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WO2025191488A1 - Système de détection brillouin amélioré - Google Patents

Système de détection brillouin amélioré

Info

Publication number
WO2025191488A1
WO2025191488A1 PCT/IB2025/052626 IB2025052626W WO2025191488A1 WO 2025191488 A1 WO2025191488 A1 WO 2025191488A1 IB 2025052626 W IB2025052626 W IB 2025052626W WO 2025191488 A1 WO2025191488 A1 WO 2025191488A1
Authority
WO
WIPO (PCT)
Prior art keywords
detection assembly
frequency
signal
filter
vipa
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2025/052626
Other languages
English (en)
Inventor
Emanuele PONTECORVO
Fabrizio GALA
Giulia ZANINI
Claudia TESTI
Giancarlo RUOCCO
Li Zhang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Crestoptics SpA
Fondazione Istituto Italiano di Tecnologia
Original Assignee
Crestoptics SpA
Fondazione Istituto Italiano di Tecnologia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Crestoptics SpA, Fondazione Istituto Italiano di Tecnologia filed Critical Crestoptics SpA
Publication of WO2025191488A1 publication Critical patent/WO2025191488A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/636—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/636—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties
    • G01N2021/638—Brillouin effect, e.g. stimulated Brillouin effect

Definitions

  • the present invention relates to the microscopy field, in particular the Brillouin microscopy.
  • the present invention more specifically, relates to a detection assembly for a Brillouin microscopy apparatus integrating solutions aimed at improving the alignment and stability of the analysis signal and increasing the resolution and the contrast while observing the sample.
  • Brillouin microscopy is a technique which allows to study the mechanical properties of a biological sample [1 ] starting from the inelastic scattering of the incident light, which as result has a shift in frequency of the same, characteristic of the system under examination.
  • the Brillouin shift v B is given by the relation: having v s sound speed in the medium under examination, n refractive index of the medium, E wavelength of the incident light, and 6 scattering angle.
  • This shift is very small, in the order of few GHz, and then very close to the elastic line which is many orders of magnitude more intense (generally by a factor in the order of 10 4 ⁇ 10 6 ).
  • the Brillouin analysis then requires a spectrometer having a high spectral resolution and a high contrast.
  • a typical Brillouin microscopy apparatus is schematized in Figure 1 and consists of a light source 1 , a microscope 3, a module 2 for scanning the sample and a detection assembly 10.
  • laser monochromatic light source
  • the peculiarities of the detection assembly 10 of such system indeed allow to discriminate the Brillouin signal from the elastic one; such group, designated with reference 10, generally results to be composed of a collection fibre 4 carrying the confocality of the final image, a spectrometer 5 (the main element) and a detector 6 (generally a camera) configured for a connection with processing means 7.
  • the spectrometer used in such apparatus consisted of an interferometer of Fabry-Perot type, a selection which on one side guarantees a high spectral resolution and an optimum contrast, on the other it suffers from a low throughput and very high measurement periods of time due to the system for scanning the interferometric cavity, by making its use in biological applications very complex.
  • a typical scheme of a double VIPA spectrometer comprising a Lyot filter in the pupil P is schematized in Figure 2 (the path of the light signal is shown according to two mutually orthogonal planes).
  • the two VIPAs, designated with reference Vi and V2 are aligned orthogonally to each other so as to obtain the final spectral dispersion of the Brillouin signal along a diagonal direction (135° of the horizontal axis if the etalons have the same dispersive power), by keeping instead the elastic signal along the horizontal and vertical axes.
  • each VIPA generally two adjustable rectangular slits Si, S2 are positioned, cutting the elastic signal in the two vertical and horizontal directions, respectively, by letting instead the dispersed Brillouin signal to pass along the diagonal which then can be detected by the camera C with high contrast.
  • the optical system of the spectrometer further comprises a series of asperical, spherical or cylindrical lenses, respectively designated as LA, LS, LC, to modify suitably the shape or to move the spectrum of the light signal as it moves in the optical system.
