Apparatus for amplifying signal using self-referenced quantitative phase imaging

Abstract

The invention relates to a quantitative phase imaging device (1) comprising:-an optical imaging system (3) for imaging an object (14) in an image plane,-a light source (2) for emitting optical radiation (I) onto a Fourier plane (C) of the optical imaging system,-an optical spatial filter (4) in the Fourier plane (C), the invention relates to an optical imaging system comprising:-an optical imaging system comprising a minimum attenuation region (41) and a maximum attenuation region (42),-a wavefront sensor (5) for measuring the intensity and phase of optical radiation passing through the optical imaging system in an image plane to quantify the phase caused by an object, where at least part of the optical radiation has interacted with the object, the light source, the optical imaging system and the optical spatial filter are such that the optical radiation (I) is focused in a maximum attenuation region when the object is not present.

Description

Technical Field

[0001] The present invention relates to a quantitative phase imaging device, in particular for characterizing nano-objects. Background Art

[0002] When observing a transparent or semi-transparent object in a conventional transmission electron microscope (i.e. in a bright-field optical microscope), the contrast observed in the image is usually low. This makes studying the object more complicated, especially when the object measures less than one micrometer in size.

[0003] Phase contrast microscopy, developed by Frederik Zernike, is an imaging method that involves exploiting phase changes of light waves passing through the object to be imaged. A disadvantage of phase contrast microscopy is that it introduces halos around the observed object in the image. Phase contrast microscopy is based on the measurement of the intensity of light waves and the assumption that some phase information is transmitted in the intensity by interference phenomena. Therefore, its main disadvantage is that it does not quantify the phase of the light wave, nor does it allow the contribution of the intensity to be isolated from that of the phase. However, as described below, accurate measurement of 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 the light that illuminates and interacts with the object to be imaged. It measures the optical phase or a parameter proportional to this wave. In most cases, it measures the light intensity at the same time. Using an additional digital camera with diffractive, refractive or reflective optics, it measures the total field resulting from the sum of the incident field and the field scattered by the object (e.g. a semi-transparent, non-absorbing, low-scattering object).

[0005] Quantitative phase microscopy generates higher contrast than that obtained with conventional microscopy for transparent or semi-transparent objects, thus enabling smaller objects to be studied in greater detail than with conventional microscopy. In particular, quantitatively measuring the phase allows a measure of the density or mass of the object being observed to be obtained.

[0006] Quantitative phase microscopy techniques include:

[0007] - "reference arm" techniques, based on measuring the interference between a reference wave and the wave interacting with the object to be imaged, such as holography, and

[0008] - Self-referencing techniques, which are based on phase gradient measurements. In particular, self-referencing techniques can be implemented using a wavefront analyzer that images the object. In particular, the wavefront analyzer can be based on the use of an optical mask upstream of the camera (e.g. a Hartmann hole array, a Shack-Hartmann lenslet array, a grating, a modified Hartmann mask, a diffuser or a metasurface).

[0009] Unlike reference arm techniques that require complex and vibration-sensitive optical components, self-referenced quantitative phase imaging techniques are simple to implement and can achieve good phase sensitivity.

[0010] Quantitative phase microscopy has many applications, especially in biology, for imaging low-scattering subcellular components while obtaining values ​​of biophysical features. However, observation at the molecular level is complicated if not impossible due to weak light-matter interactions of semi-transparent nano-objects.

[0011] Quantitative phase microscopy also makes it possible to detect and characterize nanoparticles or monitor chemical reactions. The detection limit of an object or a stream directly depends on the phase sensitivity of the method used.

[0012] Quantitative phase microscopy also makes it possible to map refractive index changes caused by thermal disturbances. It can therefore map temperature distributions to micrometer-scale spatial precision. Increasing phase sensitivity will increase temperature sensitivity. This will also make it possible to more sensitively map refractive index changes caused by electrical, acoustic or magnetic disturbances.

[0013] In optical metrology, especially in quality control of lithography or nanoprinting, quantitative phase microscopy can be used to measure surface profiles, since thickness variations induce local changes in the measured phase shift. Good phase sensitivity is essential to quantify thickness variations at the sub-nanometer level.

[0014] However, a common drawback of known quantitative phase microscopy techniques is that the amplitude of the field scattered by the observed object is very small compared to the amplitude of the incident field. This results in a weak phase signal, making it difficult to characterize nano-objects, especially when they are in motion.

[0015] Modifications to the reference arm technology have been proposed in an attempt to overcome this disadvantage, but these modifications remain complex to implement and are characterized by limited sensitivity to vibrations of the reference arm of the optical assembly.

[0016] Therefore, there is an urgent need to improve the detectability of self-referenced quantitative phase imaging technology, that is, there is a need for a self-referenced quantitative phase imaging technology that can detect at least one object, especially translucent objects and / or objects smaller than 100 nanometers, especially such moving objects, and also has high contrast. Summary of the invention

[0017] The present invention provides a quantitative phase imaging device, comprising:

[0018] - an imaging optical system for imaging an object in an image plane,

[0019] - a light source for emitting optical radiation onto at least a portion of the Fourier plane of the imaging optical system,

[0020] an optical spatial filter which extends in the Fourier plane of the imaging optical system and comprises a region in which the optical radiation is attenuated to a minimum and a region in which the optical radiation is attenuated to a maximum,

[0021] a wavefront sensor for measuring, in the image plane, the intensity and the phase of the electromagnetic field associated with the optical radiation, which was emitted by the light source, passed through the imaging optical system and the optical spatial filter, and at least partially interacted with the object, to quantify the phase caused by the object,

[0022] The light source, the imaging optical system and the optical spatial filter are arranged such that the light radiation is focused on a maximum attenuation region when the object to be imaged is not present.

[0023] The "Fourier plane" of the imaging optical system is the plane in which the Fraunhofer diffraction pattern (i.e. the Fourier transform of the object under investigation) can be observed. Thus, in the Fourier plane, the user of the device can visualize the spatial frequencies of the object under investigation. In a variant, 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 the plane in which the pupil of the optical system or the image of the pupil formed by a part of the optical system is observed.

