Apparatus and method for wide-field brillouin spectroscopy

Abstract

Optical apparatus (1) designed for wide-field Brillouin spectroscopy that comprises an illumination optical assembly (2), configured to illuminate an extended region of the sample to be analyzed (8), rather than a single point, thereby enabling wide-field measurements. The light scattered by the illuminated sample (8) is then collected by a collection and collimation optical assembly (3), which receives the scattered signal and collimates it to ensure proper propagation through the subsequent optical components. The collimated light signal is subsequently directed to a filtering optical assembly (4), which suppresses or significantly reduces the elastically scattered Rayleigh component, while transmitting the inelastically scattered Brillouin component that carries the relevant spectroscopic information. The filtered signal is then sent to a spectrometer (5), configured to introduce a variable phase delay to the light signal while maintaining a common optical path. This common-path configuration enhances stability and reduces system complexity. The apparatus includes a spectral image acquisition device (6), adapted to capture a plurality of frames. For each phase delay introduced by the spectrometer (5), the corresponding transmitted light signal is acquired. Each captured frame is therefore associated with a specific frequency component determined by the applied phase delay, allowing reconstruction of the spectral image of the sample.

Description

[0001] SPECTO S.R.L., Case:061596WO

[0002] APPARATUS AND METHOD FOR WIDE-FIELD BRILLOUIN SPECTROSCOPY

[0003] TECHNICAL FIELD

[0004] The present invention relates to an apparatus and a method for wide-field Brillouin microscopy, particularly for simultaneously acquiring multiple Brillouin spectra. Furthermore, the present invention relates to an optical device comprising said apparatus.

[0005] BACKGROUND ART

[0006] Brillouin spectroscopy is an optical technique used to study the mechanical properties of materials. This technique is based on the inelastic scattering of light, which occurs when photons interact with spontaneous acoustic waves (acoustic phonons) within a material representing the sample. While elastically scattered light (Rayleigh scattering) maintains the same frequency as the incident beam, the Brillouin spectrum reveals two sidebands, called Stokes peaks (SB) and Anti -Stokes peaks (ASB), slightly shifted — about 1-20 GHz — from the Rayleigh frequency (R). The frequency and width of these peaks contain valuable information about the viscoelastic properties of the sample. Once the material's density and refractive index are known, the Brillouin spectral bands allow the calculation of the elastic moduli that characterize the elastic tensor within the volume of the analyzed sample, without the need to apply a contact force to the sample or use markers.

[0007] Traditional methods for measuring the mechanical properties of a material require direct contact with the sample, making them invasive and limited to superficial analysis. Brillouin spectroscopy, on the other hand, uses light as a probe, avoiding any physical contact. This approach, when applied to microscopy or endoscopy techniques, allows for three-dimensional analysis of biological systems, such as cells and tissues, with optical resolution below one micron. Furthermore, as biomechanical properties are closely related to various diseases, including atherosclerosis, cancer, and glaucoma, Brillouin spectroscopy represents a promising diagnostic technology for the biomedical field.

[0008] In Brillouin microscopy, the sample is illuminated by a laser beam focused on a point. The sample is scanned in various directions of space (XYZ) using a motorized device (stage) or through systems with galvanometric mirrors. For each illuminated point on the sample, aSPECTO S.R.L., Case:061596WO

[0009] Brillouin spectrum is acquired, providing information about the mechanical properties of the illuminated region. At the end of the scan, a false-color image can be generated that represents, for example, the longitudinal modulus of the sample, from which information about stiffness and viscosity can be derived.

[0010] However, the acquisition of a single Brillouin spectrum, for example using the VIPA spectrometer commonly used in microscopy, typically requires a camera integration time of about 100 ms. This means that acquiring an entire image composed of more than one million (105) points (spectra) typically takes over 5 minutes.

[0011] This limits the application of Brillouin microscopy to the analysis of biological phenomena on long time scales or on fixed samples.

[0012] Recently, new methods called " Line -scanning Brillouin microscopy" have been introduced, allowing the acquisition of an entire illuminated line of the sample [Zhang, J. et al. Nature Methods, 20(5), pp.677-681, 2023; and Bevilacqua, C. et al. Nature Methods, 20(5), pp.755-760, 2023], More specifically, this technique enables scanning along a single direction, namely the transverse direction to the illumination line. This method has been achieved through the extension of the VIPA spectrometer, which disperses an entire scattering line along its axis. In this way, unlike the acquisition of individual Brillouin spectra at each point of the sample, the acquisition can be accelerated by simultaneously detecting Brillouin spectra along the laser’s illumination line. However, although scanning times are reduced, this method does not allow for instantaneous acquisition.

