JP2010210506A - Measuring device and method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To speedily measure distribution of transmittance in an object to be observed. <P>SOLUTION: One illustrated embodiment of a measuring device includes: observation means (11, 13, 15, 17) for acquiring both of dark field images and bright field images from the object (25a) to be observed; a storage means for storing information indicating scattering efficiency of the object to be observed beforehand; and an arithmetic operation means for calculating the distribution of transmittance of the object to be observed, based on the dark field images and bright field images acquired by the observation means and information stored by the storage means in advance. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a measuring apparatus and a measuring method for measuring the transmittance distribution of a colorless and transparent object (phase object) such as an achromatic cell colony.

  When culturing unstained cells in a petri dish and observing the growth process of colonies in the petri dish, an observation apparatus that can visualize a phase object is used. Among these types of observation apparatuses, the observation apparatus described in Patent Document 1 is particularly useful in that it has a wide field of view and can overlook the degree of colony growth.

  However, cell colonies grow not only in the direction along the bottom of the petri dish but also in the thickness direction. In order to measure the thickness distribution of such a colony, for example, as in the thickness measurement method described in Patent Document 2, the transmittance distribution of an object to be observed is measured and converted into the thickness distribution. Conceivable.

JP 2007-178426 A Japanese Patent Laid-Open No. 11-230897

  However, as described in Patent Document 2, it is usually necessary to scan an object to be observed with a laser beam to measure the transmittance distribution, and therefore it takes a very long time.

  Therefore, an object of the present invention is to provide a measuring apparatus and a measuring method capable of measuring the transmittance distribution of an object to be observed at high speed.

  One aspect illustrating the measuring apparatus of the present invention is an observation means for acquiring both a dark field image and a bright field image from an observation object, a storage means for storing in advance information indicating the scattering efficiency of the observation object, And a calculation means for calculating a transmittance distribution of the object to be observed based on a dark field image and a bright field image acquired by the observation means and information stored in advance by the storage means.

  In addition, one aspect illustrating the measurement method of the present invention includes an observation procedure for acquiring both a dark field image and a bright field image from an object to be observed, and a storage procedure for previously storing information indicating the scattering efficiency of the object to be observed. And a calculation procedure for calculating the transmittance distribution of the object to be observed based on the dark field image and the bright field image acquired by the observation procedure and the information stored in advance by the storage means.

  ADVANTAGE OF THE INVENTION According to this invention, the measuring apparatus and measuring method which can measure the transmittance | permeability distribution of to-be-observed object at high speed are implement | achieved.

The figure explaining the optical system part of a measuring device. The graph which shows the luminance distribution of the bright part and dark part which mutually adjoin the stripe image I. The block diagram which shows the structure of a controller. The figure explaining the dark part of the stripe image I. FIG. The figure explaining the bright part of the stripe image I. FIG. The flowchart of the preliminary measurement by CPU37. The flowchart of this measurement by CPU37.

[Embodiment]
Hereinafter, embodiments of the measuring apparatus of the present invention will be described.

  First, the configuration of the measuring apparatus will be described.

  FIG. 1 is a diagram illustrating an optical system portion of the measuring apparatus. As shown in FIG. 1, the measuring apparatus includes a transmissive liquid crystal panel 11, a transmissive stage (specimen stage) 13, an imaging optical system 15, and an image sensor 17.

  On the stage 13, a petri dish 25, which is a culture vessel transparent to visible light, is placed as a sample. The petri dish 25 is filled with a culture solution 25b transparent to visible light, and colonies 25a of unstained cells inhabit the culture solution 25b. This colony 25a is an object to be observed of this embodiment.

  The transmissive liquid crystal panel 11 is a transmissive liquid crystal panel with a backlight, for example, and can collectively illuminate the entire area of the petri dish 25 placed on the stage 13. The light emission pattern of the transmissive liquid crystal panel 11 is controlled by a controller (not shown), and a strip-shaped dark portion (non-light-emitting portion 1d) and a strip-shaped bright portion (light-emitting portion 1b) having the same width are alternately arranged as shown in FIG. Set to the specified pattern. The brightness of all the non-light emitting portions 1d is substantially uniform, and the brightness of all the light emitting portions 1b is also substantially uniform. Therefore, the transmissive liquid crystal panel 11 functions as a striped surface light source.

  The imaging optical system 15 captures light emitted from the transmissive liquid crystal panel 11 and transmitted through the entire area of the petri dish 25, and forms an entire image of the petri dish 25 on the imaging surface of the image sensor 17. The object-side numerical aperture NA of the imaging optical system 15 is sufficiently small (for example, NA = 0.01), and the maximum angle θ of incident light with respect to the optical axis is sufficiently small to be regarded as sin θ = θ.

