How to identify the properties of particles in a medium

Abstract

A method for identifying a property of at least one particle (P) in a medium (M), comprising the steps of: emitting a coherent light beam (6) to illuminate a sample (5) of said medium (M); recording an interference image (10) of a first portion of said light beam (6) scattered by said at least one particle (P) and a second portion of said light beam (6) not scattered by said particle (P); calculating (S1) for positions (13) in said sample (5) an electric field (E1) of said first portion from said interference image (10); generating (S2) for each of said positions (13) a representation (16) comprising a phase value (ξ) determined from the calculated electric field (E1) and the estimated electric field (E2) of the second portion, and an intensity value (I) determined from the intensity (I1) of said first portion and said phase value (ξ); and identifying (S3) said property therefrom.

Description

Detailed Description of the Invention

[0001] The present invention relates to a method for determining a property of at least one particle in a medium.

[0002] The background of the invention is in the field of observing the properties of one or more particles in colloids, such as emulsions, cell cultures and bacteria in a medium, for example to study the initiation of bacterial infection in urinary tract infections. One may also observe plastic particles in seawater, impurities in liquid food or medicine, or cells, their organelles, etc. in body fluids. The properties may be either particle-specific, such as size, shape, organization, propulsion mechanism, dry mass, or medium-related, such as particle position, velocity or diffusion in the medium. One may also determine properties such as distribution, distance, interactions of particles in the medium.

[0003] For example, it is known to observe the distribution or movement of bacteria in a medium by digital holographic microscopy. In this method, a sample containing said bacteria is illuminated with coherent light to obtain an interference image of the bacteria. From this interference image, a three-dimensional model of the bacteria and their distribution in the medium can then be calculated, for example by applying a reconstruction algorithm, such as a forward or backward propagation or a projection algorithm, to the interference image. However, experiments have shown that the three-dimensional model thus obtained is often qualitatively inadequate for a detailed analysis, due to an unavoidable background signal in the interference image. The background signal is generated by a part of the light scattered in the medium, i.e. not by the particles, but by other (usually smaller) objects in the medium (for example by Mie scattering), which impairs the contrast between the light scattered by the particles and the light not scattered by the particles.

[0004] To address this issue, Cheong FC, et. al. "Rapid, High-Throughput Tracking of Bacterial Motility in 3D via Phase-Contrast Holographic Video Microscopy", Biophysical Journal Vol.108, March 2015, pp,1248-1256 proposes to digitally introduce a phase shift between the light scattered by the medium and the light that is neither scattered by particles nor by the medium in the reconstruction algorithm. This reduces the background signal and improves the contrast.

[0005] Nevertheless, the contrast achieved by current holographic microscopy and related analytical methods may still be too low to fully resolve the properties of each particle, e.g., its location in the medium. Low contrast may lead to the false identification of artifacts as particles in the three-dimensional model. This occurs especially in the analysis of samples with high density of particles and / or small size or weak scattering particles. On the other hand, particles with high refractive index interact with light in a lens-like manner, so that the particles are located at the particle focus and appear to have different sizes and other properties, rather than at their true location. This is known as the "lensing effect". In this case, the contrast achieved by current holographic microscopy is not enough to resolve the true particle properties.

[0006] It is an object of the present invention to provide a method for characterizing the properties of at least one particle in a medium, which allows the properties to be more accurately characterized.

[0007] The object is to provide a method for characterizing the properties of at least one particle in a medium, comprising: providing a sample of a medium containing the at least one particle; Using a light source to emit a coherent light beam, irradiating the sample with the light beam, and causing a first portion of the light beam to be scattered by at least one particle to form a scattered light beam; Recording, using a camera, an interference image between the scattered light beam and a second portion of the light beam that has not been scattered by the at least one particle; For each of a plurality of positions three-dimensionally distributed within the sample, calculating, by a processor, the electric field of the first portion of the light beam at that position from the interference image; For each of the positions, generating, using a processor, a display of the sample covering the position, the display including a phase value specified from the calculated electric field and an estimated electric field of the second portion of the light beam at the position, the intensity of the first portion of the light beam, and an intensity value specified from the specified phase values at both the positions; Identifying, by a processor, the above properties using the above display. The method includes the above steps to achieve the goal.