[0024] When an object to be imaged by the imaging optical system exists, incident light radiation emitted from the light source that is not diffracted and / or scattered by the object is focused on the maximum attenuation area of ​​the spatial filter, while light radiation that is diffracted and / or scattered by the object is at least partially focused on the minimum attenuation area.

[0025] Thus, the optical spatial filter specifically attenuates the intensity of incident optical radiation that is not diffracted and / or scattered by the object, while keeping the intensity of radiation diffracted and / or scattered by the object almost unchanged outside the maximum attenuation area. The present invention makes it possible to attenuate the contribution of the incident electromagnetic field associated with the incident optical 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 phase and intensity measurements, and taking into account the complex transmittance of the maximum and minimum attenuation zones, an analytical model or digital processing makes it possible to determine the quantitative value of the phase measured when the optical spatial filter is not present with an increased signal-to-noise ratio. Therefore, relevant quantitative parameters such as the dry mass of the object, the refractive index value of the object or the thickness of the object can be calculated based on the quantized value of the phase.

[0026] The regions of maximum and minimum attenuation preferably influence the phase of the wave differently.

[0027] The attenuation caused by the "maximum attenuation region" in the wavelength of the light radiation emitted by the light source is usually lower than that of the "minimum attenuation region".

[0028] Wavefront Sensor

[0029] The wavefront sensor measures at least one change, in particular the gradient of the phase of the optical radiation passing through the object, the imaging optical system and the optical spatial filter, or a characteristic parameter of the phase, such as the optical path difference traversed by two light beams. The phase caused by the object can be determined from the characteristic parameter by integrating the measured gradient.

[0030] The wavefront sensor also measures the intensity of the radiation.

[0031] The wavefront sensor preferably comprises a light radiation detector (e.g. a camera), in particular a two-dimensional sensor for sampling the intensity. Optionally, the wavefront sensor comprises a wavefront analysis mask, which is arranged in front of the detector along the optical path of the light radiation to determine the spatial distribution of the phase gradient of the light radiation or a signal proportional to the phase.

[0032] In particular, a wavefront analysis mask can simultaneously measure the phase and intensity of the detected optical radiation.

[0033] The wavefront analysis mask preferably comprises diffractive, periodic or aperiodic optical elements and / or refractive optical elements.The wavefront analysis mask may be placed close to the detector or close to a plane conjugate to the plane in which the detector extends.

[0034] The wavefront analysis mask can in particular be selected from a mask comprising a lenslet array (e.g. a Shack-Hartmann mask), a modified Hartmann mask (e.g. described by P. Bon et al. in Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells, Opt. Express, 17, 13080-13094, 2009), and a thin diffuser (e.g. described by P. Berto, H. Rigneault and M. Guillon in Wavefront sensing with a thin diffuser, Opt. Lett. 42, 5117-5120, 2017).

[0035] The optical radiation detector preferably includes a digital camera for collecting optical radiation which has preferably interacted with a wavefront analysis mask.

[0036] The digital camera may include an optical sensor (e.g., CMOS or CCD) for collecting optical radiation and measuring the intensity of the optical radiation.

[0037] Optical spatial filter

[0038] The maximum attenuation region and optionally the minimum attenuation region are adapted to attenuate the intensity of the optical radiation.

[0039] Preferably, the minimum attenuation region does not attenuate the intensity of the optical radiation to maintain the maximum light intensity for measurement.

[0040] The maximum attenuation region causes a greater attenuation of the optical radiation intensity than the minimum attenuation region.

[0041] The maximum attenuation region and / or the minimum attenuation region can cause a phase shift respectively between the optical radiation incident on the region and the optical radiation attenuated in the region, so as to amplify the signal-to-noise ratio of the measured phase and intensity signals.

[0042] In a variant, the optical spatial filter is adapted to reflect optical radiation, and the maximum attenuation region is semi-reflective. The minimum attenuation region can be semi-reflective or preferably reflective. The minimum and maximum attenuation regions can have different thicknesses to cause different phase shifts in the optical radiation they attenuate.

[0043] In another variant, the spatial filter is adapted to transmit optical radiation, and the maximum attenuation region is semi-transparent. The minimum attenuation region can be semi-transparent or preferably transparent. The greater the intensity attenuation, the lower the transmittance. The maximum attenuation region can have a complex transmittance t defined by the equation t = t 0* e -iβ where 0 < t0 ≤ 0.7, the amplitude t0 is measured over at least a portion of the wavelength spectrum of the optical radiation emitted by the light source, and optionally a phase shift β such that -1 rad[π] ≤ β ≤ 1 rad[π], in particular to amplify the absolute value of the signal in terms of phase. The amplitude t0 of the complex transmittance can be measured by focusing a light beam on the maximum attenuation region and measuring the attenuation of the light beam intensity, thereby obtaining the transmittance T = t0 2 . The phase shift β can be measured by interferometry. Preferably, T ≤ 0.15 (e.g., T = 0.1). The phase shift β can be equal to 0.

[0044] The optical spatial filter may include a transparent support and a semi-transparent coating partially covering the support, and the maximum attenuation region is defined by the superposition of the support and the semi-transparent coating.

[0045] The transparent support may be made of glass.

[0046] The semi-transparent coating may be of radius r fs of the form of at least one disk, the radius r fs Preferably less than 0.1*r p , where r p is the maximum radius of the disk in which the collected spatial frequencies are distributed in the Fourier plane. As an example, r fs ≤100μm. In the variant where the imaging optical system is telecentric, the Fourier plane coincides with the pupil plane. p It represents the radius of the image of the aperture pupil of the imaging optical system in the Fourier plane.

[0047] In a variant, the translucent coating is annular. It can have any shape corresponding to the angular spectrum of the illumination.

[0048] The semi-transparent coating may be located at the center of the optical axis of the optical spatial filter.

[0049] The translucent coating may have a thickness of less than 100 nanometers.

[0050] The translucent coating may include a translucent layer of a metal selected from gold, silver, aluminum, chromium, titanium and alloys thereof, such as gold.

