Bidirectional scattering microscope

Abstract

To provide a bidirectional scattering microscope having a wide dynamic range which can observe and measure observation target objects at one time by using a complex amplitude image of front scattering light and a complex amplitude image of back scattering light.SOLUTION: The present invention includes: detection means for a first complex amplitude image of front scattering light of an observation target object and a second complex amplitude image of back scattering light of an observation target object; and image separation means for separating the first complex amplitude image and the second complex amplitude image from a complex image including the first complex amplitude image and the second complex amplitude image obtained by the detection means.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a bidirectional scattering microscope capable of observing an object by forward scattering and backward scattering. [Background technology]

[0002] Scattering microscopes detect scattered light resulting from the refractive index distribution of an object to be observed, and are widely used for observing transparent samples (e.g., biological samples such as cells) for which contrast cannot be obtained using bright-field microscopes (e.g., Patent Document 1). Among these, quantitative phase imaging (QPI), which acquires a complex amplitude image of forward scattered light generated by an object to be observed, is being developed for observing macroscopic structures in particular (e.g., Patent Document 2).

[0003] Furthermore, in order to observe biological cells, the above-mentioned QPI is beginning to be used to measure the degree of cell growth by measuring the dry mass of biomolecules, to measure pseudo-fluorescence images using machine learning, and to make infrared photothermal measurements.

[0004] On the other hand, for microstructures, interferometric scattering microscopy (iSCAT), which acquires backscattered light from the object being observed, is being developed and is being used for highly sensitive observation of microparticles such as single proteins and viruses (e.g., Patent Document 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2005-062155 A [Patent Document 2] JP 2008-292939 A [Patent Document 3] Special Publication No. 2019-520612 Summary of the Invention [Problem to be solved by the invention]

[0006] However, as described above, the size range of structures that can be observed (measured) differs between observation devices that use forward scattered light and observation devices that use backscattered light, and there has been a demand for a scattering microscope with a wide dynamic range that can simultaneously observe and measure objects over a wide range, for example, from 100 μm to 10 nm.

[0007] The present invention has been made in consideration of the above technical background, and has an object to provide a bidirectional scattering microscope with a wide dynamic range that can observe and measure an object at the same time using a complex amplitude image based on forward scattered light and a complex amplitude image based on backscattered light. [Means for solving the problem]

[0008] A two-way scattering microscope according to one embodiment of the present invention has the following configuration.

[0009] (1) A bidirectional scattering microscope according to aspect 1 of the present invention is characterized in that it comprises a detection means for acquiring a first complex amplitude image formed by forward scattered light of an object to be observed and a second complex amplitude image formed by backscattered light of the object to be observed, and an image separation means for separating the first complex amplitude image and the second complex amplitude image from a composite image including the first complex amplitude image and the second complex amplitude image acquired by the detection means.

[0010] (2) Aspect 2 of the present invention is characterized in that, in the bidirectional scattering microscope of aspect 1, the detection means acquires the first complex amplitude image and the second complex amplitude image for one of the objects to be observed at the same time.

[0011] (3) Aspect 3 of the present invention is characterized in that, in the bidirectional scattering microscope of aspect 2, the detection means has a photoelectric conversion element, and the first complex amplitude image and the second complex amplitude image are acquired in the same image frame by one of the photoelectric conversion elements through spatial frequency multiplexing using interference measurement.

[0012] (4) A fourth aspect of the present invention is characterized in that, in the two-way scattering microscope of any one of aspects 1 to 3, the optical path of the forward scattered light and the optical path of the backscattered light are common between the object of observation and the detection means.

[0013] (5) Aspect 5 of the present invention is characterized in that, in the bidirectional scattering microscope of any one of aspects 1 to 4, a half mirror is provided between the object to be observed and the detection means, which transmits the forward scattered light toward the detection means and reflects the observation light for generating the backscattered light toward the object to be observed.

