Quantitative phase microscope

Abstract

An illumination system 110 irradiates a sample S placed on a sample plane SP with a plurality of optically distinguishable illumination beams 2_1 to 2_N from different axially asymmetric directions. An imaging device 120 captures a plurality of images IMG1 to IMGN while distinguishing a plurality of object beams 4_1 to 4_N obtained by transmission of the plurality of illumination beams 2_1 to 2_N through the sample S.

Description

Quantitative phase microscopy

[0001] The present disclosure relates to quantitative phase microscopy.

[0002] Measurement targets in cell biology, such as living cells, are phase objects, and conventional microscopes cannot provide sufficient absorption contrast. Fluorescence microscopes specifically and selectively stain specific structures in living cells and observe them as amplitude objects. However, there are several limitations when using fluorescence microscopes to observe living cells. First, because the fluorescence is weaker than the excitation light, the excitation light required to obtain a sufficient fluorescence signal can damage living cells (phototoxicity). Second, fluorescent molecules are stochastically destroyed, limiting the observation time (photobleaching). Third, the need to stain specific structures limits the scope of application.

[0003] Therefore, it is important to also effectively utilize label-free microscopes that do not use fluorescence. Transmitted light microscopes are commonly used for non-fluorescent cell observation. When light passes through biological tissue, the amplitude of the light wave remains almost constant, while the light intensity remains almost constant. However, the phase of the light wave changes due to differences in refractive index. Phase contrast observation, which converts this phase change into intensity, is effective for visualizing biological tissue, and Zernike phase contrast microscopy (ZPC) and differential interference contrast microscopy are widely used in cell biology. These microscopy methods are powerful and can robustly visualize cells with relatively simple implementation, but they also have their own limitations. ZPC requires a limited illumination aperture, which limits spatial resolution. On the other hand, DIC converts the phase difference in the shear direction of a prism into an intensity difference, so information in the direction perpendicular to the shear is lost, limiting positioning accuracy. Furthermore, these methods are not designed to quantitatively obtain the refractive index distribution as the absolute value of the phase difference of transmitted light.

[0004] Therefore, quantitative phase microscopy (QPI), a method for quantitatively obtaining phase difference, has attracted attention in recent years and is being applied in a wide range of fields, including biomedicine and neuroscience. Phase difference is proportional to the difference in optical path length, which is the product of refractive index and path length, and is therefore proportional to thickness at a scale where the refractive index can be considered constant. Furthermore, since the relationship between dry mass and refractive index is nearly constant for materials in biological samples, dry mass can be estimated by integrating the phase difference.

[0005] The most classic method of QPI is the interferometer technique. Interferometric microscopy, which is applied to cell biology, has the advantage of being highly sensitive, but it is sensitive to the influence of vibrations from the culture medium, dishes, and equipment required for culturing cells, making it difficult to apply to actual experiments.

[0006] Therefore, QPI, which does not use coherent light interference, has been developed. A technique called phase retrieval has attracted attention. This technique theoretically formulates the imaging process of conventional microscope optical systems and calculates the phase difference of a sample from the resulting images. In particular, techniques such as the transport of intensity equation (TIE) that simultaneously solves defocused images of a sample (Non-Patent Documents 1 and 2), differential phase contrast microscopy (DPC) that simultaneously solves images obtained using a special illumination pattern (Non-Patent Document 3), and Fourier ptychography, which combines images illuminated from various angles to obtain broad frequency information (Non-Patent Document 4). Because these techniques do not use an interferometer, they can obtain phase difference information robustly even against external disturbances. However, faithfully reconstructing actual sample information requires imaging under multiple conditions, which limits the time resolution.

[0007] In TIE techniques, single-shot phase retrieval is effective for achieving high temporal resolution while simultaneously reducing motion artifacts, and several studies have been reported. The common idea is to simultaneously capture images under multiple conditions and then effectively separate them using an observation system. For example, a method has been developed in which a beam splitter is used to copy the light transmitted through the sample, and multiple focal planes are observed using different camera fields of view (Non-Patent Documents 5, 6) or multiple cameras (Non-Patent Document 7). This method has the advantage of minimizing phototoxicity by estimating the phase difference using all light passing through the sample. However, alignment and calibration tend to be complicated, and achieving high performance is economically expensive.

