WO2013124888A1

WO2013124888A1 - 3d shape measurement apparatus

Info

Publication number: WO2013124888A1
Application number: PCT/JP2012/001150
Authority: WO
Inventors: Kazuki Yamamoto
Original assignee: Sekisui Integrated Research Inc.
Priority date: 2012-02-21
Filing date: 2012-02-21
Publication date: 2013-08-29
Also published as: JP5590692B2; JP2014508914A

Abstract

Provided is a 3D shape measurement apparatus that can easily determine whether the object to be measured is actually located in the field of view. A polarization splitting element 13 splits light to be measured containing phase information on a measurement sample S into a first polarized beam L4-1 and a second polarized beam L4-2 different in polarization direction from the first polarized beam L4-1. A first optical system 14 telecentric on one side focuses the first and second polarized beams L4-1 and L4-2 individually. A pinhole plate 15 is provided with a first pinhole 15a and a second pinhole 15b. The first pinhole 15a forms a first projection beam L5-1 from the first polarized beam L4-1 focused by the first optical system 14. The second pinhole 15b forms a second projection beam L5-2 from the second polarized beam L4-2 focused by the first optical system 14. A second optical system 16 forms an image G of interference fringes by superimposing the first projection beam L5-1 and the second projection beam L5-2 with optical axes thereof inclined to each other.

Description

3D SHAPE MEASUREMENT APPARATUS

This invention relates to 3D shape measurement apparatuses.

With the recent progress in research on mesenchymal stem cells and the like, the need has arisen to measure the 3D shape of a cultured cell as the cultured cell is alive without pretreating it. An example of such a measurement method known in the art is a method in which many confocal microscopic images or interferogram images of a cultured cell are acquired and the 3D shape of the cultured cell is reconstructed from these images. However, this method needs multi shot images in order to reconstruct the 3D shape. Therefore, there arises a problem in that the process of measuring the 3D shape is burdensome.

To cope with this problem, a technique called a digital holographic microscopy has been developed. With the use of a quantitative phase microscope described in Patent Literature 1 as an example of the above technique, the 3D shape of a cultured cell can be measured from one shot image of interference fringes.

Fig. 3 shows a schematic block diagram of a quantitative phase microscope 100 described in Patent Literature 1. As shown in Fig. 3, the quantitative phase microscope 100 includes an objective lens 102, a total reflection mirror 103, a transmissive polarization splitting element 104, a condensing lens 105, a spatial filter 106, a half-wave plate 107, and a complex lens 108, which are arranged in this order between a measurement sample S and an image pickup device 101.

In the quantitative phase microscope 100, light H101 to be measured having passed through the measurement sample S is converted into parallel light H102 by the objective lens 102. The light H102 is reflected toward the transmissive polarization splitting element 104 by the total reflection mirror 103.

The light H102 is split, in the transmissive polarization splitting element 104, into a beam H103a traveling straight ahead and through the element 104 and a beam H103b refracted to the beam H103a. These beams H103a and H103b are linearly polarized beams whose polarization directions are orthogonal to each other.

Next, the linearly polarized beams H103a and H103b are converted into converging beams H104 (H104a and H104b), respectively, by the condensing lens 105 and focused on an aperture 106a and a pinhole 106b, respectively, of the spatial filter 106.

The converging beam H104a passing through the aperture 106a is emitted as an object beam H105 holding the same phase information as the light H101 to be measured.

On the other hand, the converging beam H104b passing through the pinhole 106b is converted into a reference beam H106 which is devoid of light scattered at slight angles when having passed through an object to be measured, such as a cell, contained in the measurement sample S but predominantly includes transmitted light not having passed through any object to be measured, such as a cell, and having information indicative of a uniform phase. The polarization direction of the reference beam H106 is rotated by the half-wave plate 107 disposed behind the spatial filter 106 so that the reference beam H106 has the same polarization direction as the object beam H105.

The object beam H105 and the reference beam H106 are superimposed at the complex lens 108 to form interference fringes. The image pickup device 101 takes an image of these interference fringes. The phase information of the light to be measured is quantified from the taken image of interference fringes using a fringe analysis method, such as Fourier transform, in an information processor.

JP-A-2008-292939

The quantitative phase microscope 100 can measure from a single image of interference fringes the thickness or the like of an object to be measured contained in a measurement sample without damaging the object to be measured.

