A measurement system for stimulated raman scattering (SRS) spectroscopy or microscopy

Abstract

A spectroscopic measurement system, comprising: a laser source (110) configured to generate a pump beam (BP) and a Stokes beam (BS), a modulator (120) configured to modulate one of the beams (BP, BS), a beam overlap system (130) configured to spatially and temporally overlap the beams (BP, BS), a sample holder (140) configured to receive the overlapped beams, at least one detector (150) configured to detect a signal resulting from Stimulated Raman Scattering (SRS), and a lock-in amplifier (160) configured to generate an output signal based on an input signal from the detector (150) and a reference signal (REF), characterized in that the drive signal for the modulator (120) and the reference signal (REF) for the lock-in amplifier (160) are both provided by the internal reference oscillator (INT_TRG) of the lock-in amplifier (160).

Description

[0001] A SPECTROSCOPIC MEASUREMENT SYSTEM

[0002] TECHNICAL FIELD

[0003] The present invention relates to spectroscopic measurement systems, specifically to systems employing Stimulated Raman Scattering (SRS) spectroscopy or microscopy.

[0004] BACKGROUND

[0005] Phase-sensitive detection using a lock-in amplifier is a measurement technique widely employed to extract extremely weak signals from significant background noise. This method is particularly valuable in scientific and industrial research contexts, where signals of interest may have low amplitudes that are easily obscured by noise. Lock-in amplifiers, also referred to as phase-sensitive detectors (PSD), facilitate precise determination of both the amplitude and phase of signals at a predetermined frequency.

[0006] A lock-in amplifier operates by demodulating an input signal using a reference signal having the same modulation frequency. This process involves multiplying the input signal by the reference signal, followed by passing the resultant product through a low-pass filter. The filter removes high-frequency components, leaving only the near-zero-frequency component, thereby isolating low-amplitude signals from background noise. Detailed principles of this method are described by Meade, Mike L. in "Lock-in amplifiers: principles and applications" (IEE Electrical Measurement Series, 1983).

[0007] The effectiveness of phase-sensitive detection depends on comparing the phase of the target signal with that of the reference signal. Specifically, the lock-in amplifier compares a test signal (from the investigated system) with a known reference signal. Analyzing the phase difference between these two signals enables accurate extraction of the desired information despite significant noise.

[0008] Lock-in amplifiers are extensively used in applications requiring detection of weak signals, notably spectroscopy and microscopy, due to their ability to provide precise measurements even amid considerable noise.

[0009] One prominent application of this detection method is Stimulated Raman Scattering (SRS) microscopy. In SRS microscopy, the sample is irradiated with two pulsed laser beams, the pump beam and the Stokes beam, collectively referred to as SRS beams. The difference in energy between these two beams matches the excitation energy of specific molecular vibrations in the sample. Further details on this method are provided in publications by Ploetz et al. (Applied Physics B, 87, 2007, 389-393) and Brzozowski et al. (Biotechnology Advances, 60, 2022, 108003).

[0010] Sample vibrations correspond to specific Raman bands initially identified through spontaneous Raman spectroscopy or microscopy. When irradiated with appropriately chosen wavelengths from both SRS beams, the sample experiences selective excitation, resulting in a reduction in photon count in the pump beam (Stimulated Raman Loss, SRL) and simultaneous amplification of the Stokes beam (Stimulated Raman Gain, SRG). A lock-in amplifier-based detection system records the SRS signal, enabling sensitive observation of minor variations in SRG or SRL. Typically, SRG or SRL detection involves modulating either the Stokes or pump beam at a predetermined frequency lower than the laser pulse repetition rate. This modulation induces an SRS signal at exactly this modulation frequency, enabling effective lock-in detection. In standard configurations, the unmodulated beam is directed to the detector, while the modulated beam is suppressed using optical filters. The detector records periodic intensity changes corresponding to SRG or SRL at the modulation frequency applied to the other beam. Consequently, the lock-in amplifier receives both the detector's output and the modulation drive signal as reference input, thus facilitating detection of very faint signals in SRS microscopy.

[0011] In practical applications of SRS microscopy, beam modulation typically involves using modulators such as acousto-optic modulators (AOM) or electro-optic modulators (EOM).

