Patent Application
20250035487
Aspects of this disclosure relate to methods and systems for modulation transfer techniques such as Stimulated Raman Scattering.
In optical microscopy, both spatial resolution (smallest discernible object) and contrast (discerning object from background) are critically important. Contrast is often achieved by adding exogenous dyes or stains which ‘label’ the object of interest. However, in many cases exogenous contrast agents cannot be applied or may alter the object under study. For many microscopy problems, the options for achieving label-free contrast are limited. Coherent Raman Microscopy (CRM) encompasses a range of nonlinear optical techniques which provide chemical-specific, label-free image contrast via Raman spectroscopy (molecule specific vibrational modes). It has been widely applied to biomedical applications and has found increasing use in other fields such as material sciences and geology. In standard CRM approaches, background signals due to competing nonlinear optical processes exist and limit overall image contrast. Various ‘background reduction’ schemes have been proposed and demonstrated, but these come with significant drawbacks.
Most CRM implementations make use of two ultrashort lasers whose frequency difference is set to match the frequency of the vibrational Raman mode of interest, thus achieving Raman resonance and therefore object contrast. The highest signal-to-background ratios in CRM are typically achieved using high speed (e.g. MHz) optical modulation techniques. One variant of CRM, Stimulated Raman Scattering (SRS) requires optical modulation to function and has been demonstrated using amplitude, polarization, and/or frequency modulation. Another variant, Coherent anti-Stokes Raman Scattering (CARS) microscopy can operate without modulation but achieves its high sensitivity when used in a frequency modulation configuration.
In SRS microscopy, amplitude modulation is the most widely adopted Raman-contrast technique but is not background-free due to other competing nonlinear optical processes (e.g. cross-phase modulation, two-photon absorption, thermal lensing) which also contribute to the modulated signal. This means that image brightness is often not due uniquely to Raman resonance in the object of interest, thus reducing contrast. The definitive proof of Raman contrast is to scan the Raman spectrum while imaging (cf. hyperspectral imaging). Frequency and/or Polarization modulation SRS schemes are either challenging to implement (e.g. ideally require more than two laser beams) or remove only certain types of background signals (e.g. cross-phase modulation). CARS microscopy has similar background contrast issues plus an additional coherent non-resonant background that optically interferes with the desired signal, distorting Raman spectra. Frequency and/or Polarization modulation reduces this coherent background but cannot correct for Raman spectral distortions.
Stimulated Raman Scattering (SRS) microscopy relies on modulation to achieve contrast1-3. In its initial demonstration using amplitude modulation (AM)4 it overcame the non-resonant background problem that had plagued coherent anti-Stokes Raman scattering (CARS) microscopy5. Nonetheless, due to other optical effects, both linear and nonlinear, background signals can still be present in AM-SRS6,7, leading to the development of alternative modulation schemes. Polarization modulation (PM)8, frequency modulation (FM)9,10, and time delay (i.e. linear phase)11 modulation were recognized early on as possible alternative modulation schemes12 and have been demonstrated for nonlinear optical microscopy. Indeed, these modulation schemes have a long history in optical communication and form the basis of advanced telecommunication schemes13. Beyond these traditional techniques, when using short pulses and nonlinear optics, other types of modulation schemes become possible. For example, coherent control using phase and amplitude shaping of an optical pulse can reduce background signals in coherent Raman microscopy (CRM)14,15.
In one aspect of the disclosure, there is provided a modulation method comprising steps of:
In another aspect of the disclosure, there is provided a method of imaging a sample, the method comprising:
In another aspect of the disclosure, there is provided an imaging system comprising:
In another aspect of the disclosure, there is provided a modulation scheme for suppressing noise, the method comprising:
Advantageously, the modulation scheme is based on rapidly modulating the sign of the chirp, such that only the Raman resonances are detected amplified and non-resonant background channels are substantially suppressed or removed, thereby enhancing the contrast and sensitivity in CRM.
The following detailed description refers to the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the following description to refer to the same or similar elements. While implementations of the disclosure may be described, modifications, adaptations, and other implementations are possible. For example, substitutions, additions, or modifications may be made to the elements illustrated in the drawings, and the methods described herein may be modified by substituting, reordering, or adding stages to the disclosed methods. Accordingly, the following detailed description does not limit the disclosure. Instead, the proper scope of the disclosure is defined by the appended claims.
