Apparatus for amplifying a signal in self-reference quantitative phase imaging

Abstract

In the quantitative phase imaging apparatus 1, the quantitative phase imaging apparatus 1 includes an imaging optical system 3 for imaging an object 14 on an image plane, a light source 2 for emitting light radiation I over at least a part of a Fourier plane C of the imaging optical system 3, an optical spatial filter 4 extending to the Fourier plane C of the imaging optical system 3, the optical spatial filter 4 including a region 41 where the light radiation D is minimally attenuated and a region 42 where the light radiation I is maximally attenuated, and a wavefront sensor 5 for measuring, within the image plane, the intensity and phase of an electromagnetic field related to the light radiation I;D that is emitted by the light source 2, passes through the imaging optical system 3 and the optical spatial filter 4, and at least a part of which interacts with the object 14. The light source 2, the imaging optical system 3, and the optical spatial filter 4 are configured such that when there is no object 14 to be imaged, the light radiation I is focused on the region 41 where the light radiation I is maximally attenuated. The quantitative phase imaging apparatus 1.

Description

Technical Field

[0001] The present invention relates to a quantitative phase imaging apparatus, particularly a quantitative phase imaging apparatus for characterizing nano-objects.

Background Art

[0002] When observing a transparent or semi-transparent object with a conventional transmission electron microscope, i.e., a bright-field optical microscope, the contrast observed in the image is generally low. This makes the study of the object more complicated, especially when the object is measured to be less than 1 micrometer.

[0003] Phase contrast microscopy developed by Frederik Zernike is an imaging method that involves utilizing the phase change of light waves passing through the object to be imaged. A drawback of phase contrast microscopy is the light halo introduced into the image around the observed object. This phase contrast microscopy is based on the measurement of the intensity of light waves and the assumption that part of the phase information is transmitted by the interference phenomenon with that intensity. Therefore, this phase contrast microscopy has a major drawback that it cannot quantify the phase of the wave and cannot separate the intensity contribution from the phase contribution. However, as described below, accurately measuring the phase is particularly important in many applications, especially for studying microorganisms or characterizing nanoparticles.

[0004] Quantitative phase microscopy is another imaging method based on the wavefront analysis of light that illuminates and interacts with the object to be imaged. This method measures the optical phase or a parameter proportional to this light wave. In most cases, the light intensity is measured simultaneously. A digital camera equipped with a diffractive optical element, a refractive optical element, or a reflective optical element is used to measure the entire field generated from the sum of the incident field and the field scattered by the object, for example, a semi-transparent, non-absorbing, low-scattering object.

[0005] Quantitative phase microscopy generates a greater contrast for transparent or semi-transparent objects than that obtained by conventional microscopy methods, and thus enables smaller objects than those in conventional microscopy methods to be studied in more detail. In particular, quantitatively measuring the phase enables access to the density or mass measurement of the observed object. Quantitative phase microscopy includes a "reference arm" technique based on measuring the interference between a reference wave and a wave that has interacted with an object to be imaged, such as holography, and a self-reference technique based on phase gradient measurement. In particular, the self-reference technique can be implemented using a wavefront analyzer for imaging the object. In particular, the wavefront analyzer can be based on using an optical mask, such as a Hartmann hole array, a Shack-Hartmann microlens array, a diffraction grating, a modified Hartmann mask, a diffuser, or a metasurface, upstream of the camera.

[0006] Unlike the reference arm technique that requires a complex and vibration-sensitive optical assembly, the self-reference quantitative phase imaging technique is easy to implement and can obtain good phase sensitivity.

[0007] Quantitative phase microscopy has many applications in biology, particularly for imaging low-scattering subcellular components while obtaining characteristic biophysical values. However, observation at the molecular level is complex, if not impossible, due to the weak light-matter interaction of semi-transparent nano-objects.

[0008] Quantitative phase microscopy also enables the detection and characterization of nanoparticles or the monitoring of chemical reactions. The detection limit of an object or a substance flow directly depends on the phase sensitivity of the method used.

[0009] In addition, by means of quantitative phase microscopy, it becomes possible to map the fluctuations in the refractive index resulting from thermal disturbances. Therefore, the temperature distribution can be mapped with a spatial accuracy at the micrometer level. By increasing the phase sensitivity, the temperature sensitivity can be increased. This also makes it possible to more sensitively map changes in the refractive index resulting from electrical, acoustic, or magnetic disturbances.

[0010] For optical measurement methods, particularly for quality control in lithography or nanolithography, quantitative phase microscopy can measure the surface profile because the thickness fluctuations induce local fluctuations in the phase shift measured. Good phase sensitivity is important for quantifying sub-nanometric thickness fluctuations.

[0011] However, one drawback common to known quantitative phase microscopy techniques is that the amplitude of the field scattered by the object(s) to be imaged is very small compared to the amplitude of the incident field. As a result, a weak phase signal is generated, which makes it more difficult to characterize nano-objects, especially when they are moving.