  • the final output of the spectrometer then consists only of the Brillouin signal and, specifically, of two peaks of Lorentzian shape, one corresponding to the Stokes scattering and related to the order 0 of the elastic line (placed at 0 GHz by definition and cut by the slits) and one to the anti-Stokes one related to the order 1 , as shown by way of example in Figure 3. If on one side the fact of having eliminated completely the elastic light allows to observe Brillouin peaks with a greater contrast, on the other side it removes completely the reference in camera of the incident light (given by the elastic signal) from which it is possible to calculate the shift of the Brillouin signal.
  • the calibration of the spatial dispersion of the frequencies in camera is then typically made by measuring the Brillouin signal of at least two liquids (generally water and methanol) whose shift is perfectly known in scientific literature [6], from which it is possible to reconstruct univocally a curve of pixels/frequencies required to evaluate the Brillouin shifts.
  • the Brillouin spectrometer based on double VIPA can be improved both in terms of contrast and spectral resolution and in terms of stability of the optical alignment.
  • the spatial filter FL generally used to suppress the diffraction of the slits cutting the elastic component and positioned in the pupil P of the optical system, is a circular pinhole and it has as potential disadvantage the fact of cutting the high frequencies coming from the Brillouin signal, with a consequent loss of contrast and a widening of the final line, as briefly schematized in Figure 4 and explained hereinafter.
  • both VIPAs have as input the Fourier transform of the signal to be analysed (being positioned in the focus of a lens).
  • the spectrum scattered by the VIPA (on the left in figure), comprising two elastic peaks E and two Brillouin peaks B, is filtered by the rectangular slit locking the elastic bands.
  • the double VIPA scheme works with the hypothesis that the frequency of the monochromatic light illuminating the sample remains constant overtime.
  • this is true only in first approximation; depending upon the environmental and working conditions, in fact, a spectral drift of the order of 1 GHz can be noted in 30/60 minutes, therefore unpleasant effects and artifacts can appear in the final image produced by the spectrometer during one single data taking.
  • the calibration of the spatial dispersion of the frequencies on the camera downstream of the VIPA should, strictly speaking, be re-calculated, since the origin of the reference system (that is, the position of the elastic peak) moves.
  • the currently standardized calibration is based upon the analysis of two known samples, a time-consuming process complicated to be implemented during a measurement.
  • the calibrations before and after the drift of the laser source if in the order of 1 GHz or the like, are stiff translations of one with respect to the other one, and then the direct knowledge of the value of such translation between the two of them would allow to correct the obtained value of the Brillouin shift precisely.
  • the optical system of the spectrometer In relation to the alignment of the slits, as the frequency of the incident light moves, the optical system of the spectrometer is no more aligned and, in particular, the slits used to cut the elastic peaks will be no more optimized. This can involve amplified edge diffractive effects or even the entry in camera of the elastic contribution, which could ruin the quality of the acquired piece of data, with the consequent information loss in the final fit procedure therethrough the shift map is reconstructed.
  • the misalignment is illustrated by way of example with reference to Figure 6.
  • Figure 6 shows the water Brillouin signals obtained with a double VIPA spectrometer at the beginning of the measurement (a), under the best optical alignment conditions of the spectrometer with the initial frequency of the source, and at the end of the measurement (b), wherein a shifting of the spectrum with consequent entry from the left of the elastic line can be seen clearly.
  • the technical problem placed and solved by the present invention is then to provide a solution which allows to obviate one or more of the drawbacks mentioned above with reference to the known art.
  • the invention relates to an improved detection assembly for a Brillouin microscopy apparatus allowing the optical alignment procedure of the spectrometer to be reliable, fast and simultaneous with the measurement.
  • the invention relates to a detection assembly for a Brillouin microscopy apparatus which provides more contrast and resolution while observing a sample.
  • the detection assembly of the invention is configured to detect a light signal scattered by the sample, which signal comprises an elastic component and an inelastic component, and includes a light source, monochromatic with variable frequency, incident on the sample.
  • the detection assembly further comprises a frequency filtering unit and a frequency dispersion unit, crossed by the scattered signal. In functional terms, such two units cooperate to constitute a spectrometer.
  • said filtering unit can guarantee the selective suppression/removal of determined spectral components and said dispersion unit implements the optical dispersion of said components for the purposes of detection.