[0013] Bevilacqua, C. et al. Nature Photonics, 19, pp.494-501, 2025 describe a new approach (called Fourier-transform Brillouin microscopy) for rapid spectral acquisition of scattered Brillouin light. Specifically, by utilizing a custom-built Fourier-transform spectrometer and the symmetric properties of the Brillouin spectrum, they achieved 2D spectral imaging of biological samples with an acquisition speed of up to 40,000 spectra per second and a precision of about 70 MHz over a field of view of approximately 300 x 300 pm2. The authors use a Michelson interferometer that splits the light beam to be analyzed into two arms with different optical path lengths, introducing a phase delay that generates an interferogram. The Brillouin spectrum is then obtained by applying a Fourier transform to this interferogram. This separation, however, introduces instability into the system due to thermal and mechanical drifts, and it also makes the device bulkier and slower. In fact, the spectral resolution of this device is directly proportional to the scanning distance of one of the interferometer arms.SPECTO S.R.L., Case:061596WO

[0014] The document reports an effective optical path difference of 300 mm, which results in a spectral resolution of approximately 1.5 GHz (well below that of conventional devices) and allows a 2D image to be acquired by coupling the interferometer to an array of detectors in 15.5 seconds. US 2024 / 0319094 Al describes a device for wide-field Brillouin microscopy, which includes a spectrally-selective assembly comprising a gas (or vapor) illuminated by pump light corresponding to an atomic electronic transition of the gas. Instead of relying on spatial separation to discriminate the entire Brillouin spectrum for a single point of the sample, the method described in this document exploits the narrow-linewidth transmission characteristics resulting from the interaction of the pump light with the gas, in order to isolate the scattered Brillouin light collected from multiple points of the sample for subsequent detection. While this document promises excellent performance in terms of spectral acquisition time and frequency stability, the system described is complex to operate because it requires laser frequency scanning. Moreover, rubidium gas exhibits multiple transmission and absorption bands, meaning that the Brillouin spectral peaks could be suppressed by the gas itself, creating significant uncertainty in the measurements. This system can also be used only at a specific laser wavelength (780 nm), since the transmission bands of rubidium are located around that wavelength. This restricts the applicability of the system across the entire visible range, where higher spatial resolution can be achieved.

[0015] Therefore, the object of the present invention is to provide an apparatus and a method for Brillouin spectroscopy that significantly reduces the acquisition time of an entire spectral image. Furthermore, the object of the present invention is to provide an apparatus that is compact and easy to use. Additionally, the object of the present invention is to provide a method that is more stable and precise, as well as adaptable to all wavelengths in the visible range, thereby enabling high spatial resolution.

[0016] SUMMARY

[0017] These objectives are achieved by an apparatus, an optical device, and a method for wide-field Brillouin spectroscopy according to the claims at the end of this description.

[0018] In a first aspect of the invention, an optical apparatus for wide-field Brillouin spectroscopy is provided, wherein the apparatus comprises:

[0019] an illumination optical assembly to illuminate an extended region of a sample to be analyzed;SPECTO S.R.L., Case:061596WO

[0020] a collection and collimation optical assembly to receive a scattered light signal from the illuminated sample and collimate the light signal;

[0021] a filtering optical assembly to receive the collimated light signal, suppress or reduce the elastic Rayleigh component from the light signal, and transmit the inelastic Brillouin component; a common-path spectrometer to introduce a variable phase delay to the light signal; and a spectral image acquisition device to capture a plurality of frames, wherein the light signal transmitted by the spectrometer is acquired for each phase delay introduced by the spectrometer, and wherein each frame captured by the acquisition device corresponds to a frequency determined by the phase delay introduced by the spectrometer.

[0022] In a second aspect of the invention, a method for wide-field Brillouin spectroscopy is provided, wherein the method comprises:

[0023] illuminating an extended region of a sample to be analyzed;

[0024] collecting and collimating a scattered light signal from the illuminated sample; suppressing or reducing the elastic Rayleigh component from the light signal and transmitting the inelastic Brillouin component;

[0025] introducing a variable phase delay to the light signal using a common optical path via a spectrometer; and

[0026] acquiring a spectral image of the sample by an acquisition device to capture a plurality of frames, wherein the light signal is acquired for each phase delay introduced by the spectrometer and wherein each frame captured by the acquisition device corresponds to a frequency determined by the phase delay introduced by the spectrometer.

[0027] In a third aspect of the invention, an optical device, particularly a microscope, is provided, comprising the optical apparatus according to the first aspect, wherein each frame captured by the acquisition device corresponds to a frequency determined by the phase delay introduced by the spectrometer.

[0028] Thanks to this apparatus and method, it is possible to acquire the entire spectral content of an extended region of the sample without the need for any scanning. Indeed, this technique allows for the analysis of a large area of the illuminated sample without performing a point-by-point scan. By “extended area”, it is meant a portion of the sample that is large enough to be analyzed through a single frame without the need for point-by-point scanning. In practice, this refers toSPECTO S.R.L., Case:061596WO

[0029] an area sufficiently large to encompass one or more cells, for example, a region of interest with a length ranging from 0.01 to 1 mm.