[0012] Modifications to the reference arm technique have been proposed to attempt to overcome this drawback, but they remain complex to implement and are characterized by a sensitivity limited by the vibration of the reference arm of the optical assembly. SUMMARY OF THE INVENTION PROBLEM TO BE SOLVED BY THE INVENTION

[0013] Therefore, there is a need to have a self-referencing quantitative phase imaging technique that improves detectability in self-referencing quantitative phase imaging techniques, i.e., that can detect at least one object, particularly a semi-transparent object, and / or one measurement value less than 100 nanometers, particularly a moving object of this kind, with a high degree of contrast. MEANS FOR SOLVING THE PROBLEM

[0014] The present invention relates to an imaging optical system for imaging an object on an image plane, a light source for emitting light radiation over at least a part of the Fourier plane of the imaging optical system, an optical spatial filter extending in the Fourier plane of the imaging optical system, the optical spatial filter having a region where the light radiation is attenuated to a minimum and a region where the light radiation is attenuated to a maximum, a wavefront sensor for measuring, in the image plane, the intensity and phase of an electromagnetic field related to the light radiation that has passed through the imaging optical system and the optical spatial filter associated with the light radiation emitted by the light source and at least partially interacted with the object in order to quantify the phase induced by the object, and proposes a quantitative phase imaging apparatus in which the light source, the imaging optical system, and the optical spatial filter are arranged such that, when there is no object to be imaged, the light radiation is focused on the region where the light radiation is attenuated to a maximum.

[0015] The "Fourier plane" of the imaging optical system is a plane where the Fraunhofer diffraction pattern, i.e., the Fourier transform of the object under investigation, can be observed. Therefore, in the Fourier plane, the user of the apparatus can visualize the spatial frequency of the object under investigation. In a modified embodiment, the imaging optical system is telecentric, i.e., its entrance pupil or exit pupil is at infinity, and the Fourier plane coincides with the pupil plane of the imaging optical system. The "pupil plane" of the imaging optical system is a plane where the pupil of the optical system, or an image formed by a part of the optical system, is observed.

[0016] When there is an object to be imaged by the imaging optical system, the incident light radiation coming from the light source and not diffracted and / or scattered by the object is focused on the region of maximum attenuation on the spatial filter, while the light radiation diffracted and / or scattered by the object is at least partially focused on the region of minimum attenuation.

[0017] Accordingly, the optical spatial filter specifically attenuates the intensity of the incident light radiation that is not diffracted and / or scattered by the object, while maintaining the intensity of the radiation diffracted and / or scattered by the object without substantially changing it outside the region of maximum attenuation. According to the present invention, it is possible to attenuate the contribution of the incident electromagnetic field, related to the incident light radiation emitted by the light source and not diffracted and / or scattered by the object, to the electromagnetic field detected by the wavefront sensor, thereby amplifying the phase signal. Based on the measurement of the phase and intensity, and by considering the complex transmittance in the regions of relatively large attenuation and minimum attenuation, it is possible to determine, by means of an analytical model or digital processing, the quantified value of the phase measured in the absence of the optical spatial filter with an increased signal-to-noise ratio. Therefore, it is possible to calculate related quantitative parameters such as the dry mass of the object, the refractive index value of the object, or the thickness of the object from this quantified value of the phase.

[0018] The regions of relatively large attenuation and minimum attenuation preferably have different effects on the phase of the wave.

[0019] The "area of greatest attenuation" preferably induces a lower attenuation at the wavelengths constituting the light radiation emitted by the light source than the "area of least attenuation".

[0020] Wavefront sensor The wavefront sensor measures at least one change in the phase of the light radiation that has passed through the object, the imaging optics, and the optical spatial filter, in particular the gradient, or characteristic parameters of the phase such as the optical path difference traveled by two light radiations. The phase induced by the object can be determined from the characteristic parameters by integrating the measured gradient.

[0021] The wavefront sensor further measures the intensity of the radiation.

[0022] The wavefront sensor preferably comprises an optical radiation detector, for example, a camera, in particular, a two-dimensional sensor for sampling the intensity. Optionally, the wavefront sensor comprises a wavefront analysis mask arranged in front of the detector along the optical path of the optical radiation to determine the spatial distribution of the phase of the optical radiation or the gradient of a signal proportional to said phase.

[0023] In particular, the wavefront analysis mask can simultaneously measure the phase and intensity of the detected optical radiation.

[0024] The wavefront analysis mask preferably comprises a diffractive and periodic or aperiodic optical element and / or a refractive optical element. The wavefront analysis mask can be arranged near the detector or near a plane conjugate to the plane in which the detector extends.

[0025] The wavefront analysis mask can be particularly selected from masks comprising an array of small lenses, such as the Shack-Hartmann mask, modified Maltman masks as described, for example, in "Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells" by P. Bon et al. on pages 13080 - 13094 of Optics Express (2009), and thin diffusers as described, for example, in "Wavefront sensing with a thin diffuser" by P. Berto, H. Rigneault, and M. Guillon et al. in Opt. Lett. (2017), Vol. 42, pages 5117 - 5120.

[0026] The optical radiation detector preferably comprises a digital camera for acquiring the optical radiation, which preferably interacts with the wavefront analysis mask.

[0027] The digital camera can comprise an optical sensor, for example, a CMOS or a CCD, for acquiring the optical radiation and measuring the intensity of said optical radiation.

[0028] Optical spatial filter The region of maximum attenuation, and optionally the region of minimum attenuation, are adapted to attenuate the intensity of the optical radiation.

[0029] Preferably, the region of minimum attenuation does not attenuate the intensity of the optical radiation in order to maintain the maximum intensity of the optical radiation for measurement.

[0030] The region of maximum attenuation induces a relatively large attenuation of the intensity of the optical radiation compared to the region of minimum attenuation.

[0031] The region of maximum attenuation and / or the region of minimum attenuation can induce a phase shift between the optical radiation incident on the region of maximum attenuation and the region of minimum attenuation respectively and the optical radiation attenuated by said regions so as to amplify the signal-to-noise ratio of the measured phase and intensity signals.