[0008] Note that since the electric field of a light beam is generally a complex number, it can be described by its real and imaginary parts or by its amplitude and phase. Further, a light beam has an intensity, i.e., power per unit area. The power per unit area is proportional to the square of the amplitude of the electric field.

[0009] The applicant has found that the method of the present invention provides a higher contrast in phase values ​​determined from both the light beam scattered by particles in the medium and the light beam not scattered by particles in the medium, rather than from the phase of the scattered light beam alone as is the case in the past. The higher contrast is also based on the determination of intensity values, each of which is determined from the phase value and the intensity of the first portion of the light beam. As a result, the nature of weakly scattering or small size particles in the medium, or even particles with high density, can be revealed by the display. Furthermore, artifacts can be largely eliminated, and the nature of particles exhibiting lensing can be distinguished from their focus, resulting in a more accurate determination of the nature of at least one particle in the medium.

[0010] To calculate the electric field of the first portion of the light beam, the positions may be distributed in the sample as desired, for example as a regular grid representing a contiguous cubic region in the sample, or as an irregular grid, for example representing an arbitrarily formed spatial region in the sample. However, in a preferred embodiment, the positions are in an imaginary plane perpendicular to the direction of the second portion of the light beam, and the calculating step is performed for each plane along said direction. Such a distribution of the positions allows an efficient calculation of the electric field and the determination of phase and intensity values ​​for each plane along the beam direction, i.e. in the beam direction or against. Since the planes are perpendicular to the direction of the light beam, the electric field of the second portion of the light beam can be assumed to have the same phase at all positions in the respective planes.

[0011] The interference image conveys information about both the first and second portions of the light beam. In order to easily distinguish between the first and second portions of the light beam in the calculating step, it is advantageous to normalize the interference image before the calculating step based on a reference image of a coherent light beam without scattering by at least one particle. The normalized interference image allows a more accurate calculation of the electric field of the first portion, and therefore allows a more accurate determination of both phase and intensity values, which allows an improvement in contrast.

[0012] Preferably, in the generating step, the electric field of the second portion of the light beam is estimated from values ​​of a corresponding reference image of a coherent light beam without scattering by particles, which allows the electric field of the second portion of the light beam to be estimated easily and more accurately.

[0013] Said reference image may be generated by various methods known in the art, for example as a low-pass filtered version of the interference image, or may be generated by repeating the emitting and recording steps and recording a number of the resulting interference images, or may be an average generated as a reference image. Advantageously, the reference image is generated by emitting a coherent light beam using a light source that does not include a sample of the medium containing at least one particle, and recording the reference image. In this embodiment, the reference image is easily generated using a sample of the medium, where the medium does not include said particle or sample, both of these options being encompassed by the statement "does not include a sample of the medium containing at least one particle".

[0014] A particularly high contrast can be achieved when, in the generating step, for each of the locations, a minimum difference angle between the calculated electric field phase and the estimated electric field phase is identified and the phase value is specified as part of the difference angle. According to this embodiment, the smaller the difference angle between the phases, the smaller the intensity value can be so that the contrast is further improved.

[0015] Advantageously, in the generating step, the phase value is determined as the phase of a weighted sum of the calculated and estimated electric fields. Such determination is simple and easy to compute, requiring only the calculated and estimated electric field magnitudes. The magnitudes may for example be determined from iterations or past experience of maximizing contrast, by suppression of background signals, etc.

[0016] In a particularly preferred aspect of the foregoing embodiment, in the generating step, for each of said locations, the phase value is calculated according to the following formula:

[0017]

number

[0018] where r and z are coordinates of a position, z is a coordinate in a direction opposite to the direction of the second portion of the light beam, and r is a pair of coordinates in a plane perpendicular to said opposite direction; E 1 (r,z) is the calculated electric field of the first portion of the light beam at position ξ(r,z) is the phase value at position, Re and Im represent the real and imaginary part operators. arctan2 represents the four-quadrant arctangent function. is identified according to

[0019] In this embodiment, the electric field of the second portion of the light beam is estimated to be real and has a particularly accurate amplitude in the above embodiment with a normalized interference image, whereby the first and second portions of the light beam are weighted equally and can be easily identified as having smaller intensity values ​​at locations where the difference angle between the phase of the calculated electric field and the estimated electric field is smaller, thereby further enhancing the contrast.