[0030] This enables the maximization of the acquisition speed, making it possible to obtain an entire Brillouin image, even composed of millions of points (spectra), in an almost instantaneous time, i.e., less than a second, thus significantly improving the efficiency of the spectral image acquisition process. Alternatively, with methods currently known in the art, acquiring the entire image would take on the order of several minutes, or even hours, depending on the number of spectra acquired during the scanning phase.

[0031] Moreover, the use of a common-path spectrometer allows a single shared optical path for the light signal, thereby reducing the overall system complexity. This is in contrast to Michelson interferometers used for Fourier-transform spectroscopy, where the optical waves must be separated along different paths and then carefully recombined to produce the desired interference pattern.

[0032] Because the spectrometer operates in a common-path configuration, and since the optical path difference introduced by the cavity is typically about 3 cm, one order of magnitude smaller than the ~30 cm required in a Fourier-transform system, much faster acquisition speeds can be achieved. In fact, all spectral information can be collected simultaneously within a single frame. Additionally, using a common optical path for all light waves minimizes instability effects arising from small variations in separate optical paths due to thermal or mechanical drifts, as is often the case in Fourier interferometers. This enables a spectral resolution below 0.5 GHz, compared to approximately 1.5 GHz for a Fourier-transform spectrometer, resulting in more precise and reliable spectral measurements.

[0033] Finally, in common-path spectrometers, all optical components are aligned along a single shared path, simplifying the correction of optical distortions and aberrations. In interferometers with separate arms, distortions in one path can negatively affect the interference quality and measurement accuracy, while also increasing the overall system footprint.

[0034] These and other aspects of the present invention will become more apparent in light of the following description of some preferred embodiments described below.

[0035] BRIEF DESCRIPTION OF DRAWINGS

[0036] Fig. 1 shows a schematic representation of an optical apparatus for wide-field Brillouin spectroscopy according to one example.SPECTO S.R.L., Case:061596WO

[0037] Fig. 2A schematically shows an optical apparatus in a configuration that includes a beam splitter.

[0038] Fig. 2B schematically shows the frames captured by the acquisition device and the Brillouin spectral image.

[0039] Fig. 3 A shows a sample illumination scheme according to one example.

[0040] Fig. 3B shows a sample illumination scheme according to an alternative example.

[0041] Fig. 4 shows a detail of the optical assembly for the collection and collimation of the scattered light beam.

[0042] Fig. 5A shows a schematic representation of a spectrometer consisting of an FP interferometer. Fig. 5B schematically shows the operation of the spectrometer consisting of an FP interferometer.

[0043] Fig. 6 schematically shows the operation of the spectrometer consisting of an etalon.

[0044] Fig. 7 schematically shows the operation of the spectrometer consisting of a Lyot filter.

[0045] Fig. 8 shows the transmission profile of the Lyot filter as the result of the sum of the sinusoidal transmission functions of each block consisting of a birefringent crystal.

[0046] Fig. 9 shows a flowchart of the method for wide-field Brillouin spectroscopy according to one example.

[0047] Fig. 10A shows a calibration image according to one example.

[0048] Fig. 10B shows the application of the calibration to the spectral image.

[0049] DESCRIPTION OF THE EMBODIMENTS

[0050] Figure 1 illustrates a representative diagram of an optical apparatus 1 for wide-field Brillouin spectroscopy. The optical apparatus 1 comprises several optical elements used to investigate the chemical and physical properties of a sample 8. First, the apparatus 1 includes an illumination optical assembly 2 having a light source to illuminate an extended region of the sample 8 to be analyzed. Figure 1 shows an illumination optical assembly 2 positioned laterally at an oblique angle relative to the sample 8. However, this is just an exemplary configuration. The illumination may instead follow different and specific optical paths; for example, the sample 8 can be illuminated by a light signal along an optical path at 90 degrees or 180 degrees,SPECTO S.R.L., Case:061596WO

[0051] as will be illustrated later. Following illumination by the illumination optical assembly 2, the sample 8 emits scattered light, which is received by another optical assembly, namely the collection and collimation optical assembly 3.

[0052] Advantageously, the collection and collimation optical assembly 3 is positioned along an optical path orthogonal to the illuminated surface of the sample 8. This optical assembly 3 is responsible for collecting and collimating the light signal coming from the sample 8.

[0053] Subsequently, the elastic Rayleigh scattered light is suppressed (or reduced). To this end, the optical apparatus 1 includes a filtering optical assembly 4. For example, a band-rejection filter (notch filter) can be used to eliminate the elastic Rayleigh component and transmit the inelastic Brillouin component.

[0054] The apparatus 1 further includes a spectrometer 5, positioned downstream of the filtering optical assembly 4, to analyze the transmitted light containing the Brillouin bands. The spectrometer 5 is of the "common-path" type. Unlike spectrometers based on, for example, Fourier transform methods, such as the Michelson interferometer, the present spectrometer 5 does not require the splitting of the light beam along separated optical paths. Specifically, the spectrometer 5 is configured to introduce a variable phase delay to the light signal using a common optical path. In other words, in the common-path spectrometer 5, the interfering waves travel along the same optical axis sharing the same optical environment. In contrast, in spectrometers that do not use a common path, the interfering waves travel along spatially separated paths before recombining, which increases the size, complexity of the optical setup, the instability and therefore inaccuracy in the measurements.