[0032] In a variant embodiment, the optical spatial filter is adapted to reflect the optical radiation and the region of maximum attenuation is semi-reflective. The region of minimum attenuation can be semi-reflective or preferably reflective. The region of minimum attenuation and the region of maximum attenuation can have different thicknesses in order to induce different phase shifts in the optical radiation they attenuate.

[0033] In another variant embodiment, the spatial filter is adapted to transmit the optical radiation and the region of maximum attenuation is semi-transmissive. The region of minimum attenuation can be semi-transmissive and preferably transmissive. The transmittance decreases as the intensity is attenuated. The region of maximum attenuation has the formula t = t0*e -iβIt can have a complex transmittance t defined by, where 0 < t0 ≦ 0.7, the amplitude t0 measured over at least a portion of the wavelength of the light emitted by the light source, and optionally, in particular to amplify the phase absolutely, the phase shift β can be such that -1 rad[π] ≦ β ≦ 1 rad[π]. The amplitude t0 of the complex transmittance can be measured by focusing the light beam into the region of maximum attenuation and measuring the amount of attenuation of the intensity of the light beam, whereby the transmittance T = t0 2 can be approximated. The phase shift β can be measured by an interferometric method. Preferably, T ≦ 0.15, for example T = 0.1. The phase shift β can be made equal to 0.

[0034] The optical spatial filter can comprise a transmissive support and a semi-transmissive coating that partially covers the support, and the region of maximum attenuation is defined by the overlap of the support and the semi-transmissive coating.

[0035] The transmissive support can be made of glass.

[0036] The semi-transmissive coating can be in the form of having a radius r of at least one disk fs and preferably less than 0.1*r p where r p is the maximum radius of the disk in which the spatial frequencies collected in the Fourier plane are distributed. For example, r fs ≦ 100 μm. In a modified embodiment where the imaging optical system is telecentric, the Fourier plane coincides with the pupil plane and r p represents the diameter of the image of the aperture pupil of the imaging optical system in the Fourier plane.

[0037] In one modified embodiment, the semi-transmissive coating is in the form of a ring. The non-transmissive coating can have any shape corresponding to the angular spectrum of the illumination.

[0038] The semi-transmissive coating can be arranged at the center on the optical axis of the optical spatial filter.

[0039] The semi-permeable film can have a thickness of less than 100 nm.

[0040] The semi-permeable film can comprise a semi-permeable layer of a metal selected from gold, silver, aluminum, chromium, titanium, and alloys thereof, such as gold.

[0041] The semi-permeable film can comprise an adhesion layer, made of, for example, chromium and / or titanium, which is sandwiched between the support and the semi-permeable layer and contacts the support.

[0042] The region of maximum attenuation can have a complex transmittance that varies, in particular, according to the polarization of the light radiation and / or the temperature of said region and / or can be changed by the user.

[0043] The region of maximum attenuation comprises a thermochromic material whose transmittance can vary according to temperature and / or a polarization material whose transmittance can vary according to the polarization of the light radiation.

[0044] The thermochromic material can be selected from the group formed by thermochromic liquid crystals, thermochromic dyes, optionally doped thermochromic oxides, and mixtures thereof, such as VO2, BiVO4, NbO2, and mixtures thereof.

[0045] The region of maximum attenuation and / or the region of minimum attenuation can each comprise at least one layer made of a material capable of inducing a phase shift between the light radiation incident on these regions and the light radiation attenuated by these regions. By way of example, these regions comprise a layer comprising a polymer, glass, or titanium dioxide. These regions can comprise a stack of layers made of at least two different materials, such as a stack of a layer of titanium dioxide, a layer of silicon dioxide, and another layer of titanium dioxide, or a metasurface.

[0046] The region of maximum attenuation and / or the region of minimum attenuation may each comprise a layer made of a birefringent material that induces a phase shift between the light radiation incident on the region and the light radiation attenuated by the region and that can be modulated by rotating the polarization direction of a light source.

[0047] The region of maximum attenuation and / or the region of minimum attenuation may comprise a layer of a material having a non-zero thermo-optical coefficient that induces a phase shift that varies according to temperature, in particular a liquid such as glycerol, or a polymer such as polydimethylsiloxane.

[0048] The device may comprise a thermal adjustment module for modifying the temperature of the region of maximum attenuation and / or the region of minimum attenuation in order to modify the transmittance of the region and / or the phase shift induced by the region. The thermal adjustment module may comprise an optical, electrical or magnetic heater, such as a Joule heater for example. As an example, in a variant embodiment where the region of relatively low attenuation comprises a layer of gold, optical heating of the layer changes the refractive index of the layer, resulting in fluctuations in the optical phase shift induced by the region of maximum attenuation between the light radiation incident on the region and the light radiation attenuated by the region. In a variant embodiment, the heating can be electrically induced by a resistive wire, in particular a wire made of indium tin oxide (ITO). By using the thermal adjustment module, it is possible in particular to vary in real time the phase shift induced by an optical spatial filter, thereby optimizing the signal-to-noise ratio in both the intensity signal and the phase signal.

[0049] The optical spatial filter may comprise a spatial light modulator (SLM) or a phase control diffraction grating, in particular for modulating the complex transmittance.

[0050] The spatial light modulator can be optionally connected to an opaque mask in order to attenuate part of the radiation diffracted and / or scattered by an object.