[0051] The translucent coating may include a bonding layer sandwiched between and in contact with the support and the translucent layer (eg, made of chrome and / or titanium).

[0052] The region of maximum attenuation may have a varying complex transmittance, in particular depending on the polarization state of the optical radiation and / or on the temperature of said region, and / or it may be modifiable by a user.

[0053] The region of maximum attenuation may comprise a thermochromic material, the transmittance of which may vary depending on the temperature, and / or a polarizing material, the transmittance of which may vary depending on the polarization of the optical radiation.

[0054] The thermochromic material may be selected from thermochromic liquid crystals, thermochromic colorless dyes, thermochromic oxides (which may be doped), and mixtures thereof, such as from the group consisting of VO2, BiVO4, NbO2, and mixtures thereof.

[0055] The region of maximum attenuation and / or the region of minimum attenuation may comprise at least one layer made of a material capable of inducing a phase shift between radiation incident on said region and radiation attenuated by said region, respectively. As an example, the region comprises a layer comprising a polymer, a glass or titanium dioxide. They may comprise a layer stack consisting of at least two different materials, for example a layer stack consisting of a titanium dioxide layer, a silicon dioxide layer and a further titanium dioxide layer, or a metasurface.

[0056] The maximum attenuation region and / or the minimum attenuation region may comprise a layer of birefringent material which induces a phase shift between radiation incident on said region and radiation attenuated by said region, respectively, and which may be modulated by rotating the polarization direction of the light source.

[0057] The region of maximum attenuation and / or the region of minimum attenuation may comprise a layer of a material having a non-zero thermo-optic coefficient, inducing a phase shift as a function of temperature changes, in particular a liquid such as glycerol or a polymer such as polydimethylsiloxane.

[0058] The device may include a thermal regulation module, which is used to modify the temperature of the maximum attenuation area and / or the minimum attenuation area to modify the transmittance of the area and / or the phase shift caused by the area. The thermal regulation module may include an optical, electric or magnetic heater, such as a Joule heater. As an example, in one variant, if the smaller attenuation zone includes a gold layer, the optical heating of the layer will change the refractive index of the layer, thereby causing a change in the optical phase shift between the radiation incident on the area and the radiation attenuated by the area caused by the maximum attenuation area. In one variant, the heating can be electrically induced by a resistance wire, in particular a resistance wire made of indium tin oxide (ITO) material. Using the thermal regulation module, it is possible to change the phase shift caused by the optical spatial filter in particular in real time to optimize the signal-to-noise ratio of both the intensity signal and the phase signal.

[0059] The optical spatial filter may comprise a spatial light modulator (SLM) or a phase-controlled grating, in particular to modulate the complex transmittance. The spatial light modulator may optionally be coupled with an opaque mask to attenuate part of the radiation diffracted and / or scattered by the object.

[0060] The spatial filter can have a plurality of separate regions in which the optical radiation is attenuated to a maximum extent.

[0061] The device may include a plurality of optical spatial filters. The optical spatial filters may be arranged one after another along the propagation path of the optical radiation. The optical spatial filters may be detachable, and adding or removing at least one optical spatial filter may change the equivalent complex transmittance of all the spatial filters.

[0062] Imaging optical system

[0063] The imaging optical system can conjugate an object plane with the image plane, the object being located, for example, outside the object plane but close to the plane. The arrangement allows digital refocusing of objects located outside the object plane.

[0064] The optical system may comprise an objective lens. In a variant, the optical system preferably comprises one or more additional lenses, in particular a converging lens, and / or one or more mirrors.

[0065] The optical system is preferably anastigmatic or substantially anastigmatic.

[0066] The optical system is preferably aplanatic.

[0067] The object numerical aperture NA of the optical system object Preferably between 0.12 and 1.7 to maximize the optical radiation diffracted and / or scattered by the object detected by the wavefront sensor.

[0068] The magnification of the imaging optical system is related to the image numerical aperture by the equation NA image =NA object / magnification related, when Nyquist criterion is met (>2 measurement points / PSF image ), the magnification is preferably selected to adjust the image pulse response (PSF) of the imaging optical system. image ) for in-phase sampling.

[0069] light source

[0070] The light source preferably comprises a light generator for generating optical radiation, for example selected from a lamp, in particular a halogen lamp, and a high-power laser, in particular a supercontinuum laser.

[0071] The optical radiation emitted by the light source has an angular spectrum which can be shaped by an additional optical system located upstream of the object to be imaged in order to distribute the energy of the incident optical radiation onto the area of ​​maximum attenuation.

[0072] In one embodiment, the maximum attenuation region is in the shape of a disk centered on the optical axis, and the light source may include a Additional optical system for illumination. The additional optical system can be arranged in the propagation direction of the optical radiation between the light generator and the object, in particular in order to collimate the optical radiation incident on the object.

[0073] Light sources can be configured to emit monochromatic or polychromatic radiation.

[0074] When the phase shift caused by the optical spatial filter between the radiation incident on the filter and the filtered radiation is not zero, the nominal wavelength λ nominal The light radiation preferably satisfies the condition Δβ / β nominal <50% of the spectral range Δλ. β nominal and Δβ correspond to the effects of the optical spatial filter on λ nominalThe phase shift and the phase shift variation Δβ over the spectral range. The spectral range of the optical radiation is preferably set by the way in which the phase shift caused by the optical spatial filter is generated. For the phase shift caused by the optical spatial filter generated by the refraction effect,

[0075] The radiation may be visible light, X-rays or infrared light.

[0076] The optical radiation emitted by the light source may be polarized.

[0077] The light source may comprise a filter for selecting the optical radiation of at least one wavelength emitted by the light generator and / or a filter for polarizing said optical radiation. The selection filter and / or the polarization filter may be arranged along the optical radiation path between the light source and the object to be imaged.

[0078] Other units of the device

[0079] The device may comprise an additional digital camera, in particular for measuring the intensity of the optical radiation in the Fourier plane of the imaging optical system.

[0080] An additional digital camera enables in particular the imaging of the Fourier plane in order to precisely position the optical spatial filter in this plane.

