components at the first repetition frequency fl. The processor 70 can comprise a lock-in amplifier. The processor can output a Raman spectroscopy spectrum for the sample or a pixel of an image representative of the sample.

[0075] A method of microscopy imaging will now be described with reference to figure 8, performed using the microscopy system of Fig. 1.

[0076] In a first step SI, a first train of optical pulses T1 and a second train of optical pulses T2 are generated at a first repetition frequency or pulse frequency fl.

[0077] In a second step S2, a third train of optical pulses T3 is generated at a second repetition frequency f2, different from the first repetition frequency fl .

[0078] The third train of optical pulses T3 is obtained by manipulating the first train of optical pulses Tl, in particular by removing the first repetition frequency in the first train of optical pulses Tl. In the example of fig. 2, this is achieved by the repetition frequency multiplier 30 based on extra cavity repetition frequency multiplication.

[0079] The second repetition frequency T2 is a multiple of the first repetition frequency fl.For example, if the first frequency fl is 20 MHz, the third train of optical pulses T3 can be formed by removing the frequency component of 20 MHz and its odd harmonics from the first train of optical pulses Tl.

[0080] The second train of optical pulses T2 and the third train of optical pulses T3 are directed and focused by the focusing optics 40 onto the sample in the common focal volume 5 (step S3). The second train of optical pulses T2 and the third train of optical pulses T3 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.

[0081] The second train of optical pulses T2 and the third train of optical pulses T3 are transmitted or reflected through the common focal volume to form respectively a transmitted or reflected second train of optical pulses T21 and a transmitted or reflected third train of optical pulses T31, possibly with Raman signal components resulting from Raman scattering after interaction in the sample.

[0082] It is possible to manipulate either the Stokes beam or the pump beam to remove the first frequency. With other words, the first wavelength 11 can correspond to the Stokes wavelength and the second wavelength 12 can correspond to the pump wavelength, so that the third train of optical pulses T3 is the Stokes beam and the second train of optical pulses T2 is the pump beam, but this can be the other way around.

[0083] 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 third train of optical pulses T31, but not the transmitted or reflected second train of optical pulses T21.

[0084] It is also possible to block the second train of pulses T2 from the signal transmitted or reflected through the common focal volume with a passband filter provided between the condenser 50 and the detector 60.

[0085] The skilled person understands that, since the first repetition frequency fl has been removed from the third train of optical pulses T3 before passing through the focal volume and since the transmitted or reflected train of optical pulses T21 is blocked by the detector, a signal component detected at the first repetition frequency fl on the transmitted or reflected third train of optical pulses T31 can only result from a nonlinear interaction in thesample, e.g. due to stimulated Raman scattering after interaction with the sample in the focal volume of the two excitation beams.

[0086] The stimulated Raman scattering signal is measured and detected at the first frequency fl, hence at the removed frequency.

[0087] For improved signal detection, in particular when the light source and repetition rate multiplier are fiber-based components, the detector 60 is a balanced detector, configured to receive a reference signal Tref of the third train of optical pulses T3 and to remove said reference signal Tref from the transmitted or reflected third train of optical pulses T31. The reference signal Tref is a portion of the third train of pulses which has not been transmitted or reflected through the common focal volume as a reference.

[0088] Indeed, the third train of optical pulses T3 may still comprise components at the first frequency fl not removed by the frequency rate multiplier 30. For example, extracavity repetition multiplication schemes typically allow side band suppression in the order of -35dB. This is illustrated in figure 9 showing a measured side band suppression in the third train of optical pulses for a second repetition frequency of about 40 MHz. Residual sidebands at the first repetition frequency fl of 20 MHz could inhibit or prevent the detection of the Raman signal at 20 MHz.

[0089] Figure 10 shows a detailed view of the residual sidebands at 20 MHz after the repetition frequency multiplier without balanced detection. The skilled person understands that the residual sidebands can hinder the detection of a weak SRS signal at 20 MHz due to the interaction of the second and third train of optical pulses in a common focal volume.

[0090] Therefore, a balanced detection is preferably provided, capable of noise suppression with more than 40dB. Using balance detection allows improving the detection of the signal, as can be seen on Figure 11 showing a transmitted or reflected third train of optical pulses T31 when using a balanced detector. In this example, the third train of optical pulses T3 for a second repetition frequency f2 of about 40 MHz of the first train of optical pulse T1 has underwent side band suppression in the balanced detector 60 to compensate for the residual sidebands at the first repetition frequency fl of 20 MHz. The residual sidebands are not distinguishable from the general laser noise. In this example the balance detection is measured with a bandwidth of 30 kHz. It is therefore noted that the balanced detection avoids the need for active modulation or external synchronisation to remove noise coming from inherently noisy fiber-based components.

[0091] Finally, a processor 80 is provided to process a signal component at the first repetition frequency fl in the transmitted or reflected third train of optical pulse T31.

[0092] Indeed, as will become clear, signal components at the first repetition frequency fl detected on the transmitted or reflected third train of optical pulses T31 can only result from a nonlinear interaction in the sample, e.g. due to stimulated Raman scattering after interaction with the sample in the focal volume of the two excitation beams, the first repetition frequency fl has been removed from the third train of optical pulses T3 before passing through the focal volume and since the transmitted or reflected train of optical pulses T21 is blocked by the detector.

[0093] The stimulated Raman scattering signal is measured and detected at the first frequency fl, hence at the removed frequency. Fig 12 shows an example of measured stimulated Raman scattering signal, on Deuteirumoxid (D20). The measurements were performed with a Zurich Lockin amplifier in sprectrum mode with a 39 microsecond sampling rate.