[0051] The spatial filter may have a plurality of distinct regions where the light radiation is maximally attenuated. The apparatus may comprise a plurality of optical spatial filters. The plurality of optical spatial filters may be successively arranged along the propagation path of the light radiation. The optical spatial filter can be made removable, and by adding or removing at least one of the optical spatial filters, the equivalent composite transmittance of all said spatial filters is modified.

[0052] Imaging optical system The imaging optical system can conjugate an object plane and an image plane. The object is located, for example, outside the object plane but close to the object plane. This apparatus enables an object located outside the object plane to be digitally refocused.

[0053] The optical system may comprise an objective lens. In a variant embodiment, the optical system preferably comprises one or more additional lenses, in particular converging lenses, and / or one or more mirrors.

[0054] The optical system is preferably stigmatic or substantially stigmatic.

[0055] It is desirable for the optical system to be aplanatic.

[0056] The numerical aperture NA of the optical system object is preferably between 0.12 and 1.7 in order to maximize the light radiation diffracted and / or scattered by the object detected by the wavefront sensor.

[0057] The formula NA image =NA object The magnification of the imaging optical system related to the image numerical aperture by the magnification is preferably such that it satisfies the Nyquist criterion (> 2 measurement points / PSF image ) while sampling the image impulse response (PSF image ) of the imaging optical system in phase.

[0058] Light source The light source preferably comprises a light generator that generates light radiation selected from, for example, lamps, particularly halogen lamps, and powerful lasers, particularly supercontinuum lasers.

[0059] The light radiation emitted by the light source has an angular spectrum that can be shaped by a further optical system upstream of the object to be imaged in order to distribute the energy of the incident light radiation over the region of maximum attenuation.

[0060] In one embodiment, the region of maximum attenuation is in the form of a disk centered on the optical axis, and the light source may comprise a further optical system for generating Köhler illumination. The further optical system can in particular be arranged between the light generator and the object in the propagation direction of the light radiation in order to parallelize the light radiation incident on the object.

[0061] The light source can be configured to emit monochromatic or polychromatic radiation.

[0062] When the phase shift between the light radiation incident on the filter and the filtered radiation induced by the optical spatial filter is non-zero, the light radiation of the nominal wavelength λ nominal preferably has a spectral region satisfying Δβ / β nominal <50%. β nominal and Δβ respectively correspond to the phase shift and the phase shift change induced by the optical spatial filter with respect to λ nominal and over the spectral range Δβ. The spectral range of the light radiation is preferably set by the method by which the phase shift induced by the optical spatial filter is generated. For the phase shift induced by an optical spatial filter generated by a refractive effect, it is shown in [Equation 1].

Equation

[0063] The light radiation can be visible light radiation, X-rays or infrared rays.

[0064] The light radiation emitted by the light source can be polarized.

[0065] The light source can include a filter for selecting at least one wavelength of the light radiation emitted by the light generator and / or a filter for polarizing the light radiation. The selection filter and / or the polarization filter can be arranged along the path of the light radiation between the light generator and the object to be imaged.

[0066] Other units of the device The device can further include an additional digital camera, particularly for measuring the intensity of the light radiation at the Fourier plane of the imaging optical system.

[0067] This additional digital camera can be used to image the Fourier plane, particularly for precisely positioning the optical spatial filter within this plane.

[0068] The device can include a microscope having an objective lens, and the optical spatial filter can be either remote from or not remote from the microscope. The microscope can include a light source, particularly such that the microscope itself can emit Köhler illumination. The microscope can include a sample holder. The microscope can include other elements of the optical system, particularly one or more lenses and / or one or more mirrors.

[0069] The device can include one or more additional optical elements selected, for example, from lenses, particularly converging lenses, reflecting mirrors, semi-reflecting mirrors, dichroic mirrors, and wavelength filters.

[0070] Method Furthermore, the present invention relates to a method for acquiring at least one digital image of a sample including an object by means of the device according to the present invention, the method comprising: a) emitting incident light radiation directed towards the object using a light source; b) detecting, by means of a wavefront sensor, the entire light radiation that has interacted with the object and has been transmitted by the imaging optical system and the optical spatial filter; c) processing the signal detected in step b) to quantify a parameter selected from among the phase of the radiation and / or the change in the phase of the radiation, and optionally generating a digital image of the parameter.

[0071] Preferably, the processing in step c) further comprises quantifying the intensity of the optical radiation and, preferably, generating a digital image of the intensity of the optical radiation.

[0072] The processing in step c) is quantitative, i.e., it comprises correcting the signal detected in step b) by taking into account the influence of the phase shift and attenuation induced by the optical filter on the value of the parameter.

[0073] The sample can comprise one or more objects smaller than the resolution of the imaging optics. The resolution of the imaging optics is defined by [Equation 2],

Equation

[0074] The object can be transparent or semi-transparent to the optical radiation. In a variant embodiment, the object can be opaque to the radiation.

[0075] The sample can be selected from among biological materials containing microorganisms, powders, biological culture solutions, cell or microtissue substrates, inert materials containing nanoparticles, or solutions of mixtures of particles. The microorganisms can be bacteria, vesicles or viruses.

[0076] The optical radiation can have a wavelength spectrum as described above.

[0077] Finally, the present invention relates to the use of an apparatus for studying one or more nanoparticles, a chemical reaction, an electrochemical reaction apparatus, an apparatus for analyzing a photografting reaction or a photopolymerization reaction, an apparatus for mapping a temperature distribution, or an apparatus for specifically detecting molecules by their absorption in a pump-probe system.