[0081] The device may include a microscope having an objective lens, and the optical spatial filter may or may not be remote from the microscope. The microscope may include a light source; in particular, the microscope itself may emit Illumination. The microscope may comprise a sample holder. The microscope may comprise further elements of an optical system, in particular one or more lenses and / or one or more mirrors.

[0082] The device may comprise one or more additional optical elements, for example selected from lenses (in particular converging lenses), mirrors, semi-reflecting mirrors, dichroic mirrors and wavelength filters.

[0083] method

[0084] Furthermore, the invention relates to a method for acquiring at least one digital image of a sample comprising an object by means of a device according to the invention, the method comprising:

[0085] a) Use a light source to emit incident radiation directly onto the object,

[0086] b) detecting, by means of a wavefront sensor, all optical radiation that has been transmitted by the imaging optical system and the optical spatial filter and has interacted with the object, and

[0087] c) Processing the signal detected in step b) to quantify a parameter selected from the phase of the radiation and / or a phase change of the radiation and optionally generating a digital image of said parameter.

[0088] 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.

[0089] The processing in step c) is quantitative, ie it consists in correcting the signal detected in step b) by taking into account the influence of the phase shift and attenuation caused by the optical filter on the parameter value.

[0090] The sample may include one or more objects that are smaller than the resolution of the imaging optical system. The resolution of the imaging optical system is defined as R = λ / (2NA object ), where λ is the wavelength of the light radiation, NA object is the object numerical aperture of the imaging optical system.

[0091] The object may be transparent or translucent to the optical radiation. In a variant, the object may be opaque to said radiation.

[0092] The sample can be selected from biological materials including microorganisms, powders, biological cultures, cells or microtissues, solutions of inert materials including nanoparticles or particle mixtures. The microorganisms can be bacteria, vesicles or viruses.

[0093] The optical radiation may have the wavelength spectrum mentioned above.

[0094] Finally, the invention relates to the use of the device for studying one or more nanoparticles or for analyzing chemical, electrochemical, photografting or photopolymerization reactions or for mapping temperature distributions or for detecting molecules specifically via their absorption properties in a pump-probe system. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The present invention may be better understood by reading the following detailed description of non-limiting embodiments of the invention and by studying the accompanying drawings, in which:

[0096] - Figure 1A is a schematic diagram of a portion of an exemplary device according to the present invention,

[0097] - Figure 1B is a schematic diagram of a portion of an example wavefront sensor,

[0098] - Figure 2 A and Figure 2 B schematically shows the spatial distribution of the incident field and the scattered field in the Fourier plane in the case where the device according to the present invention does not have a spatial filter and has a spatial filter, respectively, and Figure 2 C. Figure 2 D and Figure 2 E shows the complex representation of the incident field, scattered field and total field, Figure 2 C shows Figure 2 A's situation, and Figure 2 D and Figure 2 E shows Figure 2 In the case of B,

[0099] - Figure 3 A. Figure 3 B and Figure 3 C shows the digital simulation result of the phase image, and Figure 3 D. Figure 3 E and Figure 3 F shows the spherical object without filtering ( Figure 3 A and Figure 3 D) With filtering ( Figure 3 B and Figure 3 E) and filtering and quantitative processing ( Figure 3 C and Figure 3 F) Digital simulation of the intensity image of the case,

[0100] - Figure 4 A and Figure 4 B shows the simulated phase gain and intensity contrast value plotted against the transmittance and phase shift of the spatial filter, respectively.

[0101] - Figure 5 A and Figure 5 B shows the simulated relative phase and intensity error plotted against the transmittance and phase shift of the spatial filter,

[0102] - Figure 6 A and Figure 6 B shows the simulated relative phase and intensity errors, plotted against the ratio of the spatial filter radius to the optical system exit pupil radius,

[0103] - Figure 7 A is the experimental image in Fourier plane, Figure 7 B and Figure 7 C shows the image and contour of the incident light radiation in the Fourier plane with and without the filter, respectively.

[0104] - Figure 8 A. Figure 8 B and Figure 8 C each shows an intensity and phase image, and an intensity and phase contour plotted from the segments shown in the intensity and phase images,

[0105] - Fig. 9 A and Fig. 9 B shows Figure 8 A and Figure 8 A magnified view of the phase image of C.

[0106] - Fig.10 A. Fig.10 B and Fig.10 C shows three different angular spectra of the light radiation in the Fourier plane, Fig.10 D. Fig.10 E and Fig.10 F shows the corresponding optical spatial filter,

[0107] - Fig.11A is a schematic diagram of a portion of an alternative embodiment of an optical spatial filter, and Fig. 11B Shows the Fig.11A The complex representation of the incident, scattered and total fields after filtering by the optical spatial filter,

[0108] - Fig.12 A and Fig.12 B are the same object, for different values ​​of the applied variable phase shift, when Fig.11A Experimental intensity and phase images without (group 1 on the left) and with (group 2 on the right) the optical spatial filter, and

[0109] - Fig.13A and 13B They are shown respectively with Fig.12 A and 12B correspond to the intensity contrast curve and phase gain curve. DETAILED DESCRIPTION

[0110] Figure 1A An exemplary quantitative phase imaging device 1 according to the present invention is shown. The device comprises a light source 2, a telecentric imaging optical system 3, an optical spatial filter 4 and a wavefront sensor 5. A portion of the optical system 3 is housed in a microscope 9. This portion of the optical system 3 comprises an objective lens 31, a reflector 32 and a tube lens 33. The microscope 9 further comprises a sample holder 10, which is arranged in an object focal plane A of the optical system 3.

[0111] In order to image an object, a user of the device 1 places a sample 14 including the object on the sample holder 10 .

[0112] The light source 2 includes a light generator 21 and may include an optical system 22 between the light generator 21 and the object focal plane A for creating a The incident radiation I from the light source 2 is thus collimated and impinges on the sample holder 10 generally in the object focal plane A. A portion D of this radiation is then scattered by the sample 14.

[0113] In the example shown, the image focal plane B of the objective 31 corresponding to the Fourier plane of the optical system is inaccessible because it is located inside the microscope 9. Advantageously, the spatial filter 4 can be arranged in a plane C conjugate to the Fourier plane B, which is displaced by the converging lens 34 of the imaging optical system 3 after the tube lens 33.