[0020] In a preferred embodiment, in the generating step, for each of said locations, the intensity value is calculated according to the following formula:

[0021]

number

[0022] where r and z are coordinates of a position, z is a coordinate in a direction opposite to the direction of the second portion of the light beam, and r is a pair of coordinates in a plane perpendicular to said opposite direction; I 1 (r,z) is the intensity of the first portion of the light beam at position ξ(r,z) is the phase value at position, I(r,z) is the intensity value at position.) is identified according to

[0023] In this embodiment, the intensity values ​​are calculated efficiently. The additional phase shift of π further attenuates the background signal so that light scattered by particles can be more easily distinguished from light not scattered by particles in the displayed intensity value.

[0024] In order to more easily distinguish between light scattered by particles and light not scattered by particles, an intensity threshold is calculated using the intensity values ​​before the identifying step, preferably as an average of two or more of the intensity values, and it is preferred that for each of the locations, when the intensity value is smaller than the calculated intensity threshold, neither the phase value nor the intensity value contained in the representation for that location is used in the identifying step. Apart from easier discrimination, this allows for a more rapid identification of the particle distribution, since the amount of data to be considered is reduced. This embodiment allows the intensity threshold to be calculated by methods known to those skilled in the art, for example as a predefined percentage of the highest intensity value (generally, typically, expected or currently identified), the average of the intensity values ​​at several locations, the overall average of all the intensity values ​​of the representation, etc.

[0025] In a further embodiment of the invention, in the emitting step, two or more coherent light beams are emitted, and the recording, calculating and generating steps are performed for each of the two or more coherent light beams to obtain a respective representation of the sample, and the property is determined using the two or more obtained representations. Thereby, the property can be more accurately determined from the additional information provided by the two or more representations. Furthermore, if the emission is delayed in time, properties such as particle velocity, particle diffusion, electrophoresis, etc. can be studied.

[0026] According to an advantageous embodiment, two or more representations are averaged into an averaged representation, and in said identifying step, said property is identified from said averaged representation. By this averaging of several representations, background signals are efficiently attenuated in the averaged representation, so that identifying said property (e.g. three-dimensional particle distribution) of at least one particle in the medium is easier, especially if the particle is stationary.

[0027] In another embodiment, in the emitting step, each of the two or more coherent light beams is emitted using a respective one of two or more light sources. In this case, the sample can be illuminated at various angles, which provides an additional example, for example, to further facilitate differentiation of particle positions common to all light beams from the particle's focal point, each of which is differently positioned relative to these light beams. This applies a technique to increase the resolution of the display, for example, by multiplexing spatial frequency bands, known in the art as "super-resolution". Furthermore, when using a respective light source for each light beam, the coherent light beams can be emitted simultaneously, and therefore can be easily correlated.

[0028] In the above-described embodiment, the emitting step preferably emits two or more coherent light beams, each at a different frequency. This allows, for example, to obtain a colored and / or more accurate particle distribution by averaging two or more displays. Furthermore, additional information regarding particle properties, such as particle color, dispersion properties, secretion color, etc., is generated.

[0029] In a particularly preferred embodiment, in the emitting step, three coherent light beams are emitted, and the frequencies of the three coherent light beams correspond to the colors red, green, and blue. This makes it possible to easily obtain color indications by utilizing the high sensitivity of conventional camera sensors for red, green, and blue light (RGB sensors). In some cases, further particle properties, such as the oxidation state of iron in hemoglobin particles, the color of the particle's secretion such as toxins, etc., can also be identified by color indications.

[0030] The present invention will now be described in detail based on preferred exemplary embodiments with reference to the accompanying drawings, in which: FIG. 1 is a schematic side view of an in-line interferometer used in the method of the present invention; FIG. 2 is a flow chart illustrating the determination of a three-dimensional particle distribution from an interferometric image produced by the interferometer of FIG. 1 according to the present invention; Figure 3 shows the determination of phase values ​​according to the determination of Figure 2 in the complex plane; 4 and 5 show graphs of light intensity in a sample of the medium, respectively, in a direction opposite to the direction of the light beam (FIG. 4) and perpendicular to the light beam (FIG. 5).