[0078] The present invention can be better understood by reading the following detailed description of non-limiting embodiments of the present invention and examining the accompanying drawings.

Brief Description of the Drawings

[0079]

Figure 1A

Figure 1B

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Figure 6

Figure 7

Figure 8

Figure 9

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Figure 11A

Figure 11B

Figure 12

Figure 13

[0080] Figure 1A shows an example of a quantitative phase imaging apparatus 1 according to the present invention. This apparatus includes a light source 2, a telecentric imaging optical system 3, an optical spatial filter 4, and a wavefront sensor 5. A part of the optical system 3 is housed in a microscope 9. This part of the optical system 3 includes an objective lens 31, a mirror 32, and a tube lens 33. The microscope 9 also includes a sample holder 10 disposed at the object focal plane A of the optical system 3.

[0081] To image an object, a user of the apparatus 1 places a sample 14 containing the object on the sample holder 10.

[0082] The light source 2 includes a light generator 21, and may include an optical system 22 for generating Köhler illumination at the object focal plane A, and thus at the sample 14 if necessary, between the light generator 21 and the object focal plane A. Thus, the incident radiation I from the light source 2 is collimated and impinges perpendicularly on the object focal plane A on the sample holder 10. Then, a portion D of this radiation is scattered by the sample 14.

[0083] In the illustrated example, the image focal plane B of the objective lens 31, which corresponds to the Fourier plane of the optical system, is located inside the microscope 9 and thus inaccessible. Advantageously, the spatial filter 4 can be disposed in a plane C conjugate to the Fourier plane B, and this plane is shifted behind the tube lens 33 by a converging lens 34 of the imaging optical system 3.

[0084] Thus, the conjugate plane C corresponds to the Fourier plane of the optical system 3.

[0085] As shown in Figure 1B, the wavefront sensor 5 may include a digital camera 51 and a wavefront analysis mask 53, such as a Shack-Hartmann type, disposed upstream of the camera 51. The light radiation R characterized by the wavefront F reaches the wavefront analysis mask 53 before being detected by the camera 51.

[0086] The wavefront analysis mask 53 can modify the path of the light radiation and, due to the complex amplitude variations generated, can simultaneously measure the phase and intensity of the light radiation.

[0087] Thus, the wavefront sensor shown in FIG. 1B measures the total intensity and phase of the optical radiation it receives.

[0088] In the example shown in FIG. 1A, the wavefront sensor 5 is preceded by a converging lens 35 in the propagation direction of the optical radiation, and this converging lens 35 forms part of the imaging optical system 3 and can image the sample 14 onto the digital camera 51.

[0089] The apparatus 1 also includes a further camera 15 for imaging the Fourier plane C by means of a rotating mirror or a semi-transparent mirror 16 and a dual-lens optical system 17. Thus, the spatial filter 4 can be placed at the center on the optical axis and accurately positioned within the Fourier plane C, for example, using a translation stage (not shown).

[0090] The optical spatial filter 4 has a region 41 of maximum attenuation and a region 42 of minimum attenuation. In the illustrated embodiment, the spatial filter comprises a support 43 in the form of a square transmissive plate made of, for example, glass, and a disk r fs in the form of a metal coating 44 disposed at the center of the plate.

[0091] The region 42 of minimum attenuation can be defined by the portion of the support 43 not covered by the coating 44, and the region of maximum attenuation is defined by the overlap of the support 43 and the coating 44.

[0092] FIG. 2(A) schematically shows the spatial distribution in the Fourier plane of the incident and scattered electromagnetic fields obtained by the apparatus 1 according to the invention in the absence of the spatial filter 4. When the sample 14 is placed on the object focal plane A of the sample holder 10 of an apparatus without the spatial filter 4, the incident electromagnetic field corresponding to the incident radiation I not affected by the impact of the sample is E i denoted, and the scattered electromagnetic field corresponding to the scattered radiation D is E d denoted,

Number

Number

[0093] When the sample 14 is a semi-transparent and non-absorbing low-scattering object, the total electromagnetic field E t is represented by Equation (1).

Number

[0094] In this example, since the incident radiation I is collimated by the sample 14, it has a point distribution at the center of the Fourier plane C corresponding to the image of the pupil of the optical system 3.

[0095] When the sample 14 is smaller than the wavelength of the incident radiation, for example smaller than 100 nm, the radiation D is scattered quasi-isotropically. As a result, on the Fourier plane C, it is uniformly distributed over the image of the pupil of the optical system 3.

[0096] As schematically shown in FIG. 2(A), the electromagnetic fields E i and E d in the Fourier plane C can be observed by a further camera 15 that images the Fourier plane C. This electromagnetic field E d fills the Fourier plane C, and in this case, the electromagnetic field E i is concentrated at a point at the center of the Fourier plane C. More generally, the electromagnetic field E i is distributed according to the angular distribution of the light radiation I emitted by the light source 2.

[0097] In the case of a low-scattering object, as shown in FIG. 2(C), the ratio of the scattering field

Number

Number

Number

Number

[0098] For biological objects such as vesicles or viruses, or even nanoparticles, the phase shift φ is small, especially less than 0.05 rad. Thus, Taylor expansion can be used with φ, and equation (4) is obtained.

Number

[0099] Through the identification between equation (1) and equation (4), the scattered field E d can be written as the incident field E i attenuated approximately by φ and phase-shifted by π / 2 according to the following equation (5).