[0012] Fig. 1 illustrates a conventional SRS microscopy or spectroscopy measurement system. The laser source 11 generates two beams: a pump beam (BP) and a Stokes beam (BS). One of these beams (in this case, the Stokes beam BS) passes through a modulator 12, which modulates it at high frequency (typically ranging from a few kHz to 80 MHz). Both beams are then spatially and temporally overlapped in the beam overlap system 13 and directed onto a sample located on the sample holder 14, where SRS occurs. Subsequently, the unmodulated beam (here, the pump beam BP) undergoes detection in detector 15. The detector's output feeds into the lock-in amplifier 16. As its reference signal, the lock-in amplifier receives a signal from a trigger source 17, external to the amplifier, which also drives the modulator 12. Typically, the trigger source 17 is derived from the pulsed laser's internal clock 11, processed by a frequency divider. This integration ensures efficient beam modulation and accurate synchronization with the laser pulse repetition rate.

[0013] However, external trigger sources 17 employed in conventional setups introduce interference that propagates through electrical wiring and the surrounding environment, ultimately affecting the lock-in amplifier. This interference, occurring precisely at the demodulation frequency, significantly reduces demodulation efficiency and, consequently, measurement quality in SRS spectroscopy and microscopy.

[0014] SUMMARY OF THE INVENTION

[0015] It is an object of the invention to provide a solution that eliminates or reduces the drawbacks of prior-art spectroscopic measurement systems.

[0016] The invention is applicable to a wide range of spectroscopic techniques, in particular SRS spectroscopy and microscopy, including configurations where SRS is employed directly, or indirectly as the source of another effect, such as a photothermal response induced cyclically by SRS and detected with an auxiliary laser.

[0017] According to the invention, the modulator (for example, an AOM or EOM used to modulate the pump or Stokes beam in SRS spectroscopy or microscopy) is driven not by an external modulation source, but instead by the internal reference oscillator of the lock-in amplifier. This internal oscillator simultaneously provides the reference signal for the lock-in amplifier itself. By removing the need for an additional external reference source (such as a pulse generator or a processed laser clock signal), the system eliminates interference at the modulation frequency that would otherwise propagate through wiring and affect demodulation efficiency.

[0018] This approach significantly improves system sensitivity. In particular, the method allows highly sensitive detection using only a single, simple detector (for example, a semiconductor photodiode or photomultiplier) without the need for additional electronic filtering or for balanced detection schemes that rely on complex optical arrangements to suppress noise.

[0019] The invention is equally applicable to systems that employ balanced (differential) detection. In such configurations, auxiliary signals may be collected and combined with the primary signal (for example, by subtraction) to further suppress noise components not originating from SRS. While balanced detection is standard in most state-of-the-art SRS microscopy systems, the proposed solution enables comparable or superior performance even with a single detector configuration.

[0020] Accordingly, the invention encompasses both simplified systems based on a single detector, and more advanced systems employing balanced or other forms of detection aimed at further improving signal quality.

[0021] In one aspect, the invention relates to a spectroscopic measurement system, comprising: a laser source configured to generate a pump beam and a Stokes beam, a modulator configured to modulate one of the beams, a beam overlap system configured to spatially and temporally overlap the beams, a sample holder configured to receive the overlapped beams, at least one detector configured to detect a signal resulting from Stimulated Raman Scattering, and a lock- in amplifier configured to generate an output signal based on an input signal from the detector and a reference signal, characterized in that the drive signal for the modulator and the reference signal for the lock-in amplifier are both provided by the internal reference oscillator of the lock- in amplifier.

[0022] The system may further comprise a signal-conditioning circuit configured to process the internal reference oscillator signal of the lock-in amplifier by adjusting at least one parameter selected from: voltage and signal shape.

[0023] The modulator may comprise a modulation driver configured to further process the drive signal prior to modulation of the beam.

[0024] The internal reference oscillator signal of the lock-in amplifier can be routed internally within the lock-in amplifier to the reference input.

[0025] The internal reference oscillator signal of the lock-in amplifier can be provided to the reference input by connecting external ports of the lock-in amplifier, the ports being respectively dedicated to the internal reference oscillator output and to the reference input.

[0026] The detector may comprise a spectral blocking system configured to block the modulated beam and transmit the unmodulated beam.

[0027] The detector may be configured for balanced detection using more than one detection module.

[0028] The system may further comprise a scanning stage configured to move the sample holder relative to the overlapped beams.

[0029] The system may further comprise a galvanometric mirror scanner configured to scan the overlapped beams across the sample on the sample holder.

[0030] In another aspect, the invention relates to a method of spectroscopic measurement using Stimulated Raman Scattering, comprising generating a pump beam and a Stokes beam, modulating one of the beams, spatially and temporally overlapping the beams and directing them onto a sample, detecting a signal resulting from SRS, and demodulating an output from the detector with a lock-in amplifier using a reference signal, characterized in that the internal reference oscillator of the lock-in amplifier provides both the drive signal for the modulator and the reference for phase-sensitive detection in the lock-in amplifier, thereby obviating the need for an external trigger source.