[0008] In the TIE technique, an implementation has been proposed that simplifies the setup by reducing the required optical elements and irradiating multiple colors of light and observing with an RGB camera. For example, a method of obtaining images at multiple focal planes using the chromatic aberration of an objective lens (Non-Patent Document 8) can recover phase contrast in a single shot using only an inexpensive objective lens and multicolor illumination. However, it is necessary to select the appropriate wavelength and objective lens depending on the scale of observation.

[0009] In addition, a method using TIE has been proposed in which an image illuminated by multiple illumination patterns is obtained at once by using an illumination device with a color filter pattern mask placed on the pupil plane of the illumination device (Non-Patent Document 9). This is a powerful method that can extract high-resolution phase difference distributions with a simple setup, but chromatic aberration can cause artifacts, especially in high-resolution imaging, because information from different focal planes is obtained.

[0010] Therefore, a method for creating illumination patterns using polarization instead of wavelength has been proposed (Non-Patent Document 10). However, the handling of noise in the phase retrieval algorithm and the implementation method have not yet been optimized for cell biology.

[0011] Teague, M. R. Deterministic phase retrieval: “a Green's function solution”, JOSA 73, 1434.1441 (1983).Zuo, C. et al. “Transport of intensity equation: a tutorial”, Opt. Lasers Eng. 135, 106187 (2020).Tian, L. & Waller, L. “Quantitative differential phase contrast imaging in an LED array microscope”, Opt. Express 23, 11394-11403 (2015).Zheng, G., Horstmeyer, R. & Yang, C. “Wide-field, high-resolution Fourier ptychographic microscopy”, Nat. Photonics 7, 739-745 (2013).Di Martino, J. M. et al. “Single-shot phase recovery using two laterally separated defocused images”, Opt. Commun. 293, 1-3 (2013).Zuo, C., Chen, Q., Qu, W. & Asundi, A. “Noninterferometric single-shot quantitative phase microscopy”, Opt. Lett. 38, 3538-3541 (2013).Chen, C. et al. PhaseRMiC: “phase real-time microscope camera for live cell imaging. Biomed”, Opt. Express 12, 5261.5271 (2021).Waller, L., Kou, S. S., Sheppard, C. J. R. & Barbastathis, G. “Phase from chromatic aberrations”, Opt. Express 18, 22817.22825 (2010).Phillips, ZF, Chen, M. & Waller, L. “Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC)”, PLOS ONE 12, e0171228 (2017).Kalita, R. et al. “Single-shot phase contrast microscopy using polarization-resolved differential phase contrast”, J. Biophotonics 14, e202100144 (2021).

[0012] The present disclosure has been made in this context, and one exemplary purpose of an embodiment thereof is to provide a computational phase microscope (CPM) capable of detecting intracellular structures at high speed, high resolution, and high sensitivity.

[0013] An aspect of the present disclosure relates to a phase microscope, which includes an illumination system that irradiates a sample placed on a sample surface with a plurality of optically distinguishable illumination lights from different axially asymmetric directions, and an imaging device that captures a plurality of images by distinguishing between a plurality of object lights obtained when the plurality of illumination lights pass through the sample.

[0014] Any combination of the above components, and any transformation of the present invention into a method, device, system, recording medium, computer program, etc., are also valid aspects of the present invention.

[0015] According to the present disclosure, intracellular structures can be detected at high speed, with high resolution and high sensitivity.

[0016] 7 is a diagram showing the basic configuration of a phase microscope according to an embodiment; FIG. 8 is a diagram showing the optical system of a phase microscope according to an embodiment; FIG. 9 is a diagram showing a commercially available bright-field microscope; FIG. 10 is a diagram showing a polarizer mask PM; FIG. 11 is a diagram showing a polarization camera; FIG. 12 is a diagram showing the apparent illuminance distribution in each direction in a polarization camera; FIG. 13 is a diagram showing an image obtained when a sample S is observed using the phase microscope of FIG. 2; FIG. 14 is a diagram showing phase images of a portion and the entire COS-7 cell sample calculated from the four images of FIG. 7; FIG. 15 is a diagram showing a data acquisition scheme in a phase microscope; FIG. 16 is a diagram showing a time series of results of acquiring phase shifts of COS-7 cells during mitosis using a phase microscope according to an embodiment.