[0114] Therefore, the conjugate plane C corresponds to the Fourier plane of the optical system 3 .

[0115] like Figure 1B As shown, the wavefront sensor 5 may comprise a digital camera 51 and a wavefront analysis mask 53 (for example of the Shack-Hartman type) arranged upstream of the camera 51 . The radiation R is characterized by its wavefront F which reaches the wavefront analysis mask 53 before being detected by the camera 51 .

[0116] The wavefront analysis mask 53 can change the path of the optical radiation, and the resulting complex amplitude variation enables the phase and intensity of the optical radiation to be measured simultaneously.

[0117] therefore, Figure 1B The wavefront sensor shown measures the intensity and phase of all optical radiation it receives.

[0118] like Figure 1A As shown in the example in , in front of the wavefront sensor 5 in the direction of propagation of the optical radiation there is a converging lens 35 which is part of the imaging optical system 3 and allows imaging of the sample 14 onto the digital camera 51 .

[0119] The device 1 further comprises an additional camera 15 for imaging the Fourier plane C via a pivoting or semi-transparent mirror 16 and a two-lens optical system 17. Thus, the spatial filter 4 can be precisely positioned in the Fourier plane C, centered on the optical axis, for example using a translation stage (not shown).

[0120] The optical spatial filter 4 has a maximum attenuation region 41 and a minimum attenuation region 42. In the example shown, the spatial filter comprises a support 43 in the form of a transparent plate (e.g. made of glass and having a square shape), the surface of which is covered with a metal coating 44 in the shape of a disk with a radius r. fs , at the center of the plate.

[0121] The minimum attenuation region 42 may be defined by the portion of the support 43 not covered by the coating 44 , and the maximum attenuation region is defined by the superposition of the support 43 and the coating 44 .

[0122] Figure 2A schematically shows the spatial distribution of the incident and scattered electromagnetic fields in the Fourier plane obtained by the device 1 according to the present invention when the spatial filter 4 is not present. When the sample 14 is placed in the object focal plane A on the sample holder 10 of the device without the spatial filter 4, the incident electromagnetic field corresponding to the incident radiation I not affected by the sample is denoted by E i , the scattered electromagnetic field corresponding to the scattered radiation D is expressed as E d . and They represent the scattered field E d and E i The spatial Fourier transform of .

[0123] When the sample 14 is a semi-transparent, non-absorbing, low-scattering object, the total electromagnetic field E t It can be expressed by equation (1):

[0124] E t =E i +E d (1)

[0125] In this example, since the incident radiation I is collimated at the sample 14 , it has a point distribution in the center of the Fourier plane C, corresponding to the image of the pupil of the optical system 3 .

[0126] When the sample 14 is smaller than the wavelength of the incident radiation (for example smaller than 100 nm), the radiation D is scattered in a quasi-isotropic manner. It is therefore distributed uniformly over the image of the pupil of the optical system 3 in the Fourier plane C.

[0127] Field E i and E d can be observed in the Fourier plane C by an additional camera 15 which images the Fourier plane C, as Figure 2 A schematically shows the field E d fills the Fourier plane C, while the field E i In this case it is concentrated at a point in the center of the Fourier plane C. More generally, the field E i The distribution is carried out according to the angular distribution of the light radiation I emitted by the light source 2 .

[0128] In the case of low scattering objects, such as Figure 2 C, the modulus of the scattered field |E d | Compared to the norm of the incident field |E i | is smaller. For example, its value is smaller than the magnitude of the incident field |E i | is more than 1000 times smaller. Then we can consider the total field E t Equal to the incident field E i After phase shift The result after that is equation (2):

[0129]

[0130] For biological objects, such as vesicles or viruses, or even nanoparticles, the phase shift Small, especially less than 0.05 radians, so that Using Taylor expansion on , we get equation (4):

[0131]

[0132] By the notation between equation (1) and equation (4), the scattered field E d According to equation (5), the incident field E can be written as i Attenuated and phase shifted by approximately

[0133]

[0134] Figure 2 C shows the incident field E obtained using the device 1 without a spatial filter i , scattered field E d and total field E t The figure shows the scattered field E d Relative to the incident field E i near phase shift.

[0135] In the case of the device according to the invention, when the optical spatial filter 4 is present in the Fourier plane C, E i ' is the total electromagnetic field E corresponding to the incident electromagnetic field of the incident radiation I after being transmitted through the spatial filter 4 and reaching the wavefront sensor 5 t The contribution of E d ' is the contribution of the scattered electromagnetic field corresponding to the scattered radiation D after being transmitted through the spatial filter 4 to the total electromagnetic field.

[0136] When the sample 14 is a semi-transparent, non-absorbing, low-scattering object, the following equation (6) can be defined:

[0137] E t ′=E i ′+E d ′ (6)

[0138] The complex transmittance t in the smaller attenuation region is defined as t = t0e -iβ .

[0139] The radius of the smaller attenuation area r fs Preferably, the radius r of the image of the pupil of the optical system 3 in the Fourier plane isp At least 5 times smaller. Therefore, the maximum attenuation region only affects the incident field but not the scattered field. Figure 2 B. Therefore, the following equations (7) to (9) can be written as:

[0140] E′ i =tE i =t0e -iβ .E i (7)

[0141] E′ d =E d (8)

[0142] E t ′=E′ i +E′ d =t0E i e -iβ +E d (9)

[0143] Figure 2 D and Figure 2 E shows the filtered incident field E i ′, the scattered field after filtering E d and the total field E after filtering t ', where β is zero and where β is non-zero, respectively. In particular, with Figure 2 C shows the unfiltered incident field E i Compared with the amplitude of the incident field E after filtering, it can be seen that i The amplitude of the transmittance is selected so that |E i ′|=|tE i |≈|E d |In E i The best compromise is achieved between the attenuation of the phase and the signal level required for good phase reconstruction. <t0<0.7。

[0144] The area of ​​maximum attenuation is semi-transparent. Incident field E i Complete elimination of ′=0 by the maximally attenuated opaque region produces darkfield illumination but prevents the phase of the wavefront from being defined over the entire field of view.