[0031] FIG. 1 shows an in-line interferometer 1 with a light source 2 and a camera 3. The in-line interferometer 1 is used to determine the properties of at least one (microscopic) particle P in a medium M, such as the position, size, shape, structure, elasticity, dry mass, secretion, velocity, propulsion mechanism, diffusion, vesicular shape, and / or the distribution, mutual distance, interaction, etc. of one or more particles. In the illustrated embodiment, a three-dimensional particle distribution 4 (FIG. 2) of particles P in the medium M is determined. A sample 5 of the medium M containing one or more particles P is provided between the light source 2 and the camera 3 for the determination of the properties. The one or more particles P can be cells, bacteria, charged particles, microplastics, organelles, etc., and the medium M can be water, oil, body fluids (e.g. blood), liquid drugs, etc. In general, any particle P in any colloid can be analyzed.

[0032] For this purpose, the light source 2 emits a coherent light beam 6 to illuminate a provided sample 5. The light source 2 may be capable of emitting a coherent light beam 6, such as for example a laser diode.

[0033] In the sample 5, a first portion of the light beam 6 is scattered by one or more particles P, thereby generating a scattered light beam 7. However, a second portion of the light beam 6 is not scattered by the particles P and traverses the sample 5 in a beam direction 9 as an unscattered light beam 8. In the context of this specification, the scattered light beam 7 refers to scattering by one or more particles P in a medium M, whereas the unscattered light beam 8 is not scattered by the particles P in the medium M. Furthermore, scattering may mean diffraction, refraction or reflection, depending on the choice of the interferometer 1 used, which in turn depends on the properties of the particles P (e.g. their transparency, reflectivity or refractive index) and the medium M.

[0034] The scattered light beam 7 and the unscattered light beam 8 interfere with each other. At the end of the path of the light beam 6, the camera 3 records an interference image 10 (FIG. 2) of the scattered light beam 7 and the unscattered light beam 8. The camera 3 can be any analog or digital camera, for example a complementary metal oxide semiconductor (CMOS) or charge coupled device (CCD) image sensor.

[0035] It should be noted that parts of both the scattered light beam 7 and the unscattered light beam 8 may be scattered in medium M (e.g. via the Tyndall effect), e.g. by other (usually smaller) objects, and this part of the unscattered light beam 8 will deviate slightly from the beam direction 9. In the interference image 10, said parts of the scattered light beam 7 and the unscattered light beam 8 will result in unavoidable and undesirable background signals, which generally interfere with the identification of the particle distribution 4 in medium M.

[0036] Interferometer 1 may comprise one or more additional optical devices as known in the art of holographic microscopy, such as an attenuator ring for improving the signal-to-noise ratio, a microscope objective lens, a phase plate, one or more lenses (e.g., a diverging lens for magnifying interferometer image 10), etc. Additionally, interferometer 1 may be other types of interferometers than an in-line interferometer, such as, for example, an interferometer 1 that utilizes a beam splitter.

[0037] The interference image 10 recorded by the camera 3 is then transferred to a processor 11 via an interface 12. The processor 11 processes the interference image 10 to obtain properties of at least one particle P in the medium M (here, a three-dimensional particle distribution 4), as will be described with reference to Figures 2-5.

[0038] The camera 3 records an interference image 10 (based on E. coli in lysogeny solution in the example shown in FIG. 2) as a purely two-dimensional image, which encodes both the intensity and phase information of the light beam 6. This allows the processor 11 to calculate the size of the sample 5 of the medium M containing the particle P as S. 1 ~S 3It can be "reconstructed" in three steps.

[0039] First step S 1 Now, for each of the multiple locations 13 from the interference image 10, the electric field E 1 The processor 11 calculates the position 13. The positions 13 are distributed three-dimensionally in the sample 5. In the example shown in FIG. 2, the positions 13 are distributed on several imaginary planes 14. 1 , 14 2 , , , 14 in general i , and some imaginary planes 14 are perpendicular to the light beam 9 of the unscattered light beam 8. In this example, the positions 13 are located at respective imaginary planes 14 i , but this is optional. In other examples, the locations 13 may be distributed three-dimensionally within the sample 5, for example in a regular grid arrangement representing adjacent cubic regions, or in an irregular grid arrangement representing adjacent arbitrarily shaped spatial regions.