[0017] (Summary of the Embodiments) A summary of some exemplary embodiments of the present disclosure will be provided. This summary is intended to provide a simplified overview of some concepts of one or more embodiments in order to provide a basic understanding of the embodiments as a prelude to the more detailed description that follows, and is not intended to limit the scope of the invention or disclosure. This summary is not an exhaustive overview of all possible embodiments, and is not intended to identify key elements of all embodiments or to delineate the scope of some or all aspects. For convenience, the term "one embodiment" may be used to refer to one embodiment (example or variant) or multiple embodiments (examples or variants) disclosed herein.

[0018] A phase microscope according to one embodiment includes an illumination system that irradiates a sample placed on a sample surface with a plurality of optically distinguishable illumination lights from different axially asymmetric directions, and an imaging device that captures a plurality of images by distinguishing between a plurality of object lights obtained when the plurality of illumination lights pass through the sample.

[0019] In this configuration, multiple illumination beams are simultaneously irradiated from multiple axially asymmetric directions, and multiple images are simultaneously captured based on multiple object beams derived from the illumination beams. Assume that a first direction and a second direction perpendicular to the first direction are defined within the sample plane. To simplify for ease of understanding, to determine the phase gradient of a sample in the first direction, a pair of illumination beams with opposite ray vectors in the second direction are irradiated onto the sample, and the resulting two images are used to formulate and solve simultaneous equations based on imaging theory. Expanding and applying this principle, a sample can be irradiated with N (N ≥ 2) illumination beams oriented in any direction, and the resulting N images can be used to formulate simultaneous equations, allowing one-dimensional or two-dimensional phase distributions of the sample to be reconstructed. The above configuration allows for the acquisition of multiple N images required for sample phase recovery in a single shot, enabling high-speed, high-resolution, and high-sensitivity detection of intracellular structures.

[0020] In one embodiment, the illumination system may include a polarizer mask that is placed on the pupil plane and is divided into a plurality of regions corresponding to a plurality of illumination lights, with each region having a different polarization direction.

[0021] In one embodiment, the illumination system may include a color filter placed on a pupil plane, divided into a plurality of regions corresponding to a plurality of illumination lights, and having different transmission wavelengths for each region.

[0022] In one embodiment, the imaging device may include a polarization camera sensitive to different polarization directions or a color camera sensitive to different wavelengths. These cameras may be single-chip, multi-chip, or multi-layer. A single-chip camera includes, for example, a camera configured with a single sensor and a filter formed on the sensor. A multi-chip camera may include a camera configured with multiple sensors, such as a three-chip sensor, and an optical system that separates incident light into different polarizations and distributes them to multiple sensors. A multi-layer camera includes a camera including multiple stacked sensors.

[0023] In one embodiment, the phase microscope may further include a processor that processes the plurality of images to generate a phase image of the sample. The processor may solve an optimization problem for a model that includes a noise term in addition to a term for the optical properties of the sample.

[0024] (Embodiments) Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings. Note that in the description of the drawings, the same elements are given the same reference numerals, and duplicated explanations will be omitted as appropriate. Furthermore, the configurations described below are examples and do not limit the scope of the present invention in any way.

[0025] 1 is a diagram showing the basic configuration of a phase microscope 100 according to an embodiment. The phase microscope 100 includes an illumination system 110, an imaging device 120, and a processing device .

[0026] The illumination system 110 irradiates a sample S placed on a sample plane SP with a plurality of optically distinguishable illumination light beams 2_1 to 2_N (N≧2) from different axially asymmetric directions.

[0027] Optically distinguishable means that they can be optically separated and detected independently, and includes, for example, cases where the polarization is different or the wavelength is different.

[0028] The image capturing device 120 distinguishes between a plurality of object light beams 4_1 to 4_N obtained when a plurality of illumination light beams 2_1 to 2_N pass through the sample S, and captures a plurality of images IMG1 to IMGN.

[0029] The imaging device 120 includes an imaging optical system 122 and an imaging device 124. The imaging optical system 122 forms an image of the sample S on the imaging surface of the imaging device 124. The imaging device 124 separates and images the plurality of object light beams 4_1 to 4_N, and generates separate images IMG1 to IMGN.

[0030] The processing device 130 is an electronic computer such as a workstation or tablet terminal, and recovers the phase distribution of the sample S based on the multiple images IMG1 to IMGN. The sample S is irradiated with a pair of illumination light beams having ray vectors whose components parallel to the sample surface SP are in opposite directions, and the two resulting images are used to formulate and solve simultaneous equations based on imaging theory, thereby making it possible to determine the phase gradient of the sample in a direction perpendicular to the direction.