[0145] The total field E t 'Experienced with field E i ′ New phase shift associated with

[0146] Phase shift caused by sample 14 By measuring the field E t The intensity and phase of ′ are quantified.

[0147] Combining equations (1) and (8), we obtain equation (10):

[0148] E t =E t ′+(1-t0e -iβ )E i (10)

[0149] I t and I t ' are the total field E of device 1 without spatial filter 4 t The relative intensity of the total field E after filtering by the device 1 with the spatial filter 4 t ′. Equations (11) and (12) can be expressed as:

[0150]

[0151] Starting from equation (10), equation (13) can also be expressed as:

[0152]

[0153] The wavefront sensor 5 measures the relative intensity, i.e. the ratio between the reference image without the sample and the image with the sample 14 present. The intensity ratio I defined by equations (14) and (15) is m and I m ′ can therefore be measured:

[0154]

[0155] Equation (16) can then be formulated from equation (13):

[0156]

[0157] The complex transmittance through the known spatial filter 4 and based on the parameters measured in the presence of the spatial filter 4 and The phase shift caused by scattering objects from the sample 14 can be quantified and absorption I m .

[0158] The implementation of the device 1 was numerically simulated to determine the optimal conditions of use. This simulation was performed according to the method in the paper "Modeling quantitative phase image formation under tilted illuminations" by P. Bon, B. Wattellier and S. Monneret, Opt. Lett. 37, 1718 (2012).

[0159] The sample 14 under investigation is a polystyrene bead with a refractive index of 1.62 immersed in water with a refractive index of 1.33. The sample 14 is irradiated with monochromatic radiation of wavelength λ=440 nm. The objective lens 31 used has a numerical aperture of 1.4.

[0160] For the total field E t The spatial Fourier transform field of A spatial filtering simulation was performed.

[0161] Then, the radius r fs The simulated electromagnetic field in the disk with a radius of (the radius of the maximum attenuation region 41 of the spatial filter 4) is multiplied by the complex transmittance t0e of the spatial filter 4 -iβ , to simulate the interaction with the spatial filter 4. The radius is r p The field region outside the disk (the image radius of the pupil of the optical system 3 in Fourier plane C) is set to zero to simulate the finite numerical aperture of the optical system 3. It is simulated according to equation (17):

[0162]

[0163] where 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 optical radiation.

[0164] By t ′The inverse spatial Fourier transform calculation is performed to obtain the total field after filtering which can be measured on the wavefront sensor 5 .

[0165] Intensity I′ m and Phase Through and arg(E′ t ) are calculated and are respectively Figure 3 E and Figure 3 The intensity and phase diagrams of B are shown in the figure. The calculations took into account t0 = 0.1, β = 0 and Figure 3 B shows the measured phase The amplification is greater than the phase shift calculated without the spatial filter. The value (such as Figure 3 A) is more than ten times higher. Figure 3 The intensity map of E corresponds to a partial dark-field image, where the light scattered by the object (located in the center of the map) dominates the incident light.

[0166] Figure 4 A shows the non-inverting amplification, Figure 4B shows the simulated intensity contrast amplification, which is for the transmittance T of the spatial filter 4 and the phase shift β When the phase shift β caused by the spatial filter 4 approaches 0 [2π], the phase gain increases as the transmittance T decreases. This means that when the ratio When equilibrium is reached, the device 1 operates in an optimal state.

[0167] Calculated intensity I m ′ and phase is processed according to equation (18) to return to quantitative parameters:

[0168]

[0169] By comparing the actual phase and intensity values The phase and intensity values ​​obtained after filtering and quantitative processing The relative error caused by the spatial filter 4 is estimated and is particularly low. Figure 3 C and Figure 3 A and Figure 3 F and Figure 3 D can be seen that the phase and intensity values ​​obtained With actual phase and intensity values Almost identical.

[0170] This comparison reflects the correct operation of the quantitative processing used to return the value when no filter is present.

[0171] like Figure 5 As shown, modifying the complex transmittance of the spatial filter 4 results in a phase ( Figure 5 A) and strength ( Figure 5 B) relative errors are less than 10 -2 and 10 -4 .

[0172] Figure 6 The ratio of the radius of the spatial filter 4 to the image radius of the pupil of the optical system 3 in the Fourier plane C is shown. The impact on relative phase and intensity errors. In the case of Figure 6 A) and the intensity error is less than 10 -3 %( Figure 6 B) This ratio is established as the limit below which it is reasonable to assume that the presence of the spatial filter 4 does not modify the scattered field E d .

[0173] Example 1

[0174] The microscope 9 used was a commercial microscope Olympus IX71 equipped with a 60x microscope objective lens (Olympus UPlanFLN) having a numerical aperture of 0.9.

[0175] Sample 14 is viewed through the microscope 9 Illumination (halogen lamp) was illuminated and wavelength filtered.

[0176] The wavefront sensor 5 is an ID4L analyzer (four-wave lateral shearing interferometer), which consists of an Andor Zyla 5.5 camera and a 2D diffraction grating with a period of 10 μm and is optimized for a wavelength of 650 nm. The grating is re-imaged on the camera through a 2x magnification telescope consisting of two achromatic lenses. The effective distance between the diffraction grating and the camera is d = 1.2 mm.

[0177] The maximum attenuation region 41 of the spatial filter 4 is a 80 nm gold disk on a 5 nm chromium layer, with a radius r fs = 100 μm, deposited on a glass slide using electron beam evaporation. The transmittance of the optical spatial filter was experimentally measured at wavelengths λ = 440 and 550 nm as T = 7%: in the absence of a sample, the transmittance was derived from the formula T = I2 / I1 by measuring the intensity signal I1 without the filter and the intensity signal I2 with the filter. The phase shift β = 0.5 rad was measured by interferometry.