[0040] The electric field E of the scattered light beam 7 1 For each position 13, the processor 11 applies a reconstruction algorithm to the interferogram 10 to calculate the electric field E 1 Since is a complex number, its real and imaginary parts, or the phase φ 1 and amplitude are calculated, respectively. In this example, in a direction 15 opposite to the beam direction 9, the processor 11 applies a reconstruction algorithm on a plane-by-plane basis. However, this is optional and other reconstruction algorithms may be applied. Various examples of such reconstruction algorithms include forward or backward propagation or projection algorithms, such as, for example, inverse Radon transform, Fourier domain reconstruction algorithms, iterative reconstruction algorithms, etc., as known in the art.

[0041] Subsequent second process S 2Now, the processor 11 generates a representation 16 of the sample 5 covering all positions 13 and comprising, for each position 13, a respective phase value ξ (FIG. 3) and a respective intensity value I (FIGS. 4 and 5). The phase value ξ represents the phase shift of the scattered light beam 7, which is induced by scattering by particle P only. For each position 13, the phase value ξ is the electric field E of the scattered light beam 7 at that position 13. 1 and the estimated electric field E of the unscattered light beam 8 at the position 13 2 For this purpose, the electric field E of the unscattered light beam 8 is 2 may be estimated in a manner known in the art, i.e. from measurements, as will be described in more detail below. The intensity value I represents the intensity of the light beam 6. For each position 13, the intensity value I is, for example, the intensity I of the scattered light beam 7 calculated for that position 13. 1 From the electric field E 1 and from the phase value ξ determined for that position 13.

[0042] Process S 2 The third step S 3 In the process, the processor 11 executes a step S 2 The representation 16 of the sample 5 obtained in is used to identify a property (here a particle distribution 4) of at least one particle P in the medium M. This is done by evaluating the identified intensity values ​​I and / or phase values ​​ξ, as known in the art. For example, the intensity values ​​I and / or phase values ​​ξ, as well as the respective positions 13, i.e. their three-dimensional coordinates in a given coordinate system 17, may be input into a pattern recognition algorithm, a neural network, etc.

[0043] In one embodiment, said property (here particle distribution 4) of at least one particle P in medium M may be identified using processor 11 (in this embodiment with the aid of processor 11), for example by human inspection of intensity values ​​I of representation 16. A visualization of representation 16 is provided by processor 11.

[0044] Step S for generating display 16 2 According to this, both the phase value ξ and the intensity value I at each location 13 can be determined in various ways, as follows:

[0045] In the exemplary embodiment shown in FIG. 1 Phase φ 1 The estimated electric field E 2 Phase φ 2 A minimum difference angle Δφ between the first and second light beams 8 and 9 is identified, i.e., one of the two difference angles smaller than π. A phase value ξ is then determined as a fraction of said difference angle Δφ. This can be achieved in a variety of ways, for example, the phase amount ξ can be determined as a predetermined fraction (e.g., half) of the determined minimum difference angle Δφ. The phase φ of the unscattered light beam 8 is 2 It should be understood that when is assumed to be zero (as is usually defined in this case), the difference angle Δφ of the scattered light beam 7 and the calculated phase φ1 coincide. In another example, the electric field E 1 and E 2 is calculated and the estimated electric field E 1 and E 2 Phase φ 1 and φ 2 The phase φ between m With a mixed electric field E m In yet another example, the phase value ξ is mixed to obtain the mixed field E m Phase φ m and the phase φ 1 and φ 2 In particular, the electric field E 2 may be assumed to be real and have a magnitude of one, in which case the phase value ξ may be calculated as follows:

[0046]

number

[0047] (In the formula, r and z are the coordinates of the position (13), z is the coordinate in the direction 15 opposite to the direction 9 of the second part of the light beam 6, and r is the coordinate of the position 13 in a pair of coordinates in the plane x, y perpendicular to the opposite direction 15. E 1 (r,z) is the calculated electric field of the first part of the light beam 6 at the position 13. ξ(r,z) is the phase value at the position 13. Re and Im represent the operators of the real part and the imaginary part. arctan2 represents the four-quadrant inverse tangent function.) It may be specified according to.

[0048] In another embodiment, in the step S of generating 2 the phase value ξ is specified as the phase of the weighted sum of the calculated electric field E 1 and the estimated electric field E 2 In this embodiment, the magnitude of the electric field is specified, for example, by repeatedly maximizing the contrast between the high-intensity value I and the low-intensity value I in the resulting display 16 from known optical parameters of the particle P and / or the medium M.