  • the frequency filtering unit is to be meant as functional block which selects, removes or attenuates determined spectral components of a signal, with the purpose of improving the contrast, the spectral accuracy or the resolution.
  • the frequency dispersion unit is to be meant as functional block configured to separate the different spectral components of an optical signal in different positions (for example angles or different directions), so that they could be detected singularly or analysed with suitable detection means, such a sensor (for example a camera, photodetector, etc.).
  • suitable detection means such as a sensor (for example a camera, photodetector, etc.).
  • the group then comprises detection means, preferably a camera, configured to detect the signal scattered in a detection area, and a modulation unit configured to provide a reference signal inside the detection area.
  • Said reference signal is a signal with predetermined frequency, simultaneous and associated to the scattered signal.
  • the detection assembly of the invention further comprises a control circuit, in particular a closed-ring circuit, configured to acquire the position of the reference signal in the detection area, to compare it with a predetermined reference position and, based upon the detected deviation, to modify automatically one or more operating parameters of the detection assembly, preferably the frequency of the light source.
  • a control circuit in particular a closed-ring circuit, configured to acquire the position of the reference signal in the detection area, to compare it with a predetermined reference position and, based upon the detected deviation, to modify automatically one or more operating parameters of the detection assembly, preferably the frequency of the light source.
  • the modulation unit generates a reference signal with known frequency (for example, a lateral line).
  • a reference signal with known frequency (for example, a lateral line).
  • Such reference signal provides in real time information about the frequency stability of the detection assembly, fundamental aspect to have a Brillouin spectroscopy apparatus capable of obtaining reproducible and reliable measurements.
  • the reference signal when detected together with the signal coming from the sample, appears in a determinable position on the camera (or other detector).
  • the control circuit is configured to receive a piece of stability information from such reference signal and to adjust automatically one or more operating parameters of the detection assembly to compensate for frequency and/or instability drifts of the latter.
  • the optical system is stable, such reference signal remains in a constant (predetermined) position (or within a very limited range).
  • the position of such reference signal moves in a correlated way.
  • the above-mentioned “stability information” corresponds to the measurement of how much the reference signal deviates from the predetermined position. Such deviation provides a feedback which the control circuit uses to compensate or correct the instability (for example by varying the frequency of the source or by adjusting other parameters). In this way the detection assembly can be always aligned optically and it does not require manual corrections by the user.
  • the invention provides that the source can be adjusted in frequency exactly to be advantageously used as active element in a closed- ring control circuit. Such circuit allows to bring automatically the signal spectrum back in wished position and to avoid misalignments, in particular, returns of the elastic component.
  • both the frequency filtering unit and the frequency dispersion unit can include one or more interferometric filters.
  • both the filtering unit and the dispersion unit comprise an interferometric filter, for example two VIPAs in cascade, the detection assembly assumes a “double VIPA” configuration.
  • the light source can be stabilized in frequency upstream of the filtering unit, for example by locking it through a frequency locking system (lock) to an atomic transition of a rubidium, iodine, etc., vapor absorption cell.
  • a frequency locking system lock
  • Such cell can be considered (in a different optical route) also as “filter” for the signal.
  • the main source of instability typically lies in the components of the detection assembly, which can be subjected to thermal or mechanical variations, with consequent shifting in the spectral separation.
  • the frequency filtering unit comprises an isotope vapour absorption cell
  • the source has to be stabilized upstream of those specific atomic transitions.
  • the filtering unit does not comprise an isotope vapour absorption cell, or it adopts an interferometric filter, the source can be stabilized or cannot be stabilized in frequency.
  • the reference signal in the present invention, is a continuous monitoring capable of detecting instantaneously possible frequency drifts and/or fluctuations of the source or of other optical components along the path of the scattered signal.
  • the control circuit Based upon the comparison between the effective position of the reference signal and the predetermined position, the control circuit adjusts automatically at least an operating parameter of the system (for example the frequency of the source, the temperature of an optical component, the mechanical position of a dispersion element, etc.), thus bringing the system back to the initial alignment conditions and by optimizing firmly the suppression of the elastic line E and the reading of the inelastic line B.