[0178] Figure 7 A shows an image of the Fourier plane of the experimental setup acquired by the additional camera 15. The white disk corresponds to the radiation D scattered by the sample consisting of a superposition of multiple layers of paper, which is distributed over the image of the entire pupil of the optical system 3 in the Fourier plane C, and the black disk in the middle corresponds to the spatial filter 4. By comparison Figure 7 B and Figure 7 C can reveal the effect of spatial filter 4 on the intensity of incident radiation I.

[0179] The sample 14 under investigation has a refractive index of n poly = 1.62 and a diameter of e = 100 nm, immersed in a water =1.33 in water.

[0180] Figure 8 The results obtained by quantitative reconstruction starting from the filtered image using equation 16 are shown without and with spatial filter 4. Figure 8 A shows the result obtained without the spatial filter 4, Figure 8 B shows the result obtained with spatial filter 4, and Figure 8C shows the result obtained after quantitative processing based on the measurements using the spatial filter 4.

[0181] In the experiment, the gain of the phase signal was measured to be about 3 times that of the case without the spatial filter. Increase to when there is a spatial filter Close to the theoretical value simulated by the model, such as Figure 4 A. Phase values ​​are expressed in nanometers (nm) because they relate to differences in optical path difference δ, a quantity that can be related to phase (radians) by the following equation: where λ is the wavelength of the radiation.

[0182] Since the magnitude of the field scattered by the beads is very low compared to that of the incident field, the beads do not appear in the intensity image without the filter, as shown in Figure 8 As shown in A. Figure 8 B shows that the presence of spatial filter 4 produces a partial dark field configuration and causes the beads to be emphasized with a contrast of 0.2, which is also close to Figure 4 The expected theoretical value in B.

[0183] After quantitative reconstruction, Figure 8 It can be seen in C that the reconstructed intensity and phase values ​​are the same as the original ones, but the signal-to-noise ratio is significantly improved.

[0184] Fig. 9 A is Figure 8 A magnification of the phase image in B, Fig. 9 B is Figure 8 A zoom-in of the phase image in C. The noise is Fig. 9 A and Fig. 9 The calculation is done within the area delimited by the dotted line in B. Fig. 9 In A, the phase rms noise observed in the measurement without the spatial filter is And in Fig. 9 In B, the phase rms noise observed in the filtered and subsequently reconstructed measurement is

[0185] In a variant, the angular spectrum of the incident optical radiation I is a disk centered in the Fourier plane C, Fig.10 As can be seen in A, the white area corresponds to the incident light radiation I. The maximum attenuation area 41 is the disk at the center of the optical spatial filter 4, as shown in FIG. Fig.10 As shown in FIG. 4D , the grey area corresponds to the maximum attenuation area 41 .

[0186] In one variant, the angular spectrum of the incident optical radiation I is shown in the Fourier plane C by a plurality of spaced-apart circular disks, from Fig.10As can be seen in B, the white area corresponds to the incident light radiation I. The maximum attenuation area 41 consists of a plurality of disks, which are distributed on the optical spatial filter 4 in the same way as the incident light radiation I in the Fourier plane C, as shown in FIG. Fig.10 As shown in FIG. E , the gray area corresponds to the maximum attenuation area 41 .

[0187] In another variant, the angular spectrum of the incident optical radiation I is annular in the Fourier plane C, from Fig.10 As can be seen in A, the white area corresponds to the incident light radiation I. The maximum attenuation area 41 is the corresponding ring on the optical spatial filter 4, such as Fig.10 As shown in FIG. 4D , the grey area corresponds to the maximum attenuation area 41 .

[0188] Example 2

[0189] The same sample 14 and apparatus as in Example 1 were used, the only difference being the optical spatial filter 4. In this case, Fig.11A It can be seen that the maximum attenuation region 41 of the filter includes a cavity 48 containing a medium with a high thermo-optical coefficient dn / dT, such as glycerol (dn / dT = -2.7·10 -4 ), the medium is in contact with the above-mentioned 80 nanometer thick gold disk 46, and the cavity 48 and the disk 46 are located between the two glass plates 45 of the optical spatial filter 4.

[0190] By focusing the laser heating system 47, the cavity 48 makes it possible to change the incident field E by the optical spatial filter 4. i The optical spatial filter 4 is in the incident field E i The total phase shift β caused by total is the variable component β variable and a fixed component β fixed The sum of Fig. 11B It can be seen that E′ i 、E d and E t ′ are the complex representations of the filtered incident field, the filtered scattered field, and the filtered total field, respectively. Phase shift β variable Allows optimization of phase shift and intensity |E′ t | 2 The value of .

[0191] Fig.12 A and Fig.12 B shows the intensity and phase images of the experiment, respectively, when there is no filter (group 1 on the left) and when different β values ​​are obtained by heating the thermistor chamber. variable value, there is a case where an optical spatial filter 4 exists (group 2 on the right). Fig.13A and Fig. 13B The curves in show the changes in intensity and phase contrast, respectively, which are plotted against the phase shift caused by the optical spatial filter 4. It should be noted that Figure 4 The simulation results presented in A and 4B are verified in the experiment, and the transmittance T is equal to 7%.

[0192] The use of the cavity 48 and the heating system 47 enables the phase shift caused by the optical spatial filter 4 to be varied in real time in order to optimize the signal-to-noise ratio of both the intensity signal and the phase signal.

Claims

1. A quantitative phase imaging device (1), comprising: - an imaging optical system (3) for imaging an object (14) in an image plane, a light source (2) for emitting optical radiation (I) onto at least a portion of a Fourier plane (C) of the imaging optical system (3), an optical spatial filter (4) extending in the Fourier plane (C) of the imaging optical system (3) and comprising a region (41) in which the optical radiation (D) is attenuated to a minimum and a region (42) in which the optical radiation (I) is attenuated to a maximum, a wavefront sensor (5) for measuring, in the image plane, the intensity and the phase of the electromagnetic field associated with the optical radiation (I; D), emitted by the light source (2), having passed through the imaging optical system (3) and the optical spatial filter (4), and having at least partially interacted with the object (14), in order to quantify the phase caused by the object, The light source (2), the imaging optical system (3) and the optical spatial filter (4) are arranged such that when the object (14) to be imaged is not present, the optical radiation (I) is focused on the maximum attenuation region (41).