Moreover, it should be appreciated that the particular implementations shown and described herein are illustrative of the invention and are not intended to otherwise limit the scope of the present invention in any way. Indeed, for the sake of brevity, certain sub-components of the individual operating components, and other functional aspects of the systems may not be described in detail herein. Furthermore, the connecting lines shown in the various figures contained herein are intended to represent exemplary functional relationships and/or physical couplings between the various elements. It should be noted that many alternative or additional functional relationships or physical connections may be present in a practical system.
Referring to
The terms “pump beam” and “Stokes beam” are meant to be understood as they are commonly used in the field of stimulated Raman scattering, such as the CARS and SRS techniques. The expression “Stokes beam” is generally understood to refer to the one of the two laser light beams having the lower optical frequency. These names are historically related to a “Stokes” Raman scattering process where the excitation light is re-scattered at a lower frequency due to interaction with a sample, although one skilled in the art will readily understood that the origin of this terminology is not limitative to its current use on the art. The frequency difference between the two beams is tuned in order to obtain the Raman spectrum. It is the input of these two beams that results in the relatively higher SRS and CARS signals compared to spontaneous Raman.
As is known in the art of optical microscopy, the pump beam and Stokes beam are generally pulsed light beams, having a pulse width and repetition rate suitable for the purposes of the experiment being performed. Typically, light pulses in the femtosecond to picosecond duration range may be used. The tuning of the frequency difference between the two beams may be performed in any manner known to those skilled in the art.
The tunable output 16 is passed through a half-wave plate 30a and a polarizing beam splitter (PBS) 32a to control power before being sent to a variable delay stage 34 and then to a dichroic recombiner 40. The fixed output 22 is passed through a half-wave plate 30a and a polarizing beam splitter (PBS) 32b to control the power of the beam before being sent to variable delay stage 34 which includes a translation stage, and then sent to an electro-optic modulator 50 for modulating an optical property of the beams 14, 16. In the illustrated system, the electro-optic modulator 50 is embodied by a Pockels cell (350-160, Conoptics, USA) provided in the path of the Stokes beam 26. It will be readily understood that although the modulator is shown here as acting on the Stokes beam 16, in other variants it may act on the pump beam 14. In one example, the electro-optic modulator 50 is modulated at 1 MHz, and the driving waveform for the electro-optic modulator 50 is provided by a function generator, such as DS345, Stanford Research Systems, USA.
As a person of skill in the art will appreciate, in AM-SRS, one output of the electro-optic modulator 50 is blocked and the other is sent to the dichroic recombiner 40. However, in the exemplary imaging system 10, the output 60 from the electro-optic modulator 50 is spit into two outputs 62, 64 by polarizer 70, such that one output 62 is sent directly to a 50:50 beam splitter 80, and the other output 64, the previously blocked output of the electro-optic modulator 50, is sent through a half-wave plate 90 to rotate the beam 64 to the same polarization as the other output 62 and then to a grating compressor 100 followed by a computer-controlled delay stage 110 including a translation stage. The two 1040 nm beams 62, 64, are then recombined on the 50:50 beam splitter 80 before being sent to the dichroic recombiner 40 where the pulses of both the pump beam 14 and the Stokes beam 16 are recombined and transmitted to a fixed optical path length of dispersive glass 120 e.g. SF11 glass in the path of the pump beam 14 and the Stokes beam 16. By blocking one arm of the CM-SRS setup 10, it is thus possible to seamlessly switch between AM and CM-SRS.
The imaging system 10 is configured to probe a sample with the combined pump beam 14 and Stokes beam 16. In one example, a microscope 130 employs galvo mirrors (GVSM002-US, Thorlabs, USA) connected to a microscope frame (Olympus IX-71), such that, the combined pump and Stokes beams 14, 66 traverse the galvo mirrors and are focused onto the sample through a microscope objective. The interaction of the pump beam 14 and the Stokes beam 16 with the sample leads to a modulation transfer between the pump beam 14 and the Stokes beam 16, which can be detected using a homodyne or heterodyne technique such as described below.