[0014] (6) Aspect 6 of the present invention is characterized in that, in the bidirectional scattering microscope of any one of aspects 1 to 5, the image separation means is a computer that performs a Fourier transform on the data of the composite image to separate the first complex amplitude image and the second complex amplitude image. Effect of the Invention

[0015] According to the present invention, it is possible to provide a bidirectional scattering microscope having a wide dynamic range that can observe and measure an object at the same time using a complex amplitude image based on forward scattered light and a complex amplitude image based on backscattered light. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic structural diagram showing a bidirectional scattering microscope according to an embodiment of the present invention. [Diagram 2] 1A is a schematic structural diagram showing only the forward scattered light measuring section, and FIG. 1B is a schematic structural diagram showing only the backscattered light measuring section. [Diagram 3] FIG. 2 is a schematic diagram showing the flow of image processing by an image processing unit (image separating means). [Figure 4] 1 is an image showing the results of Measurement Example 1 using the two-way scattering microscope of the present invention. [Diagram 5]11 is an image showing the results of Measurement Example 2 using the two-way scattering microscope of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings. Note that the drawings used in the following description may show characteristic parts in an enlarged scale for the sake of convenience in order to make the characteristics easier to understand, and the dimensional ratios of each component may not necessarily be the same as in reality. In addition, the materials, dimensions, etc. exemplified in the following description are merely examples, and the present invention is not necessarily limited to them, and may be appropriately modified and implemented within a range that does not change the effects of the present invention.

[0018] Fig. 1 is a schematic structural diagram showing a two-way scattering microscope according to one embodiment of the present invention. Also, Fig. 2(a) is a schematic structural diagram showing only the forward scattered light measurement section of the two-way scattering microscope, and Fig. 2(b) is a schematic structural diagram showing only the backscattered light measurement section of the two-way scattering microscope. The two-way scattering microscope 10 of one embodiment of the present invention has a forward scattered light measurement section 11, a backscattered light measurement section 31, and an image processing section (image separation means) 41.

[0019] The forward scattered light measurement section 11 includes a light source 12, half mirrors 13 and 14, a condenser lens 15, a collimator lens 16, an objective lens 17, an imaging lens 18, reflecting mirrors 19 and 20, a photoelectric conversion element (detection means) 21, and a stage 22.

[0020] The backscattered light measurement unit 31 includes a light source 32 , half mirrors 33 and 14 , a condenser lens 35 , an objective lens 17 , an imaging lens 18 , a reflecting mirror 39 , a photoelectric conversion element (detection means) 21 , and a stage 22 .

[0021] Of these, the objective lens 17, the half mirror 14, the imaging lens 18, the photoelectric conversion element (detection means) 21, and the stage 22 are one member common to the forward scattered light measurement unit 11 and the backscattered light measurement unit 31.

[0022] The forward scattered light measurement unit 11 forms a measurement optical path Q1A in which the light source light L1 irradiated from the light source 12 passes through the half mirror 14, passes through the focusing lens 15, the reflecting mirror 19, and the collimating lens 16, and is incident on the object of observation S supported on the stage 22, and the forward scattered light including a first complex amplitude image obtained by irradiating the object of observation S from the front side with the light source light L1 is incident on the photoelectric conversion element 21 through the objective lens 17, the half mirror 14, and the imaging lens 18.

[0023] Furthermore, the forward scattered light measurement unit 11 forms a reference light path Q1B along which the source light L1 emitted from the light source 12 is reflected by the half mirror 14, and further reflected by the reflecting mirror 20 to enter the photoelectric conversion element .

[0024] The backscattered light measurement unit 31 forms a measurement optical path Q2A in which light source light L2 irradiated from a light source 32 passes through a half mirror 33, passes through a focusing lens 35, is reflected by the half mirror 14, and enters the observation object S supported on the stage 22 through the objective lens 17. The backscattered light including a second complex amplitude image obtained by irradiating the observation object S from the rear side with the light source light L2 passes through the objective lens 17, the half mirror 14, and the imaging lens 18 and enters the photoelectric conversion element 21.

[0025] Further, the backscattered light measurement unit 31 forms a reference light path Q2B along which the light source light L2 emitted from the light source 32 is reflected by the half mirror 33, and further reflected by the reflecting mirror 39 to enter the photoelectric conversion element .