However, the quantitative phase microscope 100 has a problem in that it is difficult to determine at a glance whether a cultured cell as an object to be measured is located in the center of the field of a quantitative phase image obtained after calculation. The reason for this is that the quantitative phase microscope 100 projects on the image pickup device the object beam H105 in which a real image formed at the aperture of the spatial filter is converted into a spatial frequency image (parallel beam) with a phase distribution by the complex lens and the quantitative phase microscope 100 generates interference fringes as a hologram obtained by the interference of the object beam H105 with the reference beam H106.
It may be considered that this problem can be avoided by allowing the real image from the objective lens or the like to interfere with the reference beam. However, the quantitative phase difference changes with a periodicity of 2p, so that a quasi-phase image obtained in the course of calculation has a large number of discrete singularities. Therefore, in the method using interference of the real image with the reference beam, if the object to be measured has a relatively small thickness of about a few microns, a precise measurement of the quantitative phase image can be made by correctively compensating for periodic shifts at the singularities according to circumstances. However, if the object to be measured has a relatively large thickness of about several tens of microns, a precise quantitative phase image is difficult to obtain from the quasi-phase image since the number of singularities in the quasi-phase image is too large.

The present invention has a principal object of providing a 3D shape measurement apparatus that can easily determine whether the object to be measured is actually located in the field of view.

A 3D shape measurement apparatus of the present invention includes a coherent parallel light source, a polarization splitting element, a first optical system telecentric on one side, a pinhole plate, a second optical system, and an operation part. The coherent parallel light source applies phase-aligned parallel light to a measurement sample. The polarization splitting element splits light including the parallel light having passed through the measurement sample and containing phase information of the measurement sample into a first polarized beam and a second polarized beam different in polarization direction from the first polarized beam. The first optical system focuses the first and second polarized beams individually. The pinhole plate is provided with a first pinhole and a second pinhole. The first pinhole forms a first projection beam from the first polarized beam focused by the first optical system. The second pinhole forms a second projection beam from the second polarized beam focused by the first optical system. The second optical system forms an image of interference fringes by superimposing the first projection beam and the second projection beam with optical axes thereof inclined to each other. The operation part calculates a quantitative differential phase image of the measurement sample from the image of interference fringes.

The distance between the centers of the first pinhole and the second pinhole is preferably different within the range of 0.2 mm to 0.5 mm from the distance mathematically determined from the distance between the polarization splitting element and the first optical system and the angle formed by the optical axis of the first polarized beam and the optical axis of the second polarized beam.

The pinhole plate is preferably formed of a polarization longitudinal slit.

The present invention can provide a 3D shape measurement apparatus that can easily determine whether the object to be measured is actually located in the field of view.

Fig. 1 is a schematic block diagram of a 3D shape measurement apparatus of a first embodiment of the present invention. Fig. 2 is a schematic plan view of an image of interference fringes. Fig. 3 is a schematic block diagram of a quantitative phase microscope described in Patent Literature 1.

Hereinafter, a description will be given of an exemplified preferred embodiment of the present invention. However, the following embodiment is simply illustrative. The present invention is not limited at all to the following embodiment.

Throughout the drawings to which the embodiment and the like refer, elements having substantially the same functions will be referred to by the same reference signs. The drawings to which the embodiment and the like refer are schematically illustrated and, therefore, the dimensional ratios and the like of objects illustrated in the drawings may be different from those of the actual objects. Different drawings may have different dimensional ratios and the like of the objects. Dimensional ratios and the like of specific objects should be determined in consideration of the following descriptions.

Fig. 1 is a schematic block diagram of a 3D shape measurement apparatus 1 of this embodiment. The 3D shape measurement apparatus 1 is an apparatus that can measure the thickness or other spatial features of a light-transmissive microscopic object to be measured, such as a cell, in a noncontact and optical manner. The object to be measured is contained in a measurement sample. Generally, besides the object to be measured, the measurement sample contains a liquid having an already known refractive index. The 3D shape measurement apparatus 1 can perform real-time analysis of, for example, biological cell samples in a living condition without the need for pretreatment. Therefore, the 3D shape measurement apparatus 1 is effectively used in fields of, for example, drug discovery, health management, national security, food industry, prevention of pollen allergy and pandemic infectious diseases, monitoring of bioterrorism, and detection of bacterial contamination.