  • an operating parameter of the system for example the frequency of the source, the temperature of an optical component, the mechanical position of a dispersion element, etc.
  • the modulation unit and the reference signal do not limit to generate additional spectral lines (lateral bands) for simple calibration or non-linearity correction purposes, as in case of acquisition of the sample signals at different frequencies to construct a pixel/frequency calibration curve.
  • the modulation unit and the reference signal according to the present invention constitute the key element of a feedback mechanism in real time, that is a control circuit which keeps stable the whole system during the data acquisition.
  • FIG. 1 shows a block diagram of a Brillouin microscopy apparatus known in the art
  • FIG. 2 shows the scheme of a double VIPA spectrometer according a configuration known in the art
  • FIG. 4 shows the spectrum of the scattered signal at a VIPA and its representation in the Fourier optics, in a spectrometer of the type illustrated in Figure 2;
  • FIG. 5 shows the scanning of a Hela cell (a) with artifacts caused by the calibration loss of the spatial dispersion during the measurement, the frequency drift of the laser source (b) and a histogram showing an artificial bi-modal distribution of the shifts obtained by measuring only the water surrounding such cell (c);
  • FIG. 6 shows the misalignment of the Brillouin signals of the water spectrum, obtained with a known double VIPA spectrometer at the beginning of the measurement (a) and at the end of the measurement (b);
  • FIG. 7 shows a block diagram of apparatus for Brillouin microscopy comprising a detection assembly according to an embodiment of the invention
  • Figure 8 shows the scheme of a double VIPA spectrometer according a configuration of the invention
  • FIG. 10 shows the scanning of the Hela cell of Figure 5 through the detection assembly of the present invention
  • FIG. 11 and Figure 12 show graphs related to numeral simulations related to the resolution and contrast performance of the optical scheme of the spectrometer, respectively, according to the known art and according to an embodiment of the present invention.
  • FIG. 7 a block diagram of an apparatus for Brillouin microscopy is illustrated comprising a detection assembly 100 according to a preferred embodiment of the invention.
  • Such embodiment relates to an exemplifying configuration of the invention, wherein a possible embodiment of a control circuit of the detection assembly 100 has been implemented.
  • the example described hereinafter relates to a detection assembly 100 comprising a modular light source 11 which is not stabilized in frequency, wherein a (frequency) filtering unit and a (frequency) dispersion unit of a spectrometer 50 comprise, each one, an interferometric filter Vi, V2, in particular a VIPA, by determining a double VIPA layout of the detection assembly 100.
  • control circuit allows to use the stability information provided by the shifting of a reference signal C on a camera 6 to modify the frequency of the light source 11 and to bring the detection assembly 100 back to the initial working conditions.
  • the invention generally, advantageously allows to implement a control circuit adapted to stabilize the detection assembly 100 and having as input the reference signal C.
  • Such control circuit then can be implemented with different configurations (layout) with respect to the now described configuration.
  • the invention is implemented by providing the presence of stabilized light sources and/or combinations of different (type and number) interferometric filters both of the filtering unit and of the dispersion unit, with respect to the herein described ones.
  • control circuit of the detection assembly of the invention 100 is configured to detect a spatial and/or spectral deviation of the reference signal C with respect to a nominal position and, based upon such deviation, to adjust automatically one or more operating parameters of the detection assembly 100.
  • control circuit allows to modify an operating parameter different from the frequency of the light source 11 , for example the temperature or the position of one or more components or optical elements of the detection assembly 100, to re-align the latter in case of shifting of the reference signal C.
  • the reference signal C has the function of monitoring signal of the instability of the detection assembly 100 and of input signal of the control circuit.
  • the closed-ring control circuit is then configured to keep the reference signal C in said predetermined position inside the detection area and to vary one or more operating parameters of the detection assembly 100 when a deviation of the above-mentioned reference signal C is detected.
  • the detection assembly 100 will be described hereinafter mainly with reference to the the distinctive technical features of the various embodiments of the invention with respect to what already illustrated previously in relation to the detection assembly of the known art and illustrated with reference to Figure 1 and Figure 2.
  • the detection assembly 100 is configured to detect a light signal scattered by a sample.