Number

[0100] Figure 2(C) shows the composite representation of the incident field E i , the scattered field E d , and the total field E t obtained using the apparatus 1 without the optical spatial filter. This figure shows a phase shift of nearly π / 2 of the scattered field E i with respect to the incident field E d .

[0101] In the case of the apparatus according to the present invention, when the optical spatial filter 4 exists within the Fourier plane C,

Number

Number

Number

[0102] When the sample 14 is a semi-transparent, non-absorbing, low-scattering object, the following equation (6) can be defined.

Number

[0103] Also, the complex transmittance t in the region with a small attenuation amount is defined as follows.

Number

[0104] The radius r of the region with relatively small attenuation fs is preferably less than at least five times the radius r of the image of the pupil of the optical system 3 on the Fourier plane. Therefore, as shown in FIG. 2(B), the region of maximum attenuation affects only the incident field and not the scattered field. Therefore, the following equations (7) to (9) can be written. p FIG. 2(D) and FIG. 2(E) show the incident field after filtering

Number

Number

Number

[0105] FIG. 2(D) and FIG. 2(E) show the incident field after filtering

Number

Number

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Number

[0106] The region of maximum attenuation is semi - transmissive. The complete cancellation of the incident field by the non - transmissive region of the maximum attenuation amount

Number

[0107] The entire field transmitted

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[0108] ​The phase shift φ induced by the sample 14 can be quantified from the measurement of the intensity and phase of the field.

Number

[0109] By combining Equation (1) and Equation (8), the following Equation (10) is obtained.

Number

[0110] I t and

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Number

Number

Number

[0111] The wavefront sensor 5 measures the relative intensity, that is, the ratio between the reference image when the sample is absent and the image when the sample 14 is present. Therefore, the intensity ratios I m and

Number

[0112] And the following formula (16) can be formulated from formula (13). [Number]

[0113] By knowing the combined transmittance of the spatial filter 4, the parameters measured when the spatial filter 4 is present [Number] and [Number] Based on, the phase shift φ and absorption I induced by the scattering object of the sample 14 m can be quantified.

[0114] The implementation of the device 1 has been digitally simulated to determine the optimal operating conditions. Such simulations were performed according to the method described in the article "Modeling quantitative phase image formation under tilted illuminations" by P. Bon, B. Wattellier, and S. Monneret in Opt. Lett. (2012) 37, p. 1718.

[0115] The tested sample 14 is polystyrene beads with an optical index of 1.62 and is immersed in water with an optical index of 1.33. The sample 14 was irradiated with monochromatic light radiation with a wavelength λ = 440 nm. The objective lens 31 used had a numerical aperture of 1.4.

[0116] Spatial filtering was simulated for the entire field E t which is the spatial Fourier transform of the entire field [Number] about.

[0117] Next, the radius r fs of the disk (the radius of the region 41 of the maximum attenuation of the spatial filter 4) was multiplied by the complex transmittance of the spatial filter 4 for the simulated electromagnetic field contained therein to simulate the interaction with the spatial filter 4. The field region outside the disk of radius (the radius of the image of the pupil of the optical system 3 on the Fourier plane C) was set to 0 to simulate the finite numerical aperture of the optical system 3. In this way, the field [Number] was simulated according to the following equation (17). [Number] Here, k is the image vector in the pupil, NA is the numerical aperture of the optical system 3, and λ is the wavelength of the light radiation. [Number] The value of the entire field after the filtering process

[0118] is measurable on the wavefront sensor 5, and this field [Number] is calculated by performing the inverse spatial Fourier transform of this field [Number] about.

[0119] [Number] And

Number

Number

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Number

[0120] FIG. 4(A) shows in-phase amplification, and FIG. 4(B) shows

Number

Number

[0121] The calculated intensity

Number

Math

Math

[0122] The actual phase value and intensity value (φ, I m ) are compared with those found after filtering and quantification (φ r , I m、r ) to estimate the relative error induced by the spatial filter 4, which is particularly low. By comparing Fig. 3(C) with Fig. 3(A), Fig. 3(F), and Fig. 3(D), the found phase value and intensity value (φ r , I m、r ) are found to be almost the same as the actual phase value and intensity value (φ, I m ).

[0123] This comparison reflects the proper functioning of the quantitative process to return to the value when the filter is absent.

[0124] As shown in Fig. 5, by modifying the complex transmittance of the spatial filter 4, a relative error of less than 10 -2 for the phase (Fig. 5(A)) and a relative error of less than 10 -4 for the intensity (Fig. 5(B)) are obtained.

[0125] Fig. 6 shows the effect on the relative phase and intensity errors of the ratio

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[0126] Example 1 The microscope 9 used was a commercially available microscope (Olympus UPlanFLN) of Olympus IX71 equipped with a 60x microscope objective lens having a numerical aperture of 0.9.

[0127] The sample 14 was illuminated by the native Köhler illumination of the microscope 9 (halogen lamp) and filtered by wavelength.

[0128] The wavefront sensor 5 was an ID4L analyzer (four-wave lateral shear interferometer) composed of an Andor Zyla 5.5 camera and a 10-μm periodic 2D diffraction grating optimized for a wavelength of 650 nm. This array was re-imaged onto the camera using a 2x magnification telescope composed of two achromatic (chromatic aberration corrected) lenses. The effective distance between the diffraction grating and the camera was d = 1.2 mm.