[0055] Additionally, the apparatus 1 includes a spectral image acquisition device 6 to capture the light signal transmitted by the spectrometer 5, specifically to capture a plurality of frames. The acquisition occurs for each phase delay introduced by the spectrometer 5. Specifically, each frame captured by the acquisition device 6 corresponds to a frequency determined by the phase delay introduced by the spectrometer 5.

[0056] It is noted that, similarly to the collection and collimation optical assembly 3, the filtering optical assembly 4, the spectrometer 5, and the acquisition device 6 are also positioned along an optical path orthogonal to the illuminated surface of the sample 8.

[0057] The optical apparatus 1 described here is therefore compact and configured for the ultra-fast acquisition of Brillouin images without the need to perform any scanning on the sample 8.SPECTO S.R.L., Case:061596WO

[0058] Advantageously, the optical apparatus 1 in Figure 1 can be part of an optical device 7, such as a microscope, for analyzing the mechanical properties of a sample 8 using the Brillouin technique. This could have significant scientific and industrial implications, as such a microscopy system would enable the analysis of the mechanical dynamics of living biological systems on a microscopic scale and on time intervals shorter than a second.

[0059] In one example, the illumination optical assembly 2 may include a laser light source 9 that emits a single-frequency light beam with a spectral linewidth of less than 100 MHz and a wavelength in the visible or near-infrared range. It is noted that due to the shift of several GHz in the Brillouin spectra, the laser source must have a spectral linewidth that is not too broad, for example, less than 100 MHz, in order to resolve these spectral bands.

[0060] With reference to Figure 2A, the laser beam, deflected by a beam splitter 10, illuminates a large area of the sample 8 through an objective 13. The objective 13 is part of the collection and collimation optical assembly 3 and therefore also serves to collect the scattered light from the sample 8 along the illuminated area. To minimize collection losses, the beam splitter 10 is used to transmit the scattered light. The beam splitter 10 can, for example, be a polarizing beam splitter (PBS). Compared to standard beam splitters (BS), which have reduced efficiency, the polarizing beam splitter (PBS) has the advantage of not losing the illumination and collection light signals.

[0061] The filtering optical assembly 4 may include a common-path notch filter to suppress (or reduce) the elastic Rayleigh light component, which would otherwise completely overshadow the inelastic Brillouin light signal. In one example, the filtering optical assembly 4 may include a cell containing a gas for the selective absorption of the elastic Rayleigh component. Specifically, the cell may contain a gas that absorbs light at a specific resonance frequency coinciding with that of the laser beam. For instance, a rubidium-containing cell can be used to absorb the background signal at wavelengths around 780 nm, or iodine can be used for wavelengths around 532 nm. Alternatively, the filtering optical assembly 4 may include one or more birefringent crystals to decouple the polarization of the Rayleigh component from that of the Brillouin component. In this way, the Rayleigh component can be suppressed using a polarizer. For example, the birefringent crystal may include YV04 or calcite. These types of filters are both common-path and therefore do not require the spatial separation of the collected image into multiple optical paths. Their main advantage lies in achieving very high levels of suppression of the Rayleigh component.SPECTO S.R.L., Case:061596WO

[0062] The Brillouin light transmitted by the filtering optical assembly 4 is spectrally analyzed through the common-path spectrometer 5. This type of spectrometer 5 introduces phase delays (p to the light signal being analyzed without the need to split the beam into two or more separate optical paths, thus using a common optical path. The signal transmitted by the spectrometer 5 is then acquired by an acquisition device 6 for each phase delay introduced by the spectrometer 5. The acquisition device 6 may be a camera or video camera (such as a CCD, EMCCD, CMOS, or a SPAD array). Consequently, each pixel of the camera or video camera detects the Brillouin scattering light signal at a specific point of the sample 8 transmitted at the frequency v(<p), determined by the phase delay (p applied by the spectrometer 5.

[0063] As shown in Figure 2B, each frame 12 captured by the acquisition device 6 corresponds to a frequency determined by the phase delay introduced by the spectrometer 5. By scanning the entire frequency range of interest for Brillouin spectroscopy (typically v = 0-50 GHz), it is possible to reconstruct a spectral image 11 of the sample 8. Specifically, the acquisition of the spectral image 11 of the sample 8 involves the detection of a set of Brillouin spectra, each associated with a different point on the sample 8 by a full phase scan performed by the spectrometer 5. A spectrum is determined for a specific pixel once the phase scan is completed with the spectrometer 5, so at the end of the scan, each pixel corresponds to a different Brillouin spectrum associated with a specific point on the sample 8.