  • the signal detected by the group 100 is a scattered signal which comprises an elastic component E and an inelastic component B (Brillouin).
  • Such scattered signal is designated with reference A in Figure 8 which as a way of example illustrates schematically two side-by-side views, orthogonal to each other, of the optical route of said signal A in the spectrometer 50, according to the present exemplifying configuration of the invention.
  • the spectrometer 50 comprises a frequency filtering unit and a frequency dispersion unit of the scattered signal A.
  • both the filtering unit and the dispersion unit comprise an interferometric filter, in particular a VIPA Vi, V2, respectively, by determining a layout of the double VIPA detection assembly 100.
  • interferometric filter is selected among one or more of an etalon, a Fabry-Perot etalon, a diffraction grating, a bi-fringe filter.
  • the frequency filtering unit can comprise, alternatively or in combination with the interferometric filter, an absorption cell with isotopic vapours, for example rubidium or iodine vapours.
  • the filtering unit is configured to suppress the (much more intense) elastic component E or to isolate the Brillouin component B.
  • the dispersion unit is an optical block configured to separate the various components in frequency present in the scattered signal A, by translating the frequency difference into a spatial separation (for example, on the detector plane of the detection means). This allows to detect distinctly the components of interest (Brillouin, elastic or others) through the detection means themselves.
  • the detection assembly 100 further comprises the light source 11 incident on the sample and detection means, in particular the camera 6, to detect the scattered signal A in a detection area.
  • the detection assembly 100 is preferably configured for a coupling with a microscope 3, in particular a confocal microscope, preferably through a scanning module 2.
  • the detection assembly 100 further comprises a modulation unit 9 configured to provide a reference signal C, which can be detected inside the detection area.
  • the reference signal C is a signal with predetermined frequency, simultaneous and associated to the scattered signal A.
  • the modulation unit 9 is preferably electro-optics, it can be of commercial type and it is advantageously guided by a micro-wave generator to guarantee the stability of the reference signal C.
  • the modulation unit is configured to generate a beat with the frequency v E of the elastic component E at a known frequency v 0 , so that the frequencies v E ⁇ v 0 are visible in the detection area of the camera 6 and positioned so as not to interfere with the peaks of the inelastic component B.
  • the detection assembly 100 further comprises a control circuit, in particular a closed-ring control circuit, configured to acquire the position of the reference signal C in the detection area and to compare said position with a predetermined reference position.
  • a control circuit in particular a closed-ring control circuit, configured to acquire the position of the reference signal C in the detection area and to compare said position with a predetermined reference position.
  • control circuit is configured to adjust automatically at least an operating parameter of the detection assembly 100.
  • said operating parameter comprises the frequency of the source 11 .
  • said reference signal C can have a higher frequency than the frequency associated to the inelastic component B and it is preferably centred between the distance between the Stokes and anti-Stokes peaks of such inelastic component B.
  • the closed-ring control circuit preferably comprises a control unit 7 operatively connected to the camera 6 and the source 11 .
  • control unit 7 is configured to adjust automatically the frequency of the source 11 (which, in the considered example, as said, is not stabilized in frequency) so that the reference signal C results in a determined position depending upon the position of the elastic component E of the scattered signal A.
  • the detection assembly 100 allows to adjust the frequency of the source 11 through the implementation of the modulation unit 9 in a closed- ring control circuit.
  • Such control has as reference the centre in frequency of the Gaussians used to fit the electro-optically modulated reference signal C. In this way no spectrum detected by the camera 6 has problems at time of the fit and the reconstruction of the Brillouin shift map.
  • the detection assembly 100 can further comprise specific spatial filtering means.
  • the spatial filtering means is configured specifically to remove the residual elastic component E of the scattered signal A, through their positioning in sequence and asymmetric with respect to the optical axis a of said signal A crossing the spectrometer 50.
  • the spatial filtering means can include various combinations of slits, masks, Lyot filters, pinhole, etc., positioned in distinct planes.
  • said spatial filtering means comprises m slits Sm coupled with corresponding m Lyot filters Fsm, with m > 0 and m ⁇ of the number of dispersion directions of the frequencies used in the detection assembly 100.