The forward propagating light from the interaction is collected using an objective lens (e.g. LUMPlanFI/IR, 40×, NA 0.8 water immersion, Olympus, Japan). The collected light was filtered by a short-pass filter 140 (e.g. BrightLine 850/310, Semrock, USA and 1064-71 NF, Iridian, Canada) to remove the Stokes beam 16 before being sent to a photo-diode 150 (e.g. FDS10X10, Thorlabs, USA) operating under reverse bias. The photo-diode signal is filtered using 1 MHz bandpass filter 160 (e.g. #3128, KR Electronics, USA) before being amplified by a transimpedance amplifier 170 (e.g. DHCPA-100 Femto Messtechnik, GmbH, Germany). Next, the output of the amplifier 170 is sent to a lock-in amplifier 180 (e.g. UHFLI, Zurich Instruments, Switzerland) which is referenced to the drive signal for the Electro-optic modulator 50. Typically, a 20 us time constant was used on the lock-in amplifier 180. Lastly, the output from the lock-in amplifier 180 is fed into a data acquisition module 190.
In one exemplary implementation, measurements of DMSO spectra and HEPG2 cells used a 60×, 1.2 NA water objective while measurements of a sweet potato used a 20×, 0.75 NA air objective. All data collection and motion of the galvanometer mirrors is synchronized using ScanImage, from Vidrio Technologies LLC, U.S.A. For all measurements, power in each 1040 nm air of the AM and CM-SRS setups is measured to be 50 mW before the microscope. For DMSO measurements, power in the tunable arm is set to 796 nm center wavelength with an average power of 100 mW. For sweet potato measurements, the tunable arm was set at 898, 912, and 929 nm center wavelength with 50 mW average power set at 50 mW. For measurements of HEPG2 cells, the tunable beam is set at a center wavelength of 800 nm and average power of 50 mW for measurements around a Raman shift of 2850 cm−1 and at a center wavelength of 844 nm and an average power of 100 mW for measurements around a Raman shift of 2200 cm−1.
In one implementation, the quadratic phase term of a short optical pulse is modulated to remove background noise in SRS microscopy, i.e. the optical chirp. This process, or chirp modulation SRS (CM-SRS) is akin to an unsharp mask in digital image processing but in this case it is applied to the spectral domain. This implementation is derived from spectral focusing coherent Raman microscopy (CRM)17-19 and maintains the ability to easily perform spectral scanning and multimodal imaging.
In SRS microscopy, background can arise from numerous terms, including cross-phase modulation, two-photon absorption, thermal effects, and variation in the linear refractive index of the media.6,7,9 PM-SRS can eliminate some of these signals but assumes a homogeneous background response. FM-SRS, and variants such as SR-GOLD20, can eliminate background signals if there are no chromatic effects and the system response only depends on the frequency differences among the excitation beams; unfortunately for many classes of samples the assumption of achromaticity breaks down. In one exemplary implementation, in CM-SRS, restrictions on background homogeneity and achromatic responses are relaxed because the excitation beams are co-polarized and the modulated beam is at a single central frequency.
Looking at
In one implementation, a single laser having dual beam capabilities or a sufficiently broad bandwidth may be used to generate both the pump beam 14 and the Stokes beam 16. In step 206, the tunable output is passed through a half-wave plate 30a and polarizing beam splitter (PBS) 32a to control the power of the beam before being sent to a variable delay stage 34 and then to a dichroic recombiner 40 (step 208). Contemporaneously, the fixed output 22 is passed through a half-wave plate 30a and polarizing beam splitter (PBS) 32b to control the power of the beam (step 210) and then sent to an electro-optic modulator 50 that employs the Pockels Effect (linear electro-optics effect). For example, using the Pockels Effect, the electro-optic modulator 50 uses a modulation reference signal from a modulation reference signal generator to cause a change in phase of the induced ordinary ray (step 212). In step 214, the output from the electro-optic modulator 50 is subjected to a temporal delay by a computer-controlled delay stage 110.
In step 216, the two outputs are then recombined. Next, in step 218, a positive chirp by changing the sign of one of the outputs. Next, in step 220, the combined pulse interrogates the particles using the chirped-modulation Coherent anti-Stokes Raman Scattering (CARS) signal.