2. The device according to claim 1, wherein the complex transmittance t of the maximum attenuation region (41) is defined by the equation t = t 0* e -iβ where 0 < t0 ≤ 0.7, the amplitude t0 is measured over at least part of the wavelength spectrum of the optical radiation (I) emitted by the light source (2), and the optional phase shift β is such that -1 rad [π] ≤ β ≤ 1 rad [π], in particular in order to amplify the absolute value of the signal in terms of phase.

3. According to the device according to any one of claims 1 and 2, the optical spatial filter (4) includes a transparent support (43) and a translucent coating (44) partially covering the support (43), and the maximum attenuation area (41) is defined by the superposition of the support (43) and the translucent coating (44).

4. The device according to claim 3, wherein the semi-transparent coating (44) is a thin film having a radius r fs At least one disc in the form of r fs ≤100μm, the radius r fs Preferably less than 0.1*r p , where r p is the maximum radius of the disk within which the spatial frequencies collected in the Fourier plane (C) are distributed.

5. The device according to claim 3, wherein the translucent coating (44) is annular.

6. The device according to any one of claims 3 to 5, wherein the semi-transparent coating (44) is located at the center of the optical axis of the optical spatial filter (4).

7. The device according to any one of claims 3 to 6, wherein the translucent coating (44) comprises a translucent layer of a metal selected from gold, silver, aluminum, chromium, titanium and alloys thereof, such as gold.

8. Device according to the preceding claim, the translucent coating (44) comprising a bonding layer sandwiched between and in contact with the support (43) and the translucent layer (for example made of chrome and / or titanium).

9. According to the device according to any of the preceding claims, the wavefront sensor (5) comprises a light radiation detector and a wavefront analysis mask (53) arranged in front of the detector along the optical path of the light radiation so as to determine the spatial distribution of the phase gradient of the light radiation (I; D; R) or the spatial distribution of a signal proportional to the phase.

10. The device according to the preceding claim, the wavefront analysis mask (53) being selected from masks comprising lenslet arrays, such as Shack-Hartmann masks, modified Hartmann masks and thin diffusers.

11. According to the device described in any one of claims 9 and 10, the optical radiation detector (I; D; R) preferably comprises a digital camera for collecting optical radiation (I; D; R), which optical radiation preferably has interacted with a wavefront analysis mask (53).

12. The device according to the preceding claim, the digital camera (51) comprising a light sensor (eg CMOS or CCD) for collecting optical radiation (I; D; R) and measuring the intensity (I; D; R) of said optical radiation.

13. The device according to any of the preceding claims, comprising an additional digital camera (15), in particular for measuring the light radiation intensity (I; D) in a Fourier plane (C) of the imaging optical system (3).

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

15. A device according to any of the preceding claims, wherein the maximum attenuation zone (41) has a variable complex transmittance, in particular depending on the polarization state of the optical radiation (I) and / or the temperature of the zone (41), and / or is modifiable by a user.

16. Device according to the preceding claim, the zone of maximum attenuation (41) comprising a thermochromic material and / or a polarizing material.

17. A device according to the preceding claim, wherein the thermochromic material is selected from thermochromic liquid crystals, thermochromic leuco dyes, thermochromic oxides (possibly doped), and mixtures thereof, such as from the group formed by VO2, BiVO4, NbO2 and mixtures thereof.

18. According to the device according to any of the preceding claims, the maximum attenuation area (41) and / or the minimum attenuation area (42) includes at least one layer made of the following materials, for example a layer including a polymer, glass or titanium dioxide, a layer stack or a metasurface: the material is capable of causing a phase shift between the radiation (I; D) incident on the area and the radiation (I; D) after attenuation by the area.

19. The device according to the preceding claim, the region of maximum attenuation (41) and / or the region of minimum attenuation (42) comprising a layer of birefringent material.

20. The device according to any one of claims 18 and 19, the maximum attenuation area and / or the minimum attenuation area comprising a layer of a material with a non-zero thermo-optic coefficient, in particular a liquid (such as glycerol) or a polymer (such as polydimethylsiloxane).

21. The device according to any of the preceding claims, comprises a thermal regulation module, which is used to modify the temperature of the maximum attenuation area (41) and / or the minimum attenuation area (42) to modify the transmittance of the area (41; 42) and / or the phase shift caused by the area (41; 42).

22. An apparatus according to any preceding claim, comprising a plurality of optical spatial filters (4).

23. A device according to any of the preceding claims, wherein the light source (2) comprises a light generator (21) for generating light radiation (I), for example selected from a lamp (especially a halogen lamp) and a high-power laser (especially a supercontinuum laser).

24. The device according to the preceding claim, wherein the maximum attenuation region is in the form of a disk centered on the optical axis, and the light source (2) comprises a An additional optical system (22) for illumination, the additional optical system (22) being arranged between a light generator (21) and the object (14) in the propagation direction of the optical radiation (I), in particular for collimating the optical radiation (I) incident on the object (14).

25. A method for acquiring at least one digital image of a sample including an object (14) by means of a device (1) according to any one of the preceding claims, the method comprising: a) emitting incident optical radiation (I) directly towards the object (14) using the light source (2), b) detecting, by means of a wavefront sensor (5), all optical radiation (I; D; R) that has been transmitted by the imaging optical system (3) and the optical spatial filter (4) and that has interacted with the object (14); c) processing the signals detected in step b) to quantify a parameter selected from the phase of said optical radiation (I; D; R) and / or a phase variation of said optical radiation (I; D; R) and optionally generating a digital image of said parameter.

26. Method according to the preceding claim, the processing in step c) further comprising quantifying the intensity of said optical radiation (I; D; R) and preferably generating a digital image of the intensity of said optical radiation (I; D; R).

27. The method according to any one of claims 25 and 26, wherein: The sample (14) includes one or more objects (14) that are smaller than the resolution of the imaging optical system (3).

28. The method according to any one of claims 25 to 27, wherein the sample is selected from biological materials including microorganisms, powders, biological culture fluids, groups of cells or microtissues, inert materials including nanoparticles or solutions of particle mixtures.

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