[0094] The processor 80 can process said signal components at the first repetition frequency fl to provide a signal representative of the interaction in the sample, such a Raman spectroscopy signal, or a pixel of an image of the sample.

[0095] This method and system allow circumventing the need for expensive and noisy high-power radio frequency components such as AOMs.

[0096] The skilled person understands that no active modulation system introducing a new frequency component is used. Instead, the present invention proposes to remove a frequency component from one beam by passive ways. The unmanipulated beam is blocked and the manipulated beam is detected.

[0097] 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.

[0098] List of reference numbers:Microscopy system 1 light source system 10 light source 15 first train of optical pulses T1 at first optical center wavelengths 11, repetition frequency fl second train of optical pulses T2 at second optical center wavelengths 12. third train of optical pulses T3 reference train of optical pulses Tref transmitted or reflected second train of optical pulses T21 transmitted or reflected third train of optical pulses T31 repetition rate multiplier 30 coupler 32 amplification stage 38 focusing optics 40, dichroic mirror 41, galvanometer scanner 42, set of lenses 43, objective lens 44, condenser 50 optical detector 60, processor 80 mode-locked ytterbium pump laser 16, fiber based optical parametric oscillator 17 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

Claims

Claims1. A microscopy system (1) for analysing a sample, comprising a fiber-based light source system (10) configured for providing a first train of optical pulses (Tl) at first optical center wavelengths (11) and a second train of optical pulses (T2) at second optical center wavelengths (12), both at a first repetition frequency (fl), a fiber-based repetition rate multiplier (30) configured to receive the first train of optical pulses (Tl) and to output a third train of optical pulses (T3) at a second repetition frequency (f2) different from the first repetition frequency (fl), focusing optics (40) configured to direct the second train of optical pulses (T2) and the third train of optical pulses (T3) to a sample in a focal volume (5), a balanced detector (60) configured to detect a transmitted or reflected third train of optical pulses (T31) after interaction in the sample in the focal volume, and a processor (80) configured to process signal components at the first repetition frequency (fl) from the detected transmitted or reflected third train of optical pulses (T31) and to output a signal or a pixel representative of the interaction in the sample.

2. The microscopy system according to claim 1, wherein the microscopy system is a stimulated Raman microscopy system with a Stokes beam and a pump beam, wherein one of the first train of optical pulses (Tl) and the second train of optical pulses (T2) is the Stokes beam and the other one of the first train of optical pulses (Tl) and the second train of optical pulses (T2) is the pump beam.

3. The microscopy system (1) of any of claims 1 to 2, wherein the balanced detector is configured to detect the transmitted or reflected the third train of optical pulses (T31) and to receive simultaneously a reference train of optical pulses (Tref), the reference train of optical pulses (Tref) being a portion of the third train of pulses (T3) not transmitted or reflected through the common focal volume.

4. The microscopy system according to any of claims 1 to 3, wherein the balanced detector has an extinction ratio of at least 40 dB.

5. The microscopy system (1) according to any of claims 1 to 4, wherein the fiberbased repetition rate multiplier (30) is a passive component and / or wherein the second repetition frequency (f2) is a multiple of the first repetition frequency (fl).

6. The microscopy system (1) according to claim 5, wherein the fiber-based repetition rate multiplier is an extra cavity repetition rate multiplier.

7. The microscopy system (1) according to claim 6, wherein the repetition frequency multiplier is a chain of interferometers in cascade, preferably a fiber Mach Zehnder interferometer.

8. The microscopy system (1) according to any of claims 1 to 7, further comprising a fiber optic amplification stage configured to amplify the third train of pulses (T3) outputted from the frequency remover (30).

9. The microscopy system (1) according to any of claims 1 to 8, wherein the processor comprises a lock-in amplifier.

10. The microscopy system (1) of any of claims 1 to 9, wherein the fiber-based light source system (400) 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 cavities11. The microscopy system (1) of any of claims 1 to 10, wherein the fiber-based light source system (400) integrates both the light source (415) and the fiber based repetition rate multiplier (430).-17-12. A method of microscopy analysing of a sample, comprisingGenerating, by a fiber-based light source system, a first train of optical pulses (Tl) at first optical center wavelengths (11) and a second train of optical pulses(T2) at second optical center wavelengths (12), both at a first repetition frequency (fl), Manipulating, using a fiber based repetition rate multiplier, the first train of optical pulses (Tl) to output a third train of optical pulses (T3) at a second repetition frequency (f2) different from the first repetition frequency (fl), directing the second train of optical pulses (T2) and the third train of optical pulses (T3) 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 second train of optical pulses (T21) and a transmitted or reflected third train of optical pulses (T31), optically detecting by a balanced detector receiving a reference signal of the third train of pulses the transmitted or reflected third train of optical pulses (T31), processing the transmitted or reflected third train of optical pulses (T31) at the first repetition frequency (fl) to output a signal or a pixel representative of the interaction in the sample.

13. The method according to claim 12, comprising blocking the transmitted or reflected second optical train (T21) before or simultaneously to optically detecting the transmitted or reflected third train of optical pulses (T31).

14. The method according to any of claims 12 to 13, wherein the step of manipulating the first train of optical pulses (Tl) or the second train of optical pulses (T2) comprises passively removing the first repetition frequency from the first train of optical pulses (Tl) or the second train of optical pulses (T2).

15. The method according to any of claims 12 to 14, wherein the balanced detector has an extinction ratio of at least 40 dB.

Images