[0049] The graphs of FIGS. 4 and 5 show the intensity values I specified along the direction 15, that is, along the z-axis of the coordinate system 17 (FIG. 4), and in the direction perpendicular thereto (here, along the y-axis of the coordinate system 17, FIG. 5). Here, the solid line 18 is the following formula:

[0050]

Equation

[0051] (where r and z are the coordinates of the position (13), z is the coordinate in the direction 15 opposite to the direction 9 of the second part of the light beam 6, and r is the coordinate of the position 13 in a pair of coordinates in the plane x, y perpendicular to the opposite direction 15. I 1 (r,z) is the intensity of the first part of the light beam 6 at the position 13. ξ(r,z) is the phase value at position 13; I(r,z) is the intensity value at position 13.) denotes an intensity value I determined according to

[0052] In Fig. 4 and Fig. 5, the dashed line 19 indicates the intensity value I determined according to the state of the art (here based on interference images of silica particles in "Milli-Q" deionized water). As can be seen in Fig. 4 and Fig. 5, the solid line 18 indicates the respective maximum intensity I max , and generally shows less variation.

[0053] Alternatively, in a more general embodiment, an additional phase shift Θ between the portions of the light beams 7, 8 scattered by the medium and the unscattered light beam 8 can be introduced, fitted, modeled, simulated and iterated to maximize the contrast between high and low intensity values ​​I in the display 16. The intensity values ​​I can be calculated, for example, according to the following formula:

[0054]

number

[0055] is identified according to

[0056] In any embodiment, the processor stage S 3 Before, the intensity threshold I th This strong threshold I th is calculated using the intensity I of the display 16, where the identified intensity value I is the calculated intensity threshold I th If the determined intensity value I and phase value ξ for position 13 are smaller than 3 (e.g., it is not input to a pattern recognition algorithm). th For example, the maximum intensity value I max(FIGS. 4 and 5), a moving average, an average of two or more, or a percentage of all intensity values ​​I included in display 16. Similarly, a phase threshold is optionally calculated for the same purpose, and the intensity threshold I th may be used in the same manner.

[0057] In a further optional embodiment, a reference image 20 of the coherent light beam 6 is generated for the light beam 8 that is not scattered by the at least one particle P and is therefore unscattered. The reference image 20 may be generated in various ways known to those skilled in the art, for example as a low-pass filtered interferometric image 10. In one aspect, the reference image 20 is generated by emitting a coherent light beam 6 of a medium M containing said at least one particle P, without a sample 5, using a light source 2, and recording said reference image 20 with a camera 3. The phrase "of a medium M containing a particle P, without a sample 5" refers to another sample, including a medium M without any sample 5 or without a particle P between the light source 2 and the camera 3 of the interferometer 1.

[0058] Based on the reference image 20, the processor 11 performs the calculation step S, as known in the art. 1 Before the interferogram 10 can be normalized, so that the electric field E 1 Used as step S 1 The normalized calculated electric field E at 1 The subsequent process S 2 In the electric field E 1 It will be used as:

[0059] The electric field E of the unscattered light beam 8 2 is the corresponding value (e.g., step S 2 In the example shown in FIG. 3, the electric field E 2 is the intensity I of the unscattered light beam 8. 2, for example, as the square root of the corresponding intensity of the reference image 20. Furthermore, in order to maximize the contrast in the display 16 or to achieve a known grain size in the grain size distribution 4, for example, by simulating the light beam 8 as 0, by forward or backward propagation of the reference image 20, and / or by step S 2 and / or Process S 3 By repeating this, the electric field E 2 Phase φ 2 is 0 and some phase φ 2 We estimate the following.

[0060] In the above-described embodiment, step S 1 -S 3 is performed on a single recorded interferogram 10. However, the method is not limited to these embodiments.

[0061] In a further embodiment, several interference images 10 are recorded based on respective coherent light beams 6 emitted from respective light sources 2. Each of these interference images 10 may optionally be normalized by a common or respective separate reference image 20. In this case, the common reference image 20 may be generated by averaging these recorded interference images 10.