The 3D shape measurement apparatus 1 includes a coherent parallel light source 9, a measurement sample mount 12, a polarization splitting element 13, a first optical system 14 telecentric on one side, a pinhole plate 15, a second optical system, an image pickup element 20 having a picture plane 20a, and an operation part 21. The coherent parallel light source 9, the measurement sample mount 12, the polarization splitting element 13, the first optical system 14 telecentric on one side, and the pinhole plate 15 are arranged on an optical path between the coherent parallel light source 9 and the image pickup element 20 and in this order from the coherent parallel light source 9 toward the image pickup element 20. In the following description, the relative positions of elements and the like are the relative positions on the optical path from the coherent parallel light source 9 to the image pickup element 20. In the optical path from the coherent parallel light source 9 to the image pickup element 20, the side thereof closer to the coherent parallel light source 9 is referred to as the "upstream" side and the side thereof closer to the image pickup element 20 is referred to as the "downstream" side. The term telecentric herein is used in the meaning that the optical axis can be considered to be parallel with a principal light beam.

The coherent parallel light source 9 applies phase-aligned parallel light L1 for use in quantitative phase measurement to the measurement sample S placed on the measurement sample mount 12.

The coherent parallel light source 9 is composed of, for example, a point light source 10 originating from coherent light, such as laser, and a collimating optical system 11. The collimating optical system 11 converts diverging light L1 emitted from the point light source 10 into parallel light L2. The collimating optical system 11 may include a plurality of lenses or may be composed of a single lens.

An unshown aperture is formed in the measurement sample mount 12. The measurement sample S is placed on at least a portion of the aperture. Normally, the measurement sample S is placed so that the entire aperture is covered with the measurement sample S. Typically, the measurement sample S is a sample containing an object to be measured, such as an adherent cell put on a culture plate, and the object to be measured is immersed into a liquid. The light L3 to be measured having passed through the measurement sample S travels downstream through the measurement sample mount 12.

The light L3 to be measured contains phase information on the measurement sample S. In other words, the light L3 to be measured is light changed in phase as a result of the parallel light L2 having passed through the measurement sample S. More specifically, the light L3 to be measured is light changed in phase from the parallel light L2 to a degree depending on the refractive index and thickness of the liquid or the object to be measured in the measurement sample S.

Downstream of the measurement sample mount 12, the polarization splitting element 13 is disposed. The polarization splitting element 13 splits the light L3 to be measured into a first polarized beam L4-1 and a second polarized beam L4-2 different in polarization direction from the first polarized beam L4-1. The first polarized beam L4-1 and the second polarized beam L4-2 are preferably 90 degrees different in polarization direction. For example, the first polarized beam L4-1 can be one of a P-polarized beam and an S-polarized beam and the second polarized beam L4-2 can be the other. In Fig. 1, the first polarized beam L4-1 is indicated by the dash-and-dot line and the second polarized beam L4-2 is indicated by the broken line.

Downstream of the polarization splitting element 13, the first optical system telecentric on one side and the pinhole plate 15 are arranged. The first optical system 14 can be composed of, for example, at least one lens. The pinhole plate 15 is provided with a first pinhole 15a and a second pinhole 15b.

The first optical system 14 telecentric on one side focuses the first polarized beam L4-1 and the second polarized beam L4-2 on the respective vicinities of the first pinhole 15a and the second pinhole 15b in the pinhole plate 15. The pinhole plate 15 does not allow light scattered at slight angles by the measurement sample S to pass through it but allows only light having passed straight through the measurement sample S to selectively pass through it. More specifically, in the first polarized beam L4-1 projected by the first optical system 14, a light component having not been scattered at slight angles by the object to be measured passes through the first pinhole 15a. The beam having passed through the first pinhole 15a provides a first projection beam L5-1. In the second polarized beam L4-2 projected by the first optical system 14, a light component having not been scattered at slight angles by the object to be measured passes through the second pinhole 15b. The beam having passed through the second pinhole 15b provides a second projection beam L5-2.

The distance between the centers of the first pinhole 15a and the second pinhole 15b is preferably different within the range of about 0.2 mm to 0.5 mm from the distance mathematically determined from the distance between the polarization splitting element 13 and the first optical system 14 and the angle formed by the optical axis of the first polarized beam L4-1 and the optical axis of the second polarized beam L4-2.

Thus, the first polarized beam L4-1 passing through the first pinhole 15a and the second polarized beam L4-2 passing through the second pinhole 15b are beams having passed through slightly different portions of the measurement sample S.

The optimal pinhole diameter (mm) is approximately 0.03679 x (focal distance (mm))^1/2.

For example, if the focal distance is 80 mm, the approximated optimal pinhole diameter is 0.33 mm.

For example, if the focal distance is 50 mm, the approximated optimal pinhole diameter is 0.26 mm.