[0129] The region 41 of the maximum attenuation of the spatial filter 4 was an 80-nm gold disk on 5-nm chromium with a radius r fs = 100 μm vapor-deposited on a glass slide with an electron beam. The transmittance of the optical spatial filter was experimentally measured at T = 7%, λ = 440 nm and 550 nm. With no sample present, the intensity signal I1 without the filter and the intensity signal I2 with the filter were measured, and from this, T = signal I2 / I1 was estimated. The phase shift β was measured by the interference method.

[0130] FIG. 7(A) shows an image of the Fourier plane of the experimental apparatus acquired by an additional camera 15. The white disk corresponds to the radiation D scattered by the sample formed by the superposition of sheets over the entire image of the pupil of the optical system 3 in the Fourier plane C, and the central black disk corresponds to the spatial filter 4. Comparing FIG. 7(B) with FIG. 7(C), the effect of the spatial filter 4 on the intensity of the incident radiation I becomes clear.

[0131] The tested sample 14 was a set of polystyrene beads immersed in water with an optical index n water = 1.33 and having an optical index n poly = 1.62 and a diameter e = 100 nm.

[0132] FIG. 8 shows the results obtained without the spatial filter 4, the results obtained with the spatial filter 4, and the results of quantitative reconstruction using Equation (16) starting from the filtered images. FIG. 8(A) shows the results obtained without using the spatial filter 4, FIG. 8(B) shows the results obtained using the spatial filter 4, and FIG. 8(C) shows the results obtained using the spatial filter 4 and the results obtained following quantitative processing based on the measurements.

[0133] Experimentally, a gain of about 3 times the phase signal is measured from φ = 3.5 nm without using the spatial filter to

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[0134] Since the rate of the field scattered by the beads is very low compared to the rate of the incident field, the beads do not appear in the intensity image without a filter, as shown in Fig. 8(A). Fig. 8(B) shows that the presence of the spatial filter 4 enables the generation of a partially dark-field configuration and allows the beads to be highlighted with a contrast of 0.2, which is also close to the expected theoretical value seen in Fig. 4(B).

[0135] After quantitative reconstruction, the reconstructed intensity and phase values can be seen in Fig. 8(C). Although they are the same as the raw intensity and phase values, there is a clear gain in the signal-to-noise ratio.

[0136] Fig. 9(A) is an enlarged view of the phase image in Fig. 8(B), and Fig. 9(B) is an enlarged view of the phase image in Fig. 8(C). The noise is calculated over the regions enclosed by the dotted lines in Figs. 9(A) and 9(B). In the measurement without using the spatial filter in Fig. 9(A),

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[0137] In a modified embodiment, the angular spectrum of the incident light radiation I is a disk centered on the Fourier plane C, as shown in Fig. 10(A), and is the white region corresponding to the incident light radiation I. The region 41 of maximum attenuation is a disk at the center of the optical spatial filter 4, which is the gray region corresponding to the region 41 of maximum attenuation, as shown in Fig. 10(D).

[0138] In a modified embodiment, the angular spectrum of the incident light radiation I is shown on the Fourier plane C by a plurality of spaced disks as seen in FIG. 10(B), and the white region corresponds to the incident light radiation I. The region 41 of maximum attenuation is composed of a plurality of disks distributed over the optical spatial filter 4, similar to the incident light radiation I on the Fourier plane C as shown in FIG. 10(E), and the gray region corresponds to the region 41 of maximum attenuation.

[0139] In another modified embodiment, the angular spectrum of the incident light radiation I is a ring within the Fourier plane C as seen in FIG. 10(A), and the white region corresponds to the incident light radiation I. The region 41 of maximum attenuation is a corresponding ring on the optical spatial filter 4 as shown in FIG. 10(D), and the gray region corresponds to the region 41 of maximum attenuation.

[0140] Example 2 The same sample 14 and the same apparatus as in Example 1 were used except for the optical spatial filter 4. In this case, the filter as can be seen in FIG. 11A has a region 41 of maximum attenuation comprising a chamber 48 containing a medium such as glycerol having a high thermo-optic coefficient dn / dT, dn / dT = -2.7·10 -4 which is in contact with the gold disk 46 of the above-mentioned 80 nm thickness, and the chamber 48 and the disk 46 are between the two glass plates 45 of the optical spatial filter 4.

[0141] By means of the focused laser heating system 47, the chamber 48 was made able to change the phase shift induced by the optical spatial filter 4 in the incident field E i Next, the total phase shift β i induced by the optical spatial filter 4 on the incident field E total is the sum of a variable component β variable and a fixed component β fixed As seen in FIG. 11B, the filtered incident field

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[0142] Figures 12(A) and 12(B) show the experimental intensity and phase images without the filter (left group 1), respectively, and the experimental intensity and phase images (right group 2) for different values of β obtained by heating the thermal chamber with the optical spatial filter 4. The curves in Figures 13(A) and 13(B) show the variations in intensity and phase contrast plotted against the phase shift induced by the optical spatial filter 4. Note that the simulation results shown in Figures 4(A) and 4(B) were experimentally found for a transmittance T equal to 7%. variable By using the chamber 48 and the heating system 47, it becomes possible to change the phase shift induced by the optical spatial filter 4 in real time and optimize the signal-to-noise ratio in both the intensity signal and the phase signal.

[0143] By using the chamber 48 and the heating system 47, it becomes possible to change the phase shift induced by the optical spatial filter 4 in real time and optimize the signal-to-noise ratio in both the intensity signal and the phase signal.