[0062] Then, for each of the interference images 10, the electric field E 1 is process S 1 In the calculation, each representation 16 of sample 5 is 2 Then, for each light beam 6, 1 Calculate S 2 These representations 16 obtained by carrying out a step of generating a particle P in the medium M can be used to identify the properties of at least one particle P in the medium M, and for this purpose, these representations 16 are optionally generated by carrying out a step S 3The averaged intensities and phases may be averaged as known in the art, for example, as geometric means, arithmetic means, etc. Alternatively, the averaged intensities and phases may be averaged as known in the art, for example, as geometric means, arithmetic means, etc. 3 may be performed separately for each representation 16, for example to identify several particle distributions 4 in order to study the movement of one or more particles P in the medium M, or to identify changes in the properties of one or more particles P over time.

[0063] According to the example shown in FIG. 1 , the interferometer 1 may optionally comprise additional light sources 2′, 2″ (two in FIG. 1 ). Each light source 2, 2′, 2″ emits a respective coherent light beam 6, 6′, 6″ at the same or different frequency to illuminate the sample 5. The coherent light beams 6, 6′, 6″ are emitted simultaneously or sequentially and are scattered by at least one particle P. A respective interference image 10 is recorded for each light beam 6, 6′, 6″ either by one camera 3 or by several cameras.

[0064] In any embodiment of this example, the three frequencies of light beams 6, 6', and 6'' correspond to red, green, and blue, although other frequencies may be selected, including frequencies of light that are invisible to the human eye.

[0065] Next, step S 1 -S 3 is performed for each of the light beams 6, 6', 6'' in the processor 11 as described above, and step S 3 , for example, a colored particle distribution 4 can be determined. Further, an averaged representation 16 can optionally be calculated as described above. Thereafter, a property of at least one particle P (here, the particle distribution 4) can be determined.

[0066] Depending on the positions of the light sources 2, 2', 2'' and any optical devices in the interferometer 1, different sets of spatial frequencies may be recorded by the camera, as required. In this case, the averaged representation 16 obtained therefrom will contain more information about each position 13 and will therefore have a higher resolution (known in the art as "super-resolution").

[0067] Furthermore, the coherent light beams 6, 6', 6'' can illuminate the sample 5 at different angles, for example taking into account the respective forward or backward propagation along the respective beam direction to be applied to the interference images obtained from each. Alternatively, in other embodiments, instead of using several light sources 2, 2', 2'', only the light source 2 can emit several coherent light beams 6 at different frequencies. The different frequencies are recorded and processed as described above.

[0068] The present invention is not limited to the particular embodiments described above, but encompasses all variations, modifications and combinations thereof that fall within the scope of the appended claims. [Brief description of the drawings]

[0069] [Figure 1] FIG. 1 is a schematic side view of an in-line interferometer used in the method of the present invention. [Diagram 2] FIG. 2 is a flow chart illustrating the determination of a three-dimensional particle distribution from an interferogram produced by the interferometer of FIG. 1 in accordance with the present invention. [Diagram 3] FIG. 3 shows the determination of phase values ​​according to the determination of FIG. 2 in the complex plane. [Figure 4] 4 and 5 show graphs of light intensity in a sample of a medium, respectively, in a direction opposite to the direction of the light beam (FIG. 4) and perpendicular to the light beam (FIG. 5). [Diagram 5] 4 and 5 show graphs of light intensity in a sample of a medium, respectively, in a direction opposite to the direction of the light beam (FIG. 4) and perpendicular to the light beam (FIG. 5).

Claims

1. A method for characterizing a property of at least one particle (P) in a medium (M), comprising the steps of: Providing a sample (5) of medium (M) containing said at least one particle (P); - emitting a coherent light beam (6) using a light source (2) and illuminating a sample (5) with said light beam (6), a first portion of said light beam (6) being scattered by at least one particle (P) resulting in a scattered light beam (7); recording, by means of a camera (3), an interference image (10) of the scattered light beam (7) and a second portion of the light beam (6) that is not scattered by said at least one particle (P); For each of a plurality of positions (13) three-dimensionally distributed within the sample (5), the electric field (E １ ) by a processor (11) １ )and, For each of said positions (13), the calculated electric field (E １ ) and the estimated electric field (E ２ ) and a phase value (ξ) is determined from both the intensity (I １ generating (S16) a representation (16) of the sample (5) covering said location (13) including an intensity value (I) determined from both the phase value (ξ) at said location (13) and the determined phase value (ξ) at said location (13); ２ )and, A step (S) of identifying said property by a processor (11) using said representation (16). ３ ) and

2. The position (13) is located in an imaginary plane (14) perpendicular to the direction (9) of the second portion of the light beam (6). ｉ ) and the calculating step (S １ 2. The method of claim 1, wherein the step of:

3. The step of calculating (S) is based on a reference image (20) of the coherent light beam (6) without being scattered by the at least one particle (P), １ 3. The method of claim 1, wherein the first and second inputs are normalized before the second input.