[0026] In this embodiment, the optical path from the object to be observed S supported on the stage 22 to the objective lens 17, the half mirror 14, the imaging lens 18, and the photoelectric conversion element 21 forms a common optical path between the measurement optical path Q1A of the forward scattered light measurement unit 11 and the measurement optical path Q2A of the backscattered light measurement unit 31, and this optical path portion Q3 propagates forward scattered light including a first complex amplitude image and backscattered light including a second complex amplitude image as an optical signal multiplexed by spatial frequency multiplexing.

[0027] The photoelectric conversion element (detection means) 21 may be, for example, a CMOS camera, into which a multiplexed optical signal including the first complex amplitude image and the second complex amplitude image from the optical path portion Q3, reference light from the forward scattered light measurement unit 11 from the reference optical path Q1B, and reference light from the backscattered light measurement unit 31 from the reference optical path Q2B are simultaneously incident, and an image signal is generated by photoelectric conversion. With this configuration, the first complex amplitude image (forward scattered light) and the second complex amplitude image (backscattered light) of the observation object can be acquired in the same image frame.

[0028] The image processing unit (image separation means) 41 may be composed of, for example, a computer (PC) on which image processing software is installed, an interface, a display for image output, and the like.

[0029] Such an image processing unit 41 is connected to the photoelectric conversion element (detection means) 21 via a communication cable or the like, and receives as input a multiplexed measurement image signal including the first complex amplitude image and the second complex amplitude image output from the photoelectric conversion element 21, a reference light image signal from the forward scattered light measuring unit 11, and a reference light image signal from the backscattered light measuring unit 31.

[0030] FIG. 3 is a schematic diagram showing the flow of image processing by the image processing unit (image separating means). Using image processing software, the image processing unit 41 generates a frequency-multiplexed hologram image including image data based on forward scattered light and image data based on backward scattered light from the object of observation S, based on the difference between the complex amplitude image when the object of observation S is present and the complex amplitude image when the object of observation S is not present.

[0031] The image processing unit 41 then performs a Fourier transform on the obtained frequency multiplexed hologram image, extracts the corresponding portions of forward scattering and backward scattering from the obtained Fourier image, and performs an inverse Fourier transform to separate and generate a scattered light image (amplitude, phase) of the observation object S due to the forward scattered light measured by the forward scattered light measurement unit 11, and a scattered light image (amplitude, phase) of the observation object S due to the backward scattered light measured by the backward scattered light measurement unit 31. This makes it possible to simultaneously obtain images of one observation object S due to the forward scattered light and the backward scattered light with one bidirectional scattering microscope 10.

[0032] As described above, according to the bidirectional scattering microscope 10 of this embodiment, by being equipped with a forward scattered light measuring unit 11 and a backscattered light measuring unit 31, it becomes possible to simultaneously obtain a scattered light image (amplitude, phase) due to forward scattered light and a scattered light image (amplitude, phase) due to backscattered light for one observation object S with a single bidirectional scattering microscope 10.

[0033] Then, by sharing the optical path of each measurement light (forward scattered light, back scattered light) that has passed through the observation object S and making it incident as a spatial frequency multiplexed signal on the photoelectric conversion element (detection means) 21, it is possible to simultaneously obtain scattered light images of the forward scattered light and the back scattered light. This makes it possible to realize a bidirectional scattering microscope 10 that can collectively observe and measure an observation object using a complex amplitude image based on the forward scattered light and a complex amplitude image based on the back scattered light, something that did not exist in the past.

[0034] In addition, by using a complex amplitude image based on forward scattered light and a complex amplitude image based on backscattered light, which have different size ranges for observable (measurable) structures, the two-way scattering microscope 10 can realize a two-way scattering microscope 10 with a wide dynamic range that can observe and measure objects in a wide range, for example, from 100 μm to 10 nm.

[0035] In this embodiment, the forward scattered light measurement unit 11 and the backscattered light measurement unit 31 are provided with independent light sources 12 and 32, respectively, but it is also possible to configure the forward scattered light measurement unit 11 and the backscattered light measurement unit 31 to have one light source shared by them. In this case, for example, the light source light from one light source may be split into two by a half mirror or the like, and each light source light may be made to enter the forward scattered light measurement unit 11 and the backscattered light measurement unit 31, respectively.