[0031] By irradiating the sample with two pairs of illumination light beams whose ray vectors are orthogonal to each other, a two-dimensional phase distribution of the sample S can be obtained. In this case, the illumination system 110 irradiates the sample with N=4 illumination light beams from four directions. Alternatively, three illumination light beams whose components parallel to the sample surface differ in direction by 120° each may be irradiated, and the three images may be used to formulate simultaneous equations, which are then solved to recover the two-dimensional phase distribution. In other words, the number N of illumination light beams 4 and the irradiation directions may be determined so that a simultaneous equation can be constructed whose solution is the phase distribution of the sample S.

[0032] The multiple illumination lights do not need to be plane waves, but can have any known wavefront, i.e., a known illumination pattern. Therefore, there may be multiple light vectors contributing to the formation of a single image. If the number of illumination lights or the illumination direction are poor, the information contained in the image will be reduced, and the estimation results will deteriorate. In other words, it is advisable to determine the number N of illumination lights, the illumination direction, and each pattern so as to increase the accuracy of phase estimation.

[0033] The calculation method in the processing device 130 may be any known technique and is not particularly limited in the present disclosure.

[0034] The above is the basic configuration of the phase microscope 100.

[0035] FIG. 2 is a diagram showing an optical system of a phase microscope 100A according to one embodiment.

[0036] The illumination system 110A irradiates a plurality of illumination lights with different polarization directions from different directions onto the sample S. Fig. 2 shows a beam of one illumination light irradiated from one direction.

[0037] The illumination system 110A is a Koehler illumination system, and includes a light source LS, a collimating lens L ｃｌｍ , diffuser D, field stop FS, collector lens L ｃｌｃ , aperture stop AS, polarizer mask (polarization mask pattern) PM, condenser lens L ｃｎｄ Includes.

[0038] Light from the light source LS passes through the collimating lens L ｃｌｍ The light is collimated by the collector lens L and passes through the diffuser D and the field stop fSW. ｃｌｃ The designed polarizer mask PM and aperture stop AS are arranged on the illumination aperture plane, and the light is collected by the condenser lens L. ｃｎｄ collimates this light and illuminates the sample S.

[0039] The imaging device 120A has an objective lens L ｏｂｊ , imaging lens L ｔ , a magnifying optical system 126, and a polarization camera 124A, which is an imaging device. The imaging optical system of the imaging device 120A can be configured as an infinity correction system (ICS), but may also be a finite correction optical system.

[0040] The object light that passes through the sample S is directed to the objective lens L ｏｂｊ The light is focused by the imaging lens L ｔ The image is magnified by two lenses L １ , L ２ The light is magnified by a magnifying optical system 126 including the following components, and an image is formed on the imaging surface of the imaging device 124A.

[0041] Polarization camera 124A is a single-plate camera that can separate polarized light, and includes a wire grid polarizer WP and a camera (image sensor) C.

[0042] Figure 3 shows a commercially available bright-field microscope 100R. In other words, the phase microscope 100 shown in Figure 2 can be easily implemented by using the commercially available bright-field microscope 100R as a base and changing the condenser unit and camera port. Specifically, a polarizer mask PM is added to the pupil plane of the illumination system 110R, and the imaging device of the imaging device 120R is replaced with a camera C equipped with a wire-grid polarizer WP instead of a camera C such as a regular CMOS sensor or CCD sensor.

[0043] FIG. 4 shows a polarizer mask PM. The polarizer mask PM is divided into multiple regions corresponding to multiple illumination light sources, and includes a polarizing film with a different polarization direction for each region. The polarizer mask PM can be easily fabricated by bonding multiple polarizing films PF together. In this example, four polarizing films PF1 to PF4, each with a polarization direction that differs by 45°, are bonded together to accommodate illumination from four directions. Each polarizing film PF is approximately fan-shaped with a central angle of 90°. To prevent strong light from leaking between different polarizing films PF, black, line-shaped light-shielding portions MSK that do not allow light to pass through are present between adjacent polarizing films PF.

[0044] 5 is a diagram showing the polarization camera 124A. This polarization camera 124A has four types of grids G1 to G4, each with a polarization direction that differs by 45 degrees, arranged in units of 2 x 2 pixels (also called subpixels). Such polarization cameras are commercially available.