  • the filtering means comprises in sequence a first spatial filter and a second spatial filter, wherein said first spatial filter comprises a first slit Si coupled with a first Lyot filter Fsi, and wherein said second spatial filter comprises a second slit S2 coupled with a second Lyot filter Fs2.
  • the first spatial filter Si, Fsi and the second spatial filter S2, Fs2 are advantageously configured to filter the scattered signal according to directions orthogonal to each other.
  • the spatial filtering means can include one or more additional slits, designated by way of example with the reference S3, in order to manage diffraction phenomena in the optical path of the scattered signal A in the spectrometer 50.
  • slit or “more slits” one (or more) geometrical (even adjustable) openings are meant, positioned along the optical path with the purpose of locking physically part of the scattered signal.
  • the slit is oriented so as to cut the elastic component E which is on a specific dispersion axis, by letting the Brillouin signal B, moved in position, to pass through.
  • the frequency filtering unit and the frequency dispersion unit of the spectrometer 50 comprise, each one, a VIPA, Vi, V2, and the spatial filtering means Si, Fsi, S2, Fs2 are associated to a respective VIPA.
  • the spatial filtering means preferably comprises at least a first element in a plane in which there is the image (or its projection) and at least a second element in a substantially Fourier plane.
  • the spatial filtering means includes at least a slit (Si or S2) placed in an intermediate image plane and at least a respective Lyot filter (Fsi, Fs2) positioned in a pupil plane (Fourier plane), coupled and configured to remove selectively the elastic component E.
  • the first Lyot filter Fsi of the first spatial filter is positioned in the pupil Pi of the first VIPA Vi and the second Lyot filter Fs2 of the second spatial filter is positioned in the pupil P2 of the second VIPA V2.
  • Said slit and said Lyot filter form two spatial filtering stages in cascade so as to suppress progressively the elastic component E while maintaining the inelastic component B.
  • a mask is meant, for example a pinhole or a fissure or a customized shape, placed in a Fourier plane (pupil) of the optical system, where the signal appears in its Fourier transform.
  • the herein illustrated embodiment of the invention provides that the spectrometer 50 integrates Lyot filters Fsi and Fs2 positioned in the pupils of the VIPA Vi, V2 and shaped to exclude exclusively the elastic component E comprised in a half-plane orthogonal to the optical axis a.
  • the coupling of a slit to a Lyot filter determines two levels of spatial filtering.
  • the width of the elastic signal E was selected as 10 4 times higher than the signals to be detected at wavelengths shifted by a typical value of the water Brillouin signal.
  • the model was implemented according to the treatment followed in [7] for a Gaussian beam incident on a VIPA.
  • Figure 11 showing ( Figure 11 c) the results in camera 6 of the first numerical simulation with the optical scheme of the spectrometer 5 according to the known art
  • Figure 12 showing ( Figure 12d) the results in camera 6 of the second numerical simulation with the optical scheme of the spectrometer 50 exemplifying a configuration of the detection assembly of the invention.
  • the invention concentrates specifically on the improvement of the contrast and spectral resolution while observing the sample, thanks to the use of spatial filtering means provided in combination with a high resolution spectrometer.
  • Such configuration of the detection assembly 100 is not illustrated in an enucleated way in Figures and it is described hereinafter by using, for the elements/units/common components with the exemplifying implementation of the detection assembly 100 described above and illustrated in Figure 7 and Figure 8, the same numeral references.
  • the invention then relates to a detection assembly 100 which comprises a light source 11 (for example a laser of fixed or variable wavelength), incident on the sample and a spectrometer 50 configured for the separation of the elastic component E and of the inelastic component B of the scattered signal A from the sample.
  • a light source 11 for example a laser of fixed or variable wavelength
  • a spectrometer 50 configured for the separation of the elastic component E and of the inelastic component B of the scattered signal A from the sample.
  • the detection assembly 100 is preferably configured for a coupling with a microscope 3, in particular a confocal microscope, preferably through a scanning module 2.
  • Said spectrometer 50 includes a frequency filtering unit and a frequency dispersion unit of the scattered signal A.