For example, if the focal distance is 30 mm, the approximated optimal pinhole diameter is 0.20 mm.

The term focal distance herein refers to a focal distance of the telecentric side of the first optical system telecentric on one side. The actual pinhole diameter may be smaller than the approximated optimal pinhole diameter.

The present invention is not limited to the above structure. For example, the pinhole plate 15 may be formed of a polarization longitudinal slit. More specifically, for example, the polarization longitudinal slit can be formed using a photonic crystal wave plate array in which two quarter-wave plates having anisotropic axes orthogonal to each other are integrated.

Downstream of the pinhole plate 15, the second optical system 16 and the image pickup device 20 including a picture plane 20a are arranged. The second optical system 16 can be composed of, for example, at least one lens. The second optical system 16 functions to exactly align the image from the first projection beam L5-1 and the image from the second projection beam L5-2 with their optical axes inclined to each other on the picture plane 20a of the image pickup device 20 to superimpose the images. Furthermore, for the purpose of aligning the first projection beam L5-1 and the second projection beam L5-2 in terms of polarization direction, a half-wave plate may be disposed beside either the first pinhole or the second pinhole.

Projected on the picture plane 20a is an image G of interference fringes as shown in Fig. 2, which is generated by superimposing the first projection beam L5-1 having passed through the first pinhole 15a and the second projection beam L5-2 having passed through the second pinhole 15b by means of the second optical system 16.

The operation part 21 connected to the image pickup element 20 calculates from the image G of interference fringes a quantitative differential phase image representing an in-plane quantitative differential phase difference distribution of the measurement sample S. Furthermore, the operation part 21 determines a quantitative phase image by integral calculation based on the quantitative differential phase image and eliminates the effects of the already known refractive index of the liquid to calculate a 3D shape as a thickness distribution of the object to be measured contained in the measurement sample S.

Next, a description will be given of the operation of the 3D shape measurement apparatus 1.

First, diverging light L1 emitted from the coherent point light source 10 is converted into coherent parallel light L2 by a collimating optical system 11. The coherent parallel light L2 enters the measurement sample S. Thus, light L3 to be measured is generated which contains phase information on the measurement sample S.

The light L3 to be measured is split, by the polarization splitting element 13, into a first polarized beam L4-1 and a second polarized beam L4-2 which have different polarization directions. The first polarized beam L4-1 and the second polarized beam L4-2 are individually focused on the pinhole plate 15 by the first optical system 14.

In the first polarized beam L4-1 focused on the pinhole plate 15 by the first optical system 14, only light having passed straight through the measurement sample S without being scattered at slight angles by the measurement sample S passes through the first pinhole 15a. Light scattered at slight angles by the measurement sample S is blocked by the pinhole plate 15. Thus, a first projection beam L5-1 derived from the first pinhole 15a is generated. On the other hand, in the second polarized beam L4-2 focused on the pinhole plate 15 by the first optical system 14, only light having passed straight through the measurement sample S without being scattered at slight angles by the measurement sample S passes through the second pinhole 15b. Light scattered at slight angles by the measurement sample S is blocked by the pinhole plate 15. Thus, a second projection beam L5-2 derived from the second pinhole 15b is generated.

In this case, the distance between the centers of the first and

second pinholes

15a and 15b is different within the range of about 0.2 mm to 0.5 mm from the distance mathematically determined from the distance between the polarization splitting element 13 and the first optical system 14 and the angle formed by the optical axis of the first polarized beam L4-1 and the optical axis of the second polarized beam L4-2. Therefore, the portion of the measurement sample S having been passed through by the first polarized beam L4-1 passing through the first pinhole 15a is slightly different from the portion of the measurement sample S having been passed through by the second polarized beam L4-2 passing through the second pinhole 15b. Hence, there exists, between the first projection beam L5-1 and the second projection beam L5-2, a phase difference corresponding to a difference between the thickness of a portion of an object to be measured in the measurement sample S through which the first polarized beam L4-1 passing through the first pinhole 15a has passed and the thickness of a portion of the object to be measured in the measurement sample S through which the second polarized beam L4-2 passing through the second pinhole 15b has passed. In this condition, the images of the first projection beam L5-1 and the second projection beam L5-2 are superimposed with their optical axes inclined to each other and in a matched polarization direction to interfere with each other, so that an image G of interference fringes is formed in which a disturbance corresponding to the differential phase difference of the measurement sample S is superimposed on carrier fringes with designed pitches. The operation part 21 eliminates the component of carrier fringes from the image G of interference fringes by a digital filtering technique using Fourier transform or the like to calculate a quantitative differential phase image representing an in-plane quantitative differential phase difference distribution of the measurement sample S. Furthermore, the operation part 21 integrates the quantitative differential phase image to determine a quantitative phase image. Next, the operation part 21 eliminates the effects of the already known refractive index of the liquid to calculate a 3D shape of the object to be measured contained in the measurement sample S, such as thickness information.