[0064] Consequently, the acquisition of the spectral image 11 of the sample 8, that is, the image formed by the plurality of frames captured by the acquisition device 6, can occur in less than one second. In this spectral image 11, the sample 8 is represented with false colors, which are associated, for example, with the frequency shift, the linewidth of the Brillouin peaks, or the elastic moduli of the sample 8, and thus its viscoelastic properties.

[0065] Figures 3A and 3B illustrate two different illumination configurations. Figure 3A shows a backscattering geometry, where the wavevectors k of the illumination light and the collected light form an angle of 180 degrees. In this configuration, the collimated laser beam is focused through a tube lens 15 onto the front focal plane 14 of the illumination objective 13. This arrangement produces a collimated light beam that illuminates the sample 8. It is noted that the beam can also be non-collimated, although collimation improves performance and is considered the preferred solution.

[0066] Figure 3B, on the other hand, shows a 90-degree scattering geometry, where the sample 8 is illuminated laterally by a first objective 13 A, while the scattered light is collected by a secondSPECTO S.R.L., Case:061596WO

[0067] objective (collection objective) 13B positioned along a direction perpendicular to the incident beam.

[0068] In both configurations, the collection and collimation optical assembly 3 may include an infinity-corrected objective 13, 13B. This type of objective is designed to project parallel light rays (collimated light) when the sample 8 is in focus. This feature is crucial for integration with common-path detection modules, specifically the notch filter in the filtering optical assembly 4 and the spectrometer 5, minimizing potential artifacts caused by phase delays that could arise from different optical paths along the collection optical arm.

[0069] Figure 4 illustrates an example of an optical apparatus 1 in which the scattered light from the sample is collected using an infinity-corrected objective 13. A4f optical system, consisting of a first lens (tube lens) 15 with focal length fl and a second lens 16 with focal length 2, allows for magnification or reduction of the sample 8 image, depending on the magnification ratio M = fl / f2. This optical adjustment, which may be part of the collection and collimation optical assembly 3, enables efficient coupling with the two common-path detection modules, namely the filtering optical assembly 4 and the spectrometer 5.

[0070] As described earlier, the filtering optical assembly 4 includes a notch filter that eliminates the elastic Rayleigh light, which would otherwise overshadow the much weaker inelastic Brillouin scattered light signal. The notch filter can be implemented, for example, with a cell containing a gas such as rubidium or iodine, or it can be made with a plurality of birefringent crystals, such as YV04 or calcite.

[0071] The Brillouin scattered light signal transmitted by the filtering optical assembly 4 is analyzed by the spectrometer 5, which is also common-path, and introduces a variable phase delay (p. This delay allows for the transmission of a single frequency v(<p) associated with that phase delay. The acquisition device 6 (which may include, for example, a CCD, EMCCD, CMOS, or a SPAD array) captures a set of spectral images. Specifically, each frame acquired by the device 6 corresponds to a specific frequency v(<p) determined by the phase delay (p introduced by the spectrometer 5.

[0072] By rapidly varying the phase delay introduced by the spectrometer 5 and detecting the transmitted intensity for each frequency v(<p), it is possible to reconstruct a full spectral image 11 of the sample 8 (Figure 2B). As previously described, this image 11 is represented in false colors, which are associated with the spectral content, such as the frequency shift, the linewidths of the Brillouin peaks, or even the elastic moduli of the sample, such as the longitudinalSPECTO S.R.L., Case:061596WO

[0073] modulus. This hyperspectral image thus provides essential information about the viscoelastic properties of the analyzed sample 8.

[0074] In this configuration, acquiring a Brillouin image is possible without the need to scan the illumination beam across the sample 8. This excludes both the configuration in which the illumination beam moves across the sample 8 via beam scanning and the configuration in which the sample 8 is moved relative to the illumination beam via a motorized stage. A set of Brillouin spectra from the sample 8 is then simultaneously detected through the pixels of the acquisition device 6 by a full phase scan performed by the spectrometer 5.

[0075] In other words, each pixel of the device 6 represents a Brillouin spectrum associated with a specific point on the sample 8. Consequently, the intrinsic limit of points, or Brillouin spectra, in the image is defined by the number of pixels of the acquisition device 6. It is noted that each point in the image corresponds to a Brillouin spectrum from a point on the sample 8.

[0076] According to one example, the spectrometer 5 consists of a Fabry -Perot (FP) interferometer 18. This configuration is shown in Figure 5A. Alens 19, referred to as a "Fourier lens," is placed at the output of the Fabry -Perot interferometer 18 and upstream of the acquisition device 6. This lens 19 performs a F ourier transform on the beams transmitted through the interferometer cavity 18. Each pixel of the acquisition device 6 (e.g., a CCD in a camera or video camera) records the intensity of the light signal transmitted through the Fabry -Perot interferometer 18 for a specific frequency, determined by the optical path introduced by the interferometer cavity 18. Since each pixel of the acquisition device 6 is associated with a point on the sample 8, it is possible to obtain a Brillouin spectral image of the sample 8 by varying the phase delay introduced by the Fabry -Perot interferometer 18.