  • both the filtering unit and the dispersion unit comprise an interferometric filter, in particular a VIPA Vi, V2, respectively, by determining a layout of the double VIPA detection assembly 100.
  • interferometric filter is selected among one or more of an etalon, a Fabry-Perot etalon, a diffraction grating, a bi-fringe filter.
  • the frequency filtering unit can include, alternatively or in combination with the interferometric filter, an absorption cell with isotopic vapours, for example rubidium or iodine vapours.
  • the group 100 comprises detection means (for example a camera 6) to detect said scattered signal A in a detection area.
  • detection means for example a camera 6
  • the detection assembly further comprises spatial filtering means.
  • the spatial filtering means is configured specifically to remove the residual elastic component E of the scattered signal A, through a positioning in sequence and asymmetrical with respect to the optical axis a of said signal A crossing the spectrometer 50.
  • This configuration allows to maximize the contrast - by removing most elastic light - and to preserve the spectral resolution, by avoiding phenomena of widening the inelastic lines due to too invasive spatial filtering.
  • the main advantage in fact, lies in the geometrical and functional configuration of the spatial filtering means, which insert in a, for example double VIPA, spectrometer, by further raising the performances thereof in terms of elastic light suppression with respect to the known art.
  • the spatial filtering means can include various combinations of slits, masks, Lyot filters, pinhole, etc., positioned in distinct planes.
  • said spatial filtering means comprises m slits Sm coupled to corresponding m Lyot filters Fsm, with m > 0 and m ⁇ of the number of dispersion directions of the frequencies used in the detection assembly 100.
  • the filtering means comprises in sequence a first spatial filter and a second spatial filter, wherein said first spatial filter comprises a first slit Si coupled with a first Lyot filter Fsi, and wherein said second spatial filter comprises a second slit S2 coupled with a second Lyot filter Fs2.
  • the first spatial filter Si, Fsi and the second spatial filter S2, Fs2 are advantageously configured to filter the scattered signal according to directions orthogonal to each other.
  • the spatial filtering means can comprise one or more additional slits, by way of example designated with reference S3, to manage diffraction phenomena in the optical path of the scattered signal A in the spectrometer 50.
  • slit or “several slits” one (or more) geometrical (even adjustable) openings is meant, positioned along the optical path with the purpose of locking physically part of the scattered signal.
  • the slit is oriented so as to cut the elastic component E which is on a specific dispersion axis, by letting the Brillouin signal B, moved in position, to pass through.
  • the frequency filtering unit and the frequency dispersion unit of the spectrometer 50 can include, each one, a VIPA, Vi, V2, and the spatial filtering means Si , Fsi, S2, Fs2 are associated to a respective VIPA.
  • the spatial filtering means preferably comprises at least a first element in a plane wherein there is the image (or its projection), and at least a second element in a substantially Fourier plane.
  • the spatial filtering means includes at least a slit (Si or S2) placed in an intermediate image plane and at least a respective Lyot filter (Fsi, Fs2) positioned in a pupil plane (piano di Fourier), coupled and configured to remove selectively the elastic component E.
  • the first Lyot filter Fsi of the first spatial filter is positioned in the pupil Pi of the first VIPA Vi and the second Lyot filter Fs2 of the second spatial filter is positioned in the pupil P2 of the second VIPA V2.
  • Said slit and said Lyot filter form two spatial filtering stages in cascade so as to suppress progressively the elastic component E while maintaining the inelastic component B.
  • a mask is meant, for example a pinhole, a fissure, a customized shape, placed in a Fourier plane (pupil) of the optical system, where the signal appears in its Fourier transform.
  • the herein illustrated embodiment of the invention provides that the spectrometer 50 integrates Lyot filters Fsi and Fs2 positioned in the pupils of the VIPAs Vi, V2 and shaped to exclude exclusively the elastic component E comprised in a half-plane orthogonal to the optical axis a.
  • the coupling of a slit with a Lyot filter determines two spatial filtering levels.
  • the width of the elastic signal E was selected as 10 4 times higher than the signals to be detected at wavelengths shifted by a typical value of the water Brillouin signal.
  • the model was implemented according to the treatment followed in [7] for a Gaussian beam incident on a VIPA.