The 3D shape measurement apparatus 1 of this embodiment can perform non-invasive measurements in which are not used any fluorescent dye, any labeled substance impairing cell properties, such as gold particles, and any mechanical probe causing structural damage to cells. Therefore, using the 3D shape measurement apparatus 1, the thickness distribution of an object to be measured, such as a cell, contained in the measurement sample S, i.e., spatial features of the object to be measured, can be measured without damage to the object to be measured. In addition, using the 3D shape measurement apparatus 1, the behavior of a living cell can be visualized. Hence, the 3D shape measurement apparatus 1 is useful for observation of dynamic morphology alteration of cells.

Furthermore, in the 3D shape measurement apparatus 1, the first polarized beam L4-1 and the second polarized beam L4-2 pass through substantially the same environment. Therefore, the first polarized beam L4-1 and the second polarized beam L4-2 are subject to substantially the same effects from disturbances, such as vibrations and air currents, and these effects can be cancelled upon formation of the image G of interference fringes. Hence, the 3D shape measurement apparatus 1 is less likely to cause a reduction in measurement accuracy due to disturbances.

In this embodiment, the light to be measured is focused on the pinhole plate 15 by the first optical system 14 telecentric on one side. Light scattered at slight angles by the measurement sample S is blocked by the pinhole plate 15. The pinhole plate 15 is adapted to allow only light having passed straight through the measurement sample S to pass through the first pinhole 15a or the second pinhole 15b. The first and second projection beams L5-1 and L5-2 having passed through slightly different portions are synthetically projected with their optical axes inclined to each other to form an image G of interference fringes. Therefore, the image G of interference fringes is formed of fringes in which a disturbance due to a differential phase difference is superimposed on carrier fringes with a designed constant width. The relative position between the above fringe pattern and the differential phase difference image of the object to be measured is apparent at a glance. Therefore, unlike the digital holographic microscopy using interference of a Fourier transform image with a reference beam, this embodiment enables a person to determine at a glance the position of the object to be measured in the measurement sample S in the field of view. Furthermore, the quantitative differential phase image has a differentiated relationship with the quantitative phase image. Therefore, even for an object to be measured having a thickness of several tens of microns, the quantitative differential phase difference falls within a narrow range of values. Thus, even if a thicker object to be measured is measured in a wider range (i.e., at a lower magnification), the number of singularities does not increase so much. Hence, compared to the method using interference of a real image with a reference beam, this embodiment enables the use of lower magnification optical systems.

1...3D shape measurement apparatus
9...Coherent parallel light source
10...Point light source
11...Collimating optical system
12...Measurement sample mount
13...Polarization splitting element
14...First optical system
15...Pinhole plate
15a...First pinhole
15b...Second pinhole
16...Second optical system
20...Image pickup element
20a...Picture plane
21...Operation part

Claims

A 3D shape measurement apparatus comprising:
a coherent parallel light source for applying phase-aligned parallel light to a measurement sample;
a polarization splitting element for splitting light including the parallel light having passed through the measurement sample and containing phase information of the measurement sample into a first polarized beam and a second polarized beam different in polarization direction from the first polarized beam;
a first optical system telecentric on one side for focusing the first and second polarized beams individually;
a pinhole plate provided with a first pinhole for forming a first projection beam from the first polarized beam focused by the first optical system and a second pinhole for forming a second projection beam from the second polarized beam focused by the first optical system;
a second optical system for forming an image of interference fringes by superimposing the first projection beam and the second projection beam with optical axes thereof inclined to each other; and
an operation part for calculating a quantitative differential phase image of the measurement sample from the image of interference fringes.
The 3D shape measurement apparatus according to claim 1, wherein the distance between the centers of the first pinhole and the second pinhole is different within the range of 0.2 mm to 0.5 mm from the distance mathematically determined from the distance between the polarization splitting element and the first optical system and the angle formed by the optical axis of the first polarized beam and the optical axis of the second polarized beam.
The 3D shape measurement apparatus according to claim 1 or 2, wherein the pinhole plate is formed of a polarization longitudinal slit.