[0077] Figure 5B illustrates in detail the operation of the spectrometer 5, according to the configuration where the spectrometer 5 is a Fabry -Perot interferometer 18. The collimated light beam S, coming from point pxof the sample 8 and transmitted by the filtering optical assembly 4, enters an optical cavity bounded by two highly reflective mirrors 28, characterized by reflectivities defined by parameters / ?xand R2. The distance between the two mirrors 28 determines the optical path and, consequently, the phase delay ^introduced on the transmitted beam. By varying the separation between the two mirrors Lby an amount Az, different phase delays can be introduced, each associated with constructive interference at a specific frequency

[0078]

[0079] SPECTO S.R.L., Case:061596WO

[0080] Thus, the Fabry -Perot interferometer 18 can be considered an extremely selective narrow-band filter that transmits only a light frequency according to its transmission function:

[0081] 1

[0082] T(v) =

[0083] 1 + Fsin

[0084]

[0085] where

[0086]

[0087] Fis the finesse factor of the Fabry -Perot interferometer, cis the speed of light in a vacuum, and 0is the propagation angle of the light within the interferometer cavity.

[0088] By scanning the cavity of the Fabry -Perot interferometer by translating one or both mirrors 28 along the z-axis, for example, using a piezoelectric material or a mechanical translator, and detecting the intensity of the transmitted light for each discrete value of mirror separation, it is possible to reconstruct over time the Brillouin spectrum B(px) associated with point pxof the sample.

[0089] Similarly, a beam coming from a point on the sample pt different from pjuses the same principle, taking advantage of the transverse extension of the optical cavity to obtain the corresponding Brillouin spectrum, detected by a different pixel of the acquisition device 6. According to an alternative example, the spectrometer 5 consists of an etalon 20, i.e., a "solid" Fabry-Perot interferometer. In this case, the etalon 20 consists of an optical cavity bounded by two highly reflective surfaces 29, with reflectivity defined by parameters Fxand R2, as shown in Figure 6. Unlike the configuration of Figure 5B, the medium separating these surfaces 29 is not air, but rather a material, such as a gas, whose refractive index n varies in response to the application of a voltage or temperature gradient.

[0090] In this configuration, the Brillouin spectrum is not scanned through the physical translation of the reflective surfaces 29, but by varying the refractive index of the medium inside the cavity. This change modifies the phase delay introduced on the input light signal S(pt), thereby altering the resonance frequency of the etalon 20 cavity. Similarly to the previous case, it is then possible to reconstruct over time the Brillouin spectrum B(pt) associated with the point ptof the sample, once the phase delay introduced by the etalon 20 has been scanned.SPECTO S.R.L., Case:061596WO

[0091] According to yet another alternative example, the spectrometer 5 is constituted by a Lyot filter 21. This is shown in Figure 7. In the Lyot filter 21, a first birefringent crystal 22 (BCi) of length Lis positioned with its optical axis oriented parallel to the entrance window and at a 45-degree angle relative to the linear polarization of the incoming Raman (R) and Brillouin (SB, ASB) optical signals. The birefringent crystal 22 introduces a phase delay in the incoming signals, so that an outgoing polarizer 24 (Pi) defines a sinusoidal transmission function, the period of which is determined by the free spectral range (FSR), given by:

[0092]

[0093] where An is the birefringence of the crystal. This process is repeated N times with birefringent crystals 22, each with a length defined by L / i, where iis the i-th crystal placed within the Lyot filter 21. Liquid crystals 23 (LC), which act as variable full -wave retarders by applying a voltage, are inserted before each polarizer 24 to align all IV sinusoidal transmission functions with different FSRs via a voltage applied by a workstation or microcontroller 25.

[0094] In this way, N sinusoidal functions with different frequency periods can be summed. The result — after the alignment process — produces a transmission function characterized by very narrow transmission peaks in frequency, as illustrated in Figure 8. The FSR of this function is defined by the thickness of the IV -th birefringent crystal 22 (BCN). By tuning this transmission function using synchronized liquid crystals 23, it becomes possible to scan the frequency content of the incoming light signal, thereby resolving the Stokes and Anti-Stokes Brillouin peaks associated with point pxof the sample 8. Similarly, a beam from a different point ptof the sample will use the same principle and the transverse extension of the optical cavity of the Lyot filter 21 to obtain the associated Brillouin spectrum, detected by a different pixel of the acquisition device 6.

[0095] In one example, the spectrometer 6 comprises an optical cavity and the optical path difference introduced by the optical cavity may be smaller than 5 cm, in particular 3 cm or smaller than 3 cm.