In step 222, the combined pulse propagates through the sample, and is collected by the microscope, and the collected light is filtered by a short-pass filter 140 to remove the Stokes beam (fixed or tunable) (step 224) for detection and amplification. The lock-in amplifier 180 uses the modulation reference signal drive signal for the electro-optic modulator 50 to single out a component of the signal at a specific reference frequency and phase. In step 226, the output from the lock-in amplifier 180 is fed into a data acquisition module 190, which converts any analog signals received into digitized equivalents; and optionally processes such received and digitized signals to improve their signal-to-noise characteristics. In addition, the data acquisition module 190 provides for a temporary storage of the received and digitized signals as associated items in a digital packet identified by its time stamp on an onboard memory module; and the data acquisition module 190 optionally transmits such digital packet to a computer module for further preliminary processing.
Chirp modulation will now be explained in more detail. Generally, in spectral focusing CRM, the pump and Stokes beams are chirped to achieve the desired spectral resolution: the spectral resolution is set by the instantaneous difference frequency between the two beams, as is illustrated in
In operation, CM-SRS effectively subtracts these two different responses since they are out of phase, allowing the peak positions to be recovered, as illustrated by the CM response in
Comparing the peak height of the AM and CM-SRS spectra, it is noticeable that the absolute signal is weaker for CM-SRS: however, the absolute peak height of the CM-SRS response scales linearly with concentration of the molecules of interest, much like it does for AM-SRS microscopy. Indeed, this linear regime is extended to lower concentration limits using CM-SRS microscopy compared to standard AM-SRS approaches because noise is suppressed. As can be seen in
Beyond performing spectroscopy, CM-SRS allows for improved imaging in heterogeneous and complex media. For example, β-carotene is an important phytochemicals of many plants but is challenging to image using SRS microscopy given its large nonlinear response which is dominated by two-photon absorption: even using FM-SRS techniques it can be challenging to remove this non-resonant response because it is highly wavelength dependent and thus subject to the chromatic limitations of FM techniques21. To test the utility of CM-SRS to these types of systems, a slice of sweet potato which contains β-carotene and other compounds was imaged. The Raman response of β-carotene is distinguished by peaks near 1150 cm−1 and 1520 cm−1 22,23. To compare resonant and non-resonant responses, recorded Raman spectra across this range. Using AM-SRS, the presence of phytochemicals is evident by their strong response; however, the response is not specific and is similar at different Raman shifts, as can be seen comparing
In one implementation, in imaging phytochemicals, the Raman signal of interest is strong, yet the non-resonant optical response is much larger, highlighting that CM-SRS can work with strong optical responses. For many biologically relevant systems, however, the optical responses are weak. To test the imaging performance of CM-SRS using weak signals, the uptake of a small molecule into cells is measured. Generally, pharmaceutical drugs are small molecules and used at low concentrations at therapeutic levels. Because the molecules are small, less than one kilodalton, traditional labeling schemes are not applicable: fluorescent labels are larger than these molecules and any measured biological activity would likely reflect the label, not the pharmaceutical drug. Raman labels, however, can be used by modifying the chemical structure of molecule of interest to include groups with Raman responses in the “quiet” region of cells24,25. For example, using a model pharmaceutical drug labeled with nitrile groups, we measured the uptake of the compound into HepG2 cells. The pharmaceutical drug was loaded into the cells by incubating the cells in a 25 mM solution. To measure uptake, the cells are rinsed in fresh media before imaging. Using AM-SRS, non-resonant effects due to the heterogeneous structure of the cell dominate the image and it is not possible to observe the compound. Using CM-SRS, however, the loading of the compound into the cell is monitored over time, reaching a final concentration of 25 mM after approximately 5 hours, as can be seen in
The descriptions of the various implementations of the present disclosure have been presented for purposes of illustration, but are not intended to be exhaustive or limited to the implementations disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described implementations. The terminology used herein was chosen to best explain the principles of the implementations, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the implementations disclosed herein.
Implementations are described above with reference to block diagrams and/or operational illustrations of methods, systems. The operations/acts noted in the blocks may be skipped or occur out of the order as shown in any flow diagram. For example, two or more blocks shown in succession may be executed substantially concurrently or the blocks may sometimes be executed in the reverse order, depending upon the functionality/acts involved. While the specification includes examples, the disclosure's scope is indicated by the following claims. Furthermore, while the specification has been described in language specific to structural features and/or methodological acts, the claims are not limited to the features or acts described above. Rather, the specific features and acts described above are disclosed as example for implementations.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/051772 | 12/2/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63285808 | Dec 2021 | US |