[0045] An example of a specific implementation of the phase microscope 100 according to the embodiment will be described. An inverted microscope (IX-83, manufactured by Olympus Corporation) was used as a base bright-field microscope 100R, and the phase microscope 100 was implemented by modifying it.

[0046] Condenser Lens L ｃｎｄ A universal condenser unit (U-UCD8 with IX-ADUCD, Olympus) and an NA1.4 oil immersion top lens (U-TLO, Olympus) were used for the transmitted illumination. A red power LED (dominant wavelength 625 nm, OSR5XNE3C1S, OptoSupply) was used as the transmitted illumination light source.

[0047] The quadrant polarizing filters were fabricated from linear polarizer film (extinction ratio 9000:1 and polarization efficiency >99.98%; XP42-18, Edmund optics). This film, mounted on a transparency, was placed on a condenser filter wheel.

[0048] To prevent high-power unpolarized light from entering through the gaps in the polarizing film, a cross and disk pattern was printed twice on an OHP sheet using the highest density setting on a printer (RICOH IM C3000). Because the OHP sheet also changes the polarization, the optical element closest to sample S is the polarizing film.

[0049] Objective lens L ｏｂｊ An oil immersion objective lens (UPLSAPO100XO, manufactured by Olympus Corporation) with an NA of 1.4 was used.

[0050] The polarization camera (CS505MUP, Thorlabs) was fitted with a magnifying glass made from a 30 mm cage system (Thorlabs) and achromatic doublet lenses (AC254-30-A-ML and AC254-100-A-ML) with focal lengths of 30 mm and 100 mm, respectively, to compensate for the graining effect of the polarizing film.

[0051] Fluorescence images can be captured with this microscope using a broad-spectrum LED light source (XT720S, Excelitas Technologies) equipped with a dichroic mirror (U-FBNA for StayGold, U-FGNA for tetramethylrhodamine (TMR), Olympus).

[0052] 6 is a diagram showing the apparent illuminance distribution in each direction in a polarization camera. This illuminance distribution is obtained by removing the sample S from the imaging device 120A in FIG. 2 and using the lens L. ２ This can be achieved by adding a Bertrand lens between the polarized light camera 124A and the polarized light camera 124B. The ideal illumination distribution would be one with high intensity in only one quadrant and zero intensity in the other quadrants, but in reality, it can be seen that crosstalk exists between the channels.

[0053] In actual measurements, the image captured by the polarization camera 124A can be considered as a mixture of images of the sample S illuminated with these patterns.

[0054] 7 shows images obtained when observing a sample S using the phase microscope 100A of FIG. 2. The sample S is a COS-7 cell sample, and each image shows normalized bright-field intensity. These four images are taken in a single shot.

[0055] Figure 8 shows phase images of a portion and the entire COS-7 cell sample calculated from the four images in Figure 7. The phase difference and optical path difference (OPD) [nm] of the transmitted light passing through the sample S are calculated based on simultaneous equations. The white scale bar in the upper panel of Figure 8 represents 5 μm, and the black scale bar in the lower panel represents 10 μm.

[0056] FIG. 9 shows the data acquisition scheme in the phase microscope 100. S100 indicates the storage thread, and S120 indicates the display thread, which are parallelized. In the storage thread S100, the image from the camera is read into a global variable named "image" and written to disk as quickly as possible. The display thread S120 reads the image from the camera, performs a simple phase retrieval calculation, and displays it on the display. Using a standard PC (Personal Computer), this takes about 100 milliseconds per image, which is within an acceptable range when using a microscope.

[0057] After real-time observation in the display thread S120, the accurate phase retrieval thread S110 is executed to obtain accurate phase contrast with high resolution in a short time.

[0058] Next, a modified example will be described.

[0059] 2, a single-plate type having a single image sensor is used as the imaging device 124, but the present disclosure is not limited to this and may include multiple image sensors and an optical system that separates incident light according to its polarization direction and distributes the light to the multiple image sensors. This optical system can be implemented using a polarizing beam splitter or a combination of multiple polarizing plates.

[0060] In the embodiment, polarization is used as a characteristic for distinguishing between multiple illumination lights having different directions, but the present disclosure is not limited to this. Specifically, multiple illumination lights can be distinguished by wavelength instead of polarization.