  • Detection assembly 100 for a Brillouin microscopy apparatus configured to detect a light signal A scattered by a sample and comprising an elastic component E and an inelastic component B, which detection assembly 100 comprises
  • ⁇ detection means 6 configured to detect said scattered signal A in a detection area
  • ⁇ spatial filtering means Si, Fsi, S2, Fs2 positioned in sequence and asymmetrically with respect to the optical axis a of the scattered signal A and configured to remove said elastic component E.
  • Detection assembly 100 wherein said spatial filtering means preferably comprises in sequence a slit (Si , S2) coupled with a Lyot filter (Fsi, Fs2).
  • Detection assembly 100 wherein said frequency filtering unit and said frequency dispersion unit comprise, preferably each one, a VIPA Vi, V2 and said spatial filtering means (Si , Fsi), (S2, Fs2) are associated to a respective VIPA Vi, V2.
  • the spatial filtering means comprises preferably in sequence a first (Si, Fsi) and a second (S2, Fs2) filter configured to filter according to directions orthogonal to each other.
  • Detection assembly 100 wherein preferably a first Lyot filter Fsi is positioned in the pupil Pi of the first VIPA Vi and a second Lyot filter Fs2 is positioned in the pupil P2 of the second VIPA V2.
  • Detection assembly 100 wherein said spatial filtering means (Si, Fsi, S2, Fs2) is preferably configured to exclude exclusively the elastic component E comprised in a half-plane orthogonal to the optical axis a.
  • the present invention has been sofar described with reference to preferred embodiments. It is to be meant that other embodiments belonging to the same inventive concept can exist, as defined by the protective scope of the herebelow reported claims.

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Abstract

La présente invention concerne un ensemble de détection amélioré pour un appareil de microscopie Brillouin, ledit ensemble améliorant l'alignement et la stabilité du signal d'analyse et permettant également d'obtenir un contraste et une résolution supérieurs tout en observant un échantillon.
PCT/IB2025/052626 2024-03-14 2025-03-12 Système de détection brillouin amélioré Pending WO2025191488A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017139747A1 (fr) * 2016-02-12 2017-08-17 The General Hospital Corporation Système pour effectuer une spectroscopie
WO2019036714A1 (fr) * 2017-08-18 2019-02-21 The General Hospital Corporation Systèmes et procédés de spectroscopie brillouin et d'imagerie de tissus
US20200018685A1 (en) * 2015-12-22 2020-01-16 Canon U.S. Life Sciences, Inc. System and method of label-free cytometry based on brillouin light scattering
WO2022031815A1 (fr) * 2020-08-04 2022-02-10 University Of Maryland, College Park Systèmes et procédés de microscopie à champ complet de brillouin

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
US20200018685A1 (en) * 2015-12-22 2020-01-16 Canon U.S. Life Sciences, Inc. System and method of label-free cytometry based on brillouin light scattering
WO2017139747A1 (fr) * 2016-02-12 2017-08-17 The General Hospital Corporation Système pour effectuer une spectroscopie
WO2019036714A1 (fr) * 2017-08-18 2019-02-21 The General Hospital Corporation Systèmes et procédés de spectroscopie brillouin et d'imagerie de tissus
WO2022031815A1 (fr) * 2020-08-04 2022-02-10 University Of Maryland, College Park Systèmes et procédés de microscopie à champ complet de brillouin

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Title
GIULIANO SCARCELLI ET AL: "Multistage VIPA etalons for high-extinction parallel Brillouin spectroscopy", OPTICS EXPRESS, 23 May 2011 (2011-05-23), United States, pages 10913 - 86, XP055484512, Retrieved from the Internet <URL:https://opg.optica.org/oe/fulltext.cfm?uri=oe-19-11-10913> DOI: 10.1364/OE.19.010913 *
HASE EIJI ET AL: "Molecular imaging analysis of lipid droplets in nonalcoholic fatty liver disease by use of Brillouin scattering microscopy", 20240312, vol. 12844, 12 March 2024 (2024-03-12), pages 128440C - 128440C, XP060205031, ISSN: 1605-7422, ISBN: 978-1-5106-6947-5, DOI: 10.1117/12.3001190 *

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