[0096] Figure 9 illustrates the various steps of method 100 for widefield Brillouin spectroscopy. In the first step (S 101), the sample 8 is illuminated by a light source. Specifically, the illumination occurs over an extended region of the sample 8, rather than at a single point. Following the interaction between the light beam from the light source and the material of the sample, a light scattering effect is created, with the light being dispersed in various directions. The scattered light signal from the illuminated sample 8 is then collected and collimated in step SI 02. In otherSPECTO S.R.L., Case:061596WO

[0097] words, the light dispersed in multiple directions is collected and converted into a parallel, directional light beam. Subsequently (SI 03), the elastic Rayleigh component of the light signal is suppressed or reduced through a filtering optical group 4, allowing only the inelastic Brillouin component to pass through.

[0098] A common-path spectrometer 5 is used in step SI 04 to introduce a variable phase delay to the light signal using a common optical path. Finally, in step SI 05, a spectral image 11 of the sample 8 is acquired by an acquisition device 6 that is configured to capture a plurality of frames. Specifically, the light signal is captured for each phase delay introduced by the spectrometer 5, and each frame captured by the acquisition device 6 corresponds to a frequency determined by the phase delay introduced by the spectrometer 5. The double arrow between SI 04 and SI 05 indicates an iterative process, meaning that a frame is acquired for each phase delay.

[0099] It should be noted that all the various processes of illumination, collection, detection, etc., described previously with reference to the optical apparatus 1, also correspond to various steps of method 100.

[0100] For example, the suppression or reduction of the Rayleigh elastic component from the light signal (step SI 03) can occur by passing the collimated light signal through a cell containing a gas for the selective absorption of the Rayleigh component. Alternatively, this can be achieved using one or more birefringent crystals to decouple the polarization of the Rayleigh component from that of the Brillouin component.

[0101] Similarly, the introduction of the variable phase delay (step S 104) can occur by passing the light signal through a Fabry -Perot interferometer 18, as shown in Figures 5 A and 5B. Alternatively, it can occur through an etalon 20, as shown in Figure 6. Another option is to use a Lyot filter 21, as shown in Figure 7.

[0102] In one example, method 100 further includes the preliminary acquisition of a calibration Brillouin image 26 from a homogeneous material, dividing the spectral image 11 of the sample 8 by the calibration image 26, and generating a calibrated image 27 of the sample 8. The system calibration process for wide-field Brillouin spectroscopy is schematically shown in Figures 10A and 10B.

[0103] The spectral analysis of a large portion of sample 8 involves different propagation angles for the collimated beams coming from different points of the sample 8, collected by the infinity-corrected objective 13. These varying propagation angles 9 result in different optical paths,SPECTO S.R.L., Case:061596WO

[0104] defined by the quantity nLcos 6, and therefore, even with the same distance between the reflecting mirrors 28 of the FP 18 or the thickness of the birefringent crystals 22 of the Lyot filter 21.

[0105] The phase delay scan of the incident beams by the spectrometer 5 can generate a gradient of frequency shift, thus causing artifacts in the obtained Brillouin images. To eliminate these artifacts, method 100 involves the preliminary acquisition of a Brillouin calibration image 26 on a homogeneous material (e.g., water, ethanol, polystyrene). In theory, the spectral image of a homogeneous sample should exhibit a constant Brillouin frequency shift. However, illumination and collection over a large area of the calibration sample can produce a frequency gradient in the resulting spectral image due to the variation in the propagation angles of the collimated beams within the common-path spectrometer. By dividing the spectral image 11 acquired from the real sample 8 to be analyzed (such as cells or tissues) by the image 26 obtained from the homogeneous calibration sample, these artifacts can be compensated, and a calibrated Brillouin image 27 can be generated with contrast based on the actual frequency shifts caused by Brillouin light scattering, thus eliminating possible artifacts.

[0106] It is worth noting that the spectral resolution of the various types of common-path spectrometers described above is on the order of 500 MHz (or even lower). This spectral resolution is perfectly suitable for recording a Brillouin spectrum for each pixel. In fact, Brillouin frequencies typically range from a few hundred MHz to a few GHz, and a resolution of 0.5 GHz is sufficient to discriminate between different Brillouin lines, to separate them from the Rayleigh peak due to elastic background scattering, and to measure the linewidth of Brillouin peaks, particularly in biological samples.

[0107] This spectral resolution is different compared to that achieved with a Fourier-transform spectrometer, which is typically above I GHz due to the long scanning distance required. The main difference compared to a Fourier-transform spectrometer lies in the scanning method and the length of the optical path involved. In FT instruments, scanning is performed along a separate and considerably longer optical path, which can reach approximately 30 cm. This length is crucial in FT instruments because the spectral resolution directly depends on the scanning path length difference: the longer the optical scanning distance, the higher the achievable spectral resolution. In common-path spectrometers, by contrast, the optical path is much shorter, on the order of 3-5 cm, as in a FP system. Moreover, unlike Fourier-transform spectrometers, the spectral resolution is not directly determined by the scanning path length butSPECTO S.R.L., Case:061596WO

[0108] rather by other intrinsic parameters of the instrument, such as the flatness and parallelism of the mirrors forming the cavity.