[0061] In this case, the polarizer mask PM may be replaced with a color filter in the illumination system 110. That is, in the mask of FIG.

[0062] Furthermore, with regard to the photographing device 120, the imaging device 124 may be replaced with a color camera sensitive to a plurality of different wavelengths. For example, cameras sensitive to RGB and near-infrared are commercially available and can be used. When designing a new imaging device 124, any wavelength can be selected. The color camera may be a single-chip type, a multi-chip type, or a multi-layer type.

[0063] (Variation 3) In the embodiment, a configuration in which the polarizing mask PM is disposed on the pupil plane of the illumination system 110 has been described, but the present disclosure is not limited to this. For example, by forming an arbitrary polarization distribution or wavelength distribution on the light-emitting surface of the light source LS of Koehler illumination, it is possible to irradiate the sample with multiple illumination lights from different directions. Furthermore, the illumination system 110 may adopt a configuration other than Koehler illumination.

[0064] Next, the calculation of the phase retrieval will be explained.

[0065] (Image Formation) a. WOTF (Weak Object Transfer Function) According to the Fourier optics of two-dimensional elements, the intensity of a bright-field image captured by a general bright-field microscope 100R shown in FIG. 3 is expressed by equation (7).

[0066] where x is the spatial coordinate, u is the spatial frequency coordinate corresponding to the spatial coordinate divided by the wavelength of the light and the focal length of the lens, S(u) is the intensity distribution of the illumination source at the front focal plane of the condenser, T(x) = a(x)exp(iφ(x)) is the complex transmittance of the sample, which includes the amplitude intensity a(x) and the phase shift φ(x) due to the sample, and P(u) is the complex transmittance of the pupil, which includes the effects of the finite size of the objective, defocus from the focal plane, or any physical mask on the back focal plane of the objective.

[0067] When the absorption contrast and phase shift of the sample are weak, as in most living specimens, a(x) ≈ a0 and |φ(x)| << 1, and we obtain Eq. (8).

[0068] where tilde ~ denotes the Fourier transform from x-space, a0 is the average of the sample absorption, η(x)=ln(a(x) / a0) is the logarithmic change of the sample absorption, I norm (x) = I(x) / a0 2 - WOTF(0) is the normalized image intensity.

[0069] The transfer function of the sample absorption and phase shift is given by equation (9).

[0070] WOTF is a weak object transfer function and is expressed by equation (11).

[0071] H ph If is non-zero, the phase delay information is converted to image intensity.

[0072] The WOTF in equation (11) is H in equation (10). ph The condition for WOTF(u) to be non-zero is that the imaginary part: Im[WOTF(u)]≠0 or that it does not have two-fold rotational (C2) symmetry, i.e., WOTF(u)≠WOTF(-u).

[0073] The former condition is met by sample defocus, or by phase delay in the objective pupil as is well known in ZPC, while the latter condition can be met by non-C2 symmetric pupils or illumination as appears in differential phase contrast (DPC) microscopy or Fourier ptychography.

[0074] ・b H p To obtain the phase shift using this imaging model over a wide spatial frequency range, the WOTF must be designed appropriately. This is because, if equation (12) holds and P has no imaginary part, the phase transfer function is non-C2 symmetric H ph (u) = -H ph Because it becomes (u). Therefore, H ph (u) values ​​must cross zero between any u and -u, resulting in a single H ph Many zeros occur in

[0075] Dependence of the source intensity distribution on the transfer function Since the functional derivative of WOTF(u) (Equation (11)) with respect to the source intensity S(v) is P*(-v)P(uv), the functional derivative of the non-diffracted light WOTF(0) is P*(-v)P(-v). Similarly, H abs or H ph The functional derivative of is calculated in the same way as in equations (9) and (10). When there is no defocus or phase delay on the pupil plane of the objective lens, P*=P, so the functional derivative can be calculated as follows:

[0076] For small v, δH ph (u) / δS(v) is small, but WOTF(0) and δH abs (u) / δS(v) cannot be ignored.

[0077] This characteristic means that as the spatial low-frequency content of illumination power increases, the raw image intensity due to phase effects remains roughly the same, while the intensity due to undiffracted and absorption effects increases. In other words, illumination energy near the optical axis on the pupil plane reduces the ratio of phase information to total information in the formed image. This illumination reduces the relative phase information within the dynamic range of the camera, reducing phase sensitivity for high-speed observations limited by the camera.