[0036] Furthermore, the forward scattered light measuring unit and the backscattered light measuring unit that constitute the bidirectional scattering microscope are not limited to optical configurations such as lenses and mirrors as in this embodiment, and the optical configurations such as lenses and mirrors may be of any type as long as they are capable of acquiring a first complex amplitude image based on forward scattered light of the object being observed and a second complex amplitude image based on backscattered light.

[0037] Although the embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included in the scope of the invention and its equivalents described in the claims, as well as in the scope and spirit of the invention. EXAMPLES

[0038] In the following, an object to be observed was actually measured using the two-way scattering microscope of this embodiment having the configuration shown in FIG.

[0039] (Measurement example 1) As the observation object, two different beads (beads 1, beads 2) made of methacrylic acid methyl ester (acrylic resin) with an average particle diameter of 100 nm were used, and a first complex amplitude image by forward scattered light and a second complex amplitude image by backward scattered light were obtained by a photoelectric conversion element (detection means). Then, the input data was image-processed by an image processing unit. The measurement images of the two types of beads thus obtained and the noise evaluation are shown in FIG. 4.

[0040] According to the results shown in Figure 4, the intensity ratios of the forward scattered wave and the backscattered wave are different with respect to the incident wave. It can also be confirmed that the minimum detectable scattering cross-sections are different between the forward scattered wave and the backscattered wave.

[0041] (Measurement example 2) Using a living cell as the observation object, a first complex amplitude image by forward scattered light and a second complex amplitude image by backward scattered light were acquired by a photoelectric conversion element (detection means). Then, a static image and a dynamic image of the living cell were generated. The results are shown in FIG.

[0042] The results shown in Figure 5 confirm that it is possible to observe the dynamic behavior of living cells based on the magnitude of temporal fluctuations between a static image based on the phase of forward scattered light after background processing and a static image based on the amplitude of backscattered light after background processing. [Industrial Applicability]

[0043] According to the present invention, by including a forward scattered light measurement unit and a backscattered light measurement unit, it is possible to realize a bidirectional scattering microscope with a wide dynamic range that can observe and measure an observation object all at once. Therefore, the bidirectional scattering microscope of the present invention has industrial applicability. [Explanation of symbols]

[0044] 10. Bidirectional scattering microscope 11...Forward scattered light measurement section 12...Light source 13,14…Half mirror 15...Condenser lens 16...Collimating lens 17...Objective lens 18...Imaging lens 19,20…Reflective mirror 21...Photoelectric conversion element (detection means) 22...Stage 31...Backscattered light measuring section 32…Light source 33...Half mirror 35...Condenser lens 39…Reflective mirror 41...Image processing unit (image separation means)

Claims

1. 1. A bidirectional scattering microscope comprising: a detection means for acquiring a first complex amplitude image generated by forward scattered light of an object to be observed and a second complex amplitude image generated by backscattered light of the object to be observed; and an image separation means for separating the first complex amplitude image and the second complex amplitude image from a composite image including the first complex amplitude image and the second complex amplitude image obtained by the detection means.

2. 2. The bidirectional scattering microscope according to claim 1, wherein the detection means obtains the first complex amplitude image and the second complex amplitude image for one of the observation objects at the same timing.

3. The bidirectional scattering microscope according to claim 2, characterized in that the detection means has a photoelectric conversion element, and the first complex amplitude image and the second complex amplitude image are acquired in the same image frame by one of the photoelectric conversion elements through spatial frequency multiplexing using interference measurement.

4. 3. The bidirectional scattering microscope according to claim 1, wherein an optical path for the forward scattered light and an optical path for the back scattered light are common between the object to be observed and the detection means.

5. 3. The two-way scattering microscope according to claim 1, further comprising a half mirror disposed between the object to be observed and the detection means, the half mirror transmitting the forward scattered light toward the detection means and reflecting the observation light for generating the backscattered light toward the object to be observed.

6. 3. The bidirectional scattering microscope according to claim 1, wherein the image separation means is a computer that performs a Fourier transform on data of the composite image to separate the first complex amplitude image and the second complex amplitude image.

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