Claims

1. In a quantitative phase imaging apparatus (1), the quantitative phase imaging apparatus (1) includes an imaging optical system (3) for forming an image of an object (14) on an image plane, a light source (2) for emitting light radiation (I) over at least a part of a Fourier plane (C) of the imaging optical system (3), an optical spatial filter (4) extending to the Fourier plane (C) of the imaging optical system (3), the optical spatial filter (4) including a region (41) where the light radiation (D) is minimally attenuated and a region (42) where the light radiation (I) is maximally attenuated, a wavefront sensor (5) for measuring, within the image plane, the intensity and phase of an electromagnetic field related to the light radiation (I; D) that is emitted by the light source (2), passes through the imaging optical system (3) and the optical spatial filter (4), and at least a part of which interacts with the object (14), in order to quantify the phase induced by the object (14), wherein the light source (2), the imaging optical system (3), and the optical spatial filter (4) are arranged such that the light radiation (I) is focused on the region (41) where the light radiation (I) is maximally attenuated when no object (14) to be imaged is present. A quantitative phase imaging apparatus (1).

2. The region of maximum attenuation (41) has a complex transmittance t defined by the formula t = t ０＊ e -ｉβ and has 0 < t ０ ≦ 0.7, and the amplitude t ０ is measured over at least a part of the spectrum of the wavelength of the light radiation (I) emitted by the light source (2), and optionally, in particular for amplifying the signal absolutely over the phase, the phase shift β is such that -1 rad [π] ≦ β ≦ 1 rad [π], the apparatus according to claim 1.

3. The optical spatial filter (4) includes a transmissive support (43) and a semi-transmissive film (44) partially covering the transmissive support (43), and the region of maximum attenuation (41) is defined by an overlap of the transmissive support (43) and the semi-transmissive film (44). The apparatus according to any one of Claims 1 and 2.

4. The semi-transparent film (44) is in the form of at least one disk having a radius r ｆｓ and the radius r ｆｓ is preferably less than 0.1*r ｐ where r ｐ is the maximum radius of the disk, and the spatial frequency collected within the Fourier plane (C) in the disk is, for example, r ｆｓ ≦ 100 μm. The apparatus according to claim 3

5. The wavefront sensor (5) includes a light radiation detector and a wavefront analysis mask (53) arranged in front of the light radiation detector along an optical path of the light radiation in order to determine a spatial distribution of a phase (I; D; R) of the light radiation or a gradient of a signal proportional to the phase. The apparatus according to any one of Claims 1 to 4.

6. The wavefront analysis mask (53) is selected from an array of small lenses, such as a Shack-Hartmann mask, a modified Hartmann mask, and a mask including a thin diffuser. The apparatus according to Claim 5.

7. The device according to any one of claims 1 to 6, comprising a microscope (9) having an objective lens (31), the microscope optionally comprising a sample holder (10) and / or a light source (2), the optical spatial filter (4) being remote from the microscope (9).

8. The device according to any one of claims 1 to 7, wherein the region of maximum attenuation (41) has a complex transmittance that varies, in particular, as a function of the polarization of the light radiation (I) and / or the temperature of the region (41), and / or the complex transmittance can be changed by the user.

9. The device according to claim 8, wherein the region of maximum attenuation (41) comprises a thermochromic and / or polarizing material.

10. The device according to any one of claims 1 to 9, wherein the region of maximum attenuation (41) and / or the region of minimum attenuation (42) comprises at least one layer made of a material capable of inducing a phase shift between the light radiation (I; D) incident on the region of maximum attenuation (41) and the region of minimum attenuation (42) respectively, and the radiation (I; D) attenuated by the region, the at least one layer being, for example, a single layer comprising a polymer, glass or titanium dioxide, or a stack of multiple layers, or a metasurface.

11. The device according to claim 10, wherein the region of maximum attenuation (41) and / or the region of minimum attenuation (42) comprises a layer of a birefringent material.

12. The device according to any one of claims 10 and 11, wherein the region of maximum attenuation and / or the region of minimum attenuation comprises a layer of a material having a non-zero thermo-optical coefficient, in particular a liquid such as glycerol, or a polymer such as polydimethylsiloxane.

13. The device according to any one of claims 1 to 12, comprising a thermal adjustment module for correcting the temperature of the region of maximum attenuation (41) and / or the region of minimum attenuation (42) in order to correct the transmittance and / or the phase shift induced by the region of maximum attenuation (41) and the region of minimum attenuation (42).

14. The region of maximum attenuation is in the form of a disk centered on the optical axis, the light source (2) comprising a further optical system (22) for generating Köhler illumination, in particular the further optical system (22) being arranged between the light generator (21) and the object (14) in the propagation direction of the light radiation (I) in order to parallelize the light radiation (I) incident on the object (14), the apparatus according to claim 13.

15. In a method of acquiring at least one digital image of a sample comprising an object (14) by means of an apparatus (1) according to any one of claims 1 to 14, the method comprises a) emitting incident light radiation (I) directed towards the object (14) using a light source (2); b) detecting, by means of a wavefront sensor (5), the totality of the light radiation (I; D; R) which has interacted with the object (14) and passed through the imaging optical system (3) and the optical spatial filter (4); c) processing the signal detected in step b) to quantify a parameter selected from the phase of the light radiation (I; D; R) and / or the change in the phase of the light radiation (I; D; R), and optionally generating a digital image of the parameter.

16. The processing in step c) further comprises quantifying the intensity of the light radiation (I; D; R) and preferably generating a digital image of the intensity of the light radiation (I; D; R), the method according to claim 15.

Images