[0078] (Phase Retrieval) The propagation model is explained by equations (7) to (11). However, since the sensor in an actual camera has individual differences between pixels and dust particles, the ideal intensity obtained is equation (1).

[0079] where q(x) is the quantum efficiency of each pixel, T dus (x) = a dus (x)exp(iφ dus (x)) represents the complex transmittance of dust near the camera's image sensor. It is immediately apparent from equation (1) that the phase shift near the camera does not affect the intensity, and by weak object approximation, we obtain equation (2).

[0080] A model including a noise term in addition to the optical properties of the sample S is created and solved as an optimization problem. The acquired intensity I^ contains noise ξ related to the ideal intensity I, as I^(x) = I(x) + ξ(x,I). This noise includes dark current, read noise, and shot noise.

[0081] The most important part is the shot noise, which follows a Poisson distribution related to the number of photons corresponding to the intensity. This shot noise therefore has a variance that is a constant multiple of I(x). In the setup described above, the number of photons is large enough to ensure that the intensity distribution obtained for a static sample is Gaussian.

[0082] For constants η and φ, the background image I0(x) ≒ q(x) a 2 dus (x) a 20 WOTF(0). The shot noise term can be made negligible compared to the signal by averaging the image over a long period of time. Dividing the acquired image by this background gives a scaled, noisy, corrected image I cor^ can be obtained.

[0083] I cor (x) is a simple image transferred from the sample and is expressed as follows:

[0084] Next, we estimate the most likely sample phase shift from the acquired images. In the above setup, the images can be considered as a mixture of images illuminated with four different light source patterns. This information is combined with Bayesian inference to find the set of four acquired images J = {I j ^cor (x))x,j∈{1,2,3,4}, the phase shift map Φ = {φ(x)} is obtained by maximizing the posterior distribution Pr(φ|J). x Ask for.

[0085] j represents the intensity pattern index. Using Bayes' rule, this posterior distribution is calculated from the prior distribution and the likelihood function as follows:

[0086] Find the phase shift map Φ that maximizes the product of Pr(J|Φ). For each x, Pr(J|Φ) is calculated by multiplying ξ(x;I) by q(x)a 2 dus (x)a 2 is a Gaussian distribution with variance divided by 0WOTF(0), and σ I (x) is defined as

[0087] To analytically calculate the optimal φ(x), we assume that Pr(Φ) has variance σ for each x. φ Then we obtain the following equation:

[0088] Pr(J|Φ) The maximum value of the logarithm of Pr(Φ) argmax φ(x) Think about it.

[0089] This argmax is obtained as a solution where the functional derivative of the objective function with respect to φ(x') is 0 over the entire x' space. This functional derivative is calculated as follows:

[0090] As a result, the equation is expressed as follows:

[0091] Taking the Fourier transform from x' space to v space and considering the minimum in v space, this equation simplifies to:

[0092] However, the following formula was used:

[0093] Therefore, we obtain the final equation as follows:

[0094] The absorption equation is also derived as equation (5).

[0095] These equations are I (x) as a constant σ I Approximate to F[1 / σ 2 I (x)](v) = 1 / σ 2 I This can be analytically solved by regarding it as follows, and equation (6) is obtained.

[0096] The 2x2 matrix on the left has non-zero σ I has a non-zero determinant. Therefore, this equation has a solution. Also, Therefore, this matrix is ​​a positive Hermitian matrix with positive diagonal elements.

[0097] In one embodiment, this solution can be used as the phase retrieval result.

[0098] This solution is σ Ｉ ２ / σ η ２ and σ Ｉ ２ / σ φ ２This is the same as the result of Tikhonov regularization when σ is considered as the regularization parameter. Ｉ ２ / σ η ２ does not affect the phase, so σ Ｉ ２ / σ φ ２ The same value can be used.