[0109] The wide-field spectroscopy technique described here enables acquisition of the entire spectral content of an extended region of the sample without the need for point-by-point scanning. Instead of collecting data incrementally for each individual pixel, the full spectrum of the illuminated area is acquired simultaneously.

[0110] It should also be noted that a spectral resolution of 0.5 GHz is typically associated with an instrumental precision of approximately 10 MHz, which is fully compatible with, and generally considered standard for, Brillouin peak analysis.

[0111] The apparatus, method, and optical device described above can be subject to numerous additional modifications and variations by a person skilled in the art, in order to meet further and specific needs. All of these modifications and variations are nonetheless encompassed within the scope of protection of the present invention as defined by the appended claims.

Claims

SPECTO S.R.L., Case:061596WOCLAIMS1. Optical apparatus (1) for wide-field Brillouin spectroscopy, wherein the apparatus (1) comprises:an illumination optical assembly (2) to illuminate an extended region of a sample to be analyzed (8);a collection and collimation optical assembly (3) to receive a scattered light signal from the illuminated sample (8) and collimate said light signal;a filtering optical assembly (4) to receive the collimated light signal and suppress or reduce the elastic Rayleigh component from the light signal and transmit the inelastic Brillouin component;a common-path spectrometer (5) to introduce a variable phase delay to the light signal; and a spectral image acquisition device (6) to capture a plurality of frames, wherein the light signal transmitted by the spectrometer (5) is acquired for each phase delay introduced by the spectrometer (5) and wherein each frame captured by the acquisition device (6) corresponds to a frequency determined by the phase delay introduced by the spectrometer (5).

2. Optical apparatus (1) according to claim 1, wherein the illumination optical assembly (2) comprises a laser light source (9) that emits a single-frequency light beam with a spectral linewidth of less than 100 MHz and a wavelength in the visible or near-infrared range.

3. Optical apparatus (1) according to one of the preceding claims, wherein the spectrometer (5) comprises:a. a F abry -Perot interferometer (18); orb. an etalon (20); orc. a Lyot filter (21).

4. Optical apparatus (1) according to one of the preceding claims, wherein the collection and collimation optical assembly (3) comprises an infinity-corrected objective (13, 13B).

5. Optical apparatus (1) according to one of the preceding claims, wherein the filtering optical assembly (4) comprises:a. a cell containing a gas for the selective absorption of the elastic Rayleigh component; orb. one or more birefringent crystals to decouple the polarization of the Rayleigh component from that of the Brillouin component.SPECTO S.R.L., Case:061596WO6. A method (100) for wide-field Brillouin spectroscopy, wherein the method (100) comprises:illuminating (SI 01) an extended region of a sample to be analyzed (8);collecting and collimating (SI 02) a scattered light signal from the illuminated sample (8); suppressing or reducing (SI 03) the elastic Rayleigh component from the light signal and transmitting the inelastic Brillouin component;introducing (SI 04) a variable phase delay to the light signal using a common optical path via a spectrometer (5); andacquiring (S105) a spectral image (11) of the sample (8) by an acquisition device (6) to capture a plurality of frames, wherein the light signal is acquired for each phase delay introduced by the spectrometer (5) and wherein each frame captured by the acquisition device (6) corresponds to a frequency determined by the phase delay introduced by the spectrometer (5).

7. Method (100) according to claim 6, further comprising:preliminarily acquiring a calibration Brillouin image (26) on a homogeneous material; dividing the spectral image (11) of the sample (8) by the calibration image (26); and generating a calibrated image (27) of the sample (8).

8. Method (100) according to one of the claims comprised between 6 and 7, wherein the suppression or reduction (SI 03) of the elastic Rayleigh component from the light signal occurs by passing the collimated light signal through:a. a cell containing a gas for selective absorption of the Rayleigh component; or b. one or more birefringent crystals to decouple the polarization of the Rayleigh component from that of the Brillouin component.

9. Method (100) according to one of the claims comprised between 6 and 8, wherein the introduction (SI 04) of the variable phase delay occurs by passing the light signal through:a. a F abry -Perot interferometer (18); orb. an etalon (20); orc. a Lyot filter (21).SPECTO S.R.L., Case:061596WO10. Method (100) according to one of the claims comprised between 6 and 9, wherein the acquisition (S105) of the spectral image (11) of the sample (8) comprises the simultaneous detection of a set of Brillouin spectra of the sample (8) each associated with a different point on the sample 8 by a full phase scan performed by the spectrometer (5).

11. Method (100) according to one of the claims comprised between 6 and 10, wherein the acquisition (S105) of the spectral image (11) of the sample (8) formed by the plurality of frames captured by the acquisition device (6) occurs in a time of less than a second.

12. Optical device (7), in particular a microscope, comprising the optical apparatus (1) according to one of claims 1 to 5.

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