[0031] These and other features, aspects and advantages of the invention will become better understood with reference to the following drawings, descriptions and claims. BRIEF DESCRIPTION OF DRAWINGS

[0032] The invention will be explained in detail using exemplary embodiments with reference to the accompanying drawings, in which:

[0033] Fig. 1 illustrates a conventional SRS spectroscopy / microscopy measurement system according to the prior art;

[0034] Fig. 2 illustrates a first embodiment of a measurement system according to the present invention;

[0035] Fig. 3 illustrates a second embodiment of a measurement system according to the present invention, incorporating additional signal conditioning.

[0036] DETAILED DESCRIPTION

[0037] The following detailed description is of the best currently contemplated modes of carrying out the invention. The description is not to be taken in a limiting sense, but is made merely for the purpose of illustrating the general principles of the invention.

[0038] Fig. 2 illustrates a first embodiment of a spectroscopic measurement system according to the invention. A laser source 110 generates two beams: a pump beam BP and a Stokes beam BS. One of these beams (in this example, the Stokes beam BS) is directed through a modulator 120, which modulates the beam at high frequency. Both beams are then spatially and temporally overlapped in a beam overlap system 130 and directed onto a sample positioned on a sample holder 140, where SRS occurs.

[0039] Detection is performed on the unmodulated beam (in this case, the pump beam BP), which carries information about intensity variations caused by the SRS process. These variations appear at the modulation frequency applied to the Stokes beam BS. Detector 150 converts the incident light into an electrical signal composed of a large DC component corresponding to the transmitted pump beam, a small modulated component corresponding to the useful SRS signal (here specifically SRL), and additional noise contributions. Components 110-150 correspond to those used in standard systems such as the prior-art configuration of Fig. 1.

[0040] The detector output is provided to the IN input of lock-in amplifier 160. Unlike conventional systems, which require an external trigger 17 to drive the modulator 120 and simultaneously provide a reference input for the amplifier, the invention instead employs the internal reference oscillator (INT TRG) of the lock-in amplifier. The internal oscillator signal is routed both to the reference input REF of amplifier 160 and to the drive input of modulator 120. Consequently, the drive signal of modulator 120 and the reference signal of amplifier 160 are identical.

[0041] This arrangement eliminates interference originating from external reference sources, which in known systems (see Fig. 1) can propagate into the lock-in amplifier at the modulation frequency and severely compromise measurement accuracy. In the present system, such interference is avoided because no external reference source is required.

[0042] In the embodiment of Fig. 2, the internally generated oscillator signal (INT TRG) must meet the input requirements of modulator 120, particularly regarding signal shape and voltage. If direct compatibility cannot be achieved, the embodiment of Fig. 3 introduces an additional circuit to address this.

[0043] It should be noted that the internal reference oscillator of lock-in amplifier 160, when used simultaneously as a modulation drive and as a reference signal, is not precisely synchronized with the repetition rate of the laser pulses generated by laser source 110. The inventors, however, have found that this lack of perfect synchronization introduces significantly less measurement error than the noise caused by the external reference source in prior-art systems. Moreover, residual synchronization errors can be minimized by fine-tuning the frequency of the lock-in’s internal reference oscillator around the desired modulation frequency.

[0044] Fig. 3 illustrates a second embodiment of the invention. This embodiment corresponds to the system of Fig. 2, but additionally includes a signal-conditioning circuit 161. Circuit 161 processes the internal reference oscillator signal (INT TRG) from lock-in amplifier 160 to adapt it to the requirements of modulator 120, for example by adjusting the voltage level or converting the signal waveform, while maintaining synchronization in time.

[0045] A specific implementation of the second embodiment is an SRS microscope. In this embodiment, laser source 110 is a Fluence Lazurite laser system comprising a fiber-based Stokes laser and an Optical Parametric Oscillator (OPO). The Stokes laser generates a Stokes beam BS with an average power of 450 mW at 1029 nm, while the OPO generates a tunable pump beam BP in the 750-950 nm range with an average power of 100 mW. Both beams consist of 2 ps pulses at a repetition rate of 20 MHz.