4. The generating step (S ２ ) of the electric field (E ２ 4. The method according to claim 1, wherein the at least one particle (P) is scattered by the at least one particle (P) and the at least one particle (P) is scattered by the at least one particle (P) and the at least one particle (P) is scattered by the at least one particle (P).

5. 5. The method according to claim 3 or 4, wherein the generation of the reference image (20) is performed by emitting the coherent light beam (6) free of the sample (5) of the medium (M) containing the at least one particle (P) using the light source (2) and recording the reference image (20) using the camera (3).

6. The generating step (S ２ ) for each of the positions (13), the calculated electric field (E １ ) phase (φ １ ) and the estimated electric field (E ２ ) phase (φ ２ 6. The method according to claim 1, wherein a minimum difference angle (Δφ) between the phases (ξ) and (ξ) is identified, and the phase value (ξ) is determined as a portion of the difference angle (Δφ).

7. The generating step (S ２ ), the phase value (ξ) is calculated based on the electric field (E １ ) and the estimated electric field (E ２ 7. The method according to claim 1, wherein the phase of the signal is determined as a weighted sum of the phases of the signals.

8. The generating step (S ２ ) for each of said positions (13), the phase value (ξ) satisfies the following formula: [0010] (In the formula, r and z are coordinates of a position (13), z is a coordinate in a direction (15) opposite to the direction (9) of the second portion of the light beam (6), and r is a pair of coordinates in a plane (x, y) perpendicular to said opposite direction (15), E １ (r,z) is the calculated electric field of the first portion of the light beam (6) at the position (13); ξ(r,z) is the phase value at position (13), arctan2 represents the four-quadrant arctangent function. The method according to claim 6 or 7, characterized according to

9. The step of specifying (S ３ ), the intensity value (I) is used to determine an intensity threshold (I ｔｈ ) is calculated, preferably using the average of two or more of said intensity values ​​(I) to determine an intensity threshold (I ｔｈ ) is calculated, The step of specifying (S ３ ), for each of the positions (13), the intensity value (I) is compared to the calculated intensity threshold (I ｔｈ 9. The method according to claim 1, wherein neither the phase value (ξ) nor the intensity value (I) contained in the representation (16) of the position (13) is used when ξ is smaller than ξ.

10. The generating step (S ２ ) for each of said positions (13), the intensity value (I) is expressed by the following formula: [0025] (In the formula, r and z are coordinates of a position (13), z is a coordinate in a direction (15) opposite to the direction (9) of the second portion of the light beam (6), and r is a pair of coordinates in a plane (x, y) perpendicular to said opposite direction (15), I １ (r,z) is the intensity of the first portion of the light beam (6) at the position (13); ξ(r,z) is the phase value at position (13), I(r,z) is the intensity value at position (13). The method according to any one of claims 1 to 9, characterized according to

11. In the radiating step, two or more coherent light beams (6, 6', 6'') are emitted, The recording step and the calculating step (S １ ), and the generating step (S ２ ) is performed for each of the two or more coherent light beams (6, 6', 6''), The method according to any one of the preceding claims, further comprising obtaining a respective representation (16) of said sample (5) and determining said property using two or more of said obtained representations (16).

12. The two or more representations (16) are averaged into an averaged representation, and the identifying step (S ３ 12. The method of claim 11, wherein the property is identified from the averaged representation.

13. 13. The method according to claim 11 or 12, wherein in the emitting step, each of the two or more coherent light beams (6, 6', 6'') is emitted by a respective one of two or more light sources (2, 2', 2'').

14. The method according to any one of claims 11 to 13, wherein in the emitting step, each of the two or more coherent light beams (6, 6', 6'') is emitted at a different frequency.

15. 15. The method of claim 14, wherein in the emitting step, three coherent light beams (6, 6', 6'') are emitted, the frequencies of the three coherent light beams (6, 6', 6'') corresponding to the colors red, green, and blue, respectively.

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