[0099] Therefore, for real-time phase retrieval by observation, the absorption term can be neglected and the phase shift can be calculated as follows:

[0100] <Experiment> Measurements of COS-7 cells were performed using a phase microscope according to the embodiment. Regarding COS-7 cells: COS-7 cells were provided by the Japanese Collection of Research Bioresourses (JCRB) cell bank of the Human Science Research Resources Bank (HSRRB), a research resource bank program run by the Japan Health Science Foundation (now the National Institutes of Biomedical Innovation, Health and Nutrition). The cells were grown in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific) at 37°C and 5% CO ２ Before imaging, the cells were seeded onto 50-mm diameter glass-bottom dishes (P50G-1.5-14-F / H, MatTek), and the medium was replaced with phenol red-free Leibovitz's L15 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum. A coverslip was then placed on top and sealed with silicone (Dent Silicone V, Matsufu).

[0101] Experimental Setup: The phase-contrast microscope was based on an inverted microscope (IX-83, Evident). The condenser lens configuration consisted of a universal condenser unit (U-UCD8 with IX-ADUCD, Evident) and a 1.4 numerical aperture oil-immersion top lens (U-TLO, Evident). A red power LED (OSR5XNE3C1S, OptoSupply) with a dominant wavelength of 625 nm was used as the transmitted illumination light source. This LED was attached to an aluminum plate machined with an NC machine and fixed to the condenser lens via a 3D-printed holder. A linear polarizing film (XP42-18, Edmund Optics) with an extinction ratio of 9000:1 and polarization efficiency >99.98% was used as the polarizing filter. It was fixed to a transparency and placed in front of the condenser filter wheel.

[0102] To prevent high-power unpolarized light from penetrating through the gaps in the polarizing film and to improve the tolerances in film manufacturing, cross and disk patterns were printed twice on an OHP sheet using a printer (IM C3000, RICOH) set at maximum density. Because the OHP sheet itself also affects polarization, the polarizing film was placed at the optical element position closest to the sample.

[0103] The objective lens used was an oil immersion objective with a numerical aperture of 1.4 (UPLSAPO100XO, Evident). During observation, the dish was heated using the objective lens and a lens heater (TPiE-LH, Tokai Hit) was used. The polarization camera (CS505MUP, Thorlabs) was equipped with a magnification optical system to compensate for the coarsening effect of the polarization grid.

[0104] The magnification optical system consisted of a 30 mm cage system (Thorlabs) and achromatic doublet lenses with focal lengths of 30 mm and 100 mm (AC254-30-A-ML and AC254-100-A-ML, Thorlabs).

[0105] Experimental Results: Figure 10 shows a time series of phase shift images of COS-7 cells during mitosis acquired using a phase microscope according to an embodiment. Each image is based on the average of 20 consecutive frames. As shown in the left image, during the M phase, the system successfully visualized two centrioles, indicated by yellow arrows.

[0106] As shown in the center image, spindle fibers and chromosomes (chromatids) are observed in high resolution from prophase to anaphase. As shown in the right image, after the end of mitosis, the midbody structure formed between the two daughter cells is captured.

[0107] The phase microscope according to the embodiment is expected to be used in applications such as non-staining and non-invasive karyotype analysis.

[0108] The present invention has been described above based on the embodiments. These embodiments are merely examples, and it will be understood by those skilled in the art that various modifications are possible in the combination of the components and treatment processes, and that such modifications are also within the scope of the present invention.

[0109] The present disclosure relates to quantitative phase microscopy.

[0110] 100 Phase microscope 110 Illumination system 120 Imaging device 130 Processing device S Sample 124 Imaging device

Claims

1. A phase microscope comprising: an illumination system that irradiates a sample placed on the surface with multiple optically distinguishable illumination lights from different axially asymmetric directions; and an imaging device that captures multiple images by distinguishing between multiple object lights obtained when the multiple illumination lights pass through the sample.

2. A phase microscope according to claim 1, wherein the plurality of illumination lights have different polarizations.

3. A phase microscope according to claim 2, characterized in that the illumination system includes a polarizer mask placed on a pupil plane, divided into a plurality of regions corresponding to the plurality of illumination lights, and having a different polarization direction for each region.

4. A phase microscope according to claim 1, wherein the plurality of illumination lights have different wavelengths.

5. A phase microscope according to claim 4, characterized in that the illumination system is placed on a pupil plane, divided into a plurality of regions corresponding to the plurality of illumination lights, and includes color filters with different transmission wavelengths for each region.

6. A phase microscope according to any one of claims 1 to 5, further comprising a processing device that processes the plurality of images and generates a phase image of the sample, wherein the processing device solves, as an optimization problem, a model that includes a noise term in addition to a term for the optical properties of the sample.

Images