[0046] In this system, the Stokes beam BS is modulated at 4 MHz using an AOM-type modulator 120. The pump beam BP and the modulated Stokes beam BS are overlapped in beam overlap system 130, which includes a dichroic mirror for spatial overlap and an Optical Delay Line (ODL) in the pump path for temporal synchronization of the pulses. The overlapped beams are directed to a sample positioned on sample holder 140. The sample is scanned using galvanometric beam-scanning mirrors and / or a piezoelectric stage integrated into an inverted Nikon Ti2 microscope.

[0047] After interaction with the sample, the modulated Stokes beam BS is blocked using optical filters, and only the pump beam BP is transmitted to detector 150. The sample holder 140 is equipped with a UPLXAPO40X NA 0.95 40* Olympus air objective to focus the beams, while a Nikon MRD07620 50* NA 1.0 immersion objective is used to collect the signal. Detector 150 is a Thorlabs SM1PD1 photodiode detector.

[0048] The lock-in amplifier 160 is an SR865A unit from Stanford Research Systems. Its internal oscillator provides a 2 V peak-to-peak sine wave (INT TRG), which is converted into a 5 V TTL-level square wave using signal-conditioning circuit 161. The conditioned signal is then used to drive the AOM modulator 120. The demodulated output of lock-in amplifier 160 is processed by control software (e.g., WITec), which collects and processes image frames.

[0049] This SRS microscope can operate with SRL detection, enabling detailed analysis of molecular vibrational modes in the sample.

[0050] While the invention has been described with respect to a limited number of embodiments, it will be appreciated that many variations, modifications and other applications of the invention may be made. Therefore, the claimed invention as recited in the claims that follow is not limited to the embodiments described herein.

Claims

CLAIMS1. A spectroscopic measurement system, comprising: a laser source (110) configured to generate a pump beam (BP) and a Stokes beam (BS), a modulator (120) configured to modulate one of the beams (BP, BS), a beam overlap system (130) configured to spatially and temporally overlap the beams (BP, BS), a sample holder (140) configured to receive the overlapped beams, at least one detector (150) configured to detect a signal resulting from Stimulated Raman Scattering (SRS), and a lock-in amplifier (160) configured to generate an output signal based on an input signal from the detector (150) and a reference signal (REF), characterized in that- the drive signal for the modulator (120) and the reference signal (REF) for the lock-in amplifier (160) are both provided by the internal reference oscillator (INT TRG) of the lock-in amplifier (160).

2. The system according to claim 1, wherein the system further comprises a signal-conditioning circuit (161) configured to process the internal reference oscillator signal (INT TRG) of the lock-in amplifier (160) by adjusting at least one parameter selected from: voltage and signal shape.

3. The system according to claim 1 or 2, wherein the modulator (120) comprises a modulation driver configured to further process the drive signal prior to modulation of the beam (BP, BS).

4. The system according to any of claims 1 to 3, wherein the internal reference oscillator signal (INT TRG) of the lock-in amplifier (160) is routed internally within the lock-in amplifier (160) to the reference input (REF).

5. The system according to any of claims 1 to 3, wherein the internal reference oscillator signal (INT TRG) of the lock-in amplifier (160) is provided to the reference input (REF) by connecting external ports of the lock-in amplifier (160), the ports being respectively dedicated to the internal reference oscillator output (INT TRG) and to the reference input (REF).

6. The system according to any of claims 1 to 5, wherein the detector (150) comprises a spectral blocking system configured to block the modulated beam (BS, BP) and transmit the unmodulated beam (BP, BS).

7. The system according to any of claims 1 to 5, wherein the detector (150) is configured for balanced detection using more than one detection module.

8. The system according to any of claims 1 to 7, further comprising a scanning stage configured to move the sample holder (140) relative to the overlapped beams.

9. The system according to any of claims 1 to 7, further comprising a galvanometric mirror scanner configured to scan the overlapped beams across the sample on the sample holder (140).

10. A method of spectroscopic measurement using Stimulated Raman Scattering (SRS), comprising: generating, with a laser source (110), a pump beam (BP) and a Stokes beam (BS); modulating, with a modulator (120), one of the beams (BP, BS); spatially and temporally overlapping the beams (BP, BS) with a beam overlap system (130) and directing the overlapped beams onto a sample on a sample holder (140); detecting, with at least one detector (150), a signal resulting from SRS; and demodulating, with a lock-in amplifier (160), an output from the detector (150) using a reference signal, characterized in that an internal reference oscillator (INT TRG) of the lock-in amplifier (160) provides both a drive signal applied to the modulator (120) and the reference used for phase-sensitive detection in the lock-in amplifier (160), whereby no external trigger source is used.

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