  Although not depicted in FIG. 1, the measuring device is also provided with a focus adjustment mechanism. The user drives the focus adjustment mechanism to position the focal plane of the imaging optical system 15 near the bottom surface of the petri dish 25. In this state, the colony 25a can be observed.

  The image sensor 17 captures an entire image of the petri dish 25 formed on the imaging surface and generates an image. The pixel resolution (object side distance per pixel) of the image sensor 17 is assumed to be sufficiently larger than one cell (for example, 10 μm). However, in order to enable observation of the shape of the colony 25a, the pixel resolution is sufficiently smaller than the size of the colony 25a. Therefore, for example, the pixel resolution of the image sensor 17 is set to 20 μm. The image generated by the image sensor 17 is taken into a controller (not shown).

  Here, since the light emission pattern of the transmissive liquid crystal panel 11 is a stripe shape as described above, an image generated by the image sensor 17 is a stripe image I.

  For example, when a phase object image appears in a certain bright part of the stripe image I and a phase object image appears in the adjacent dark part, the luminance distribution of the bright part and the dark part is, for example, the graph of FIG. become that way.

  As shown on the left side of FIG. 2, in the bright part of the stripe image I, the luminance of the phase object image wb is relatively low. That is, the bright part of the stripe image I is a bright field image in which a dark image appears in a bright background. This bright field image can be used for the thickness distribution measurement of the observation object (colony 25a) (details will be described later).

  On the other hand, as shown on the right side of FIG. 2, in the dark part of the stripe image I, the luminance of the phase object image wd is relatively high. That is, the dark part of the stripe image I is a dark field image in which a bright image appears on a dark background. This dark field image can be used for both shape observation and thickness distribution measurement of the observation object (colony 25a).

  As shown in the center of FIG. 2, the phase object image is unclear in the bright and dark transition portion of the stripe image I, and thus is not suitable for shape observation or thickness distribution measurement of the observation object (colony 25a). .

  Therefore, the measuring apparatus according to the present embodiment acquires three or more (for example, four) stripe images I while changing the light / dark phase of the surface light source described above by one period, and a transition portion among the dark portions of the stripe images I is obtained. The dark field image of the whole area of the petri dish 25 is acquired by cutting out and connecting only the effective portions that are out of the range. Moreover, the measuring apparatus of this embodiment acquires the bright-field image of the whole region of the petri dish 25 by cutting out only the effective part which remove | deviated from the transition part among the bright parts of these stripe images I, and connecting them.

  FIG. 3 is a block diagram showing the configuration of the controller. The controller includes an image sensor control unit 35, a stage control unit 29, an illumination control unit 33, a memory 41, a CPU 37, a display device 27, and an input device 28.

  The image sensor control unit 35 drives the image sensor 17 in accordance with an instruction from the CPU 37 and captures the stripe image I acquired by the image sensor 17.

  The illumination control unit 33 changes the light / dark phase of the surface light source described above by giving a drive signal to the transmissive liquid crystal panel 11 in accordance with an instruction from the CPU 37.

  The memory 41 stores an operation program of the CPU 37, information necessary during the operation of the CPU 37, and the like. The information stored in the memory 41 includes parameters (B, C, C ′ values) necessary for the main measurement described later. These values of B, C, and C ′ are appropriately updated by the CPU 37.

  The input device 28 inputs various instructions from the user to the CPU 37. The instructions input from the user to the CPU 37 via the input device 28 include a preliminary measurement start instruction described later and a main measurement start instruction described later.

  The CPU 37 performs preliminary measurement and main measurement, which will be described later, by comprehensively controlling the image sensor control unit 35, the stage control unit 29, and the illumination control unit 33 in accordance with the operation program stored in the memory 41.

  The display device 27 displays information according to instructions from the CPU 37. The information displayed on the display device 27 includes information that needs to be notified to the user in preliminary measurement and main measurement, and information such as a dark field image and thickness distribution acquired by the CPU 37 in the main measurement.

  Next, the principle of the measuring apparatus will be described.

FIG. 4 is a diagram illustrating the dark part of the stripe image I. In the description, attention is paid to an arbitrary point P on the observation object (colony 25a) reflected in the dark part, and the luminance I _dark of the pixel corresponding to the point P is considered. This point P faces the non-light emitting portion 1d.

  First, each quantity regarding the point P is defined as follows.

  z: the position of the point P in the optical axis direction.

  x: The position of the point P in the stripe pitch direction.

  y: the position of the point P in the line direction of the stripe.

B: Signal amount per unit solid angle given to the luminance I _dark by the light emitting unit 1b when the object to be observed (colony 25a) does not exist (a value also including light loss in the dish 25 and the imaging optical system 15).

ω _i : The incident angle (vector amount) of the light beam toward the point P.

ω _f : An emission angle (vector amount) of a light ray emitted from the point P.

  Ω: solid angle range of light rays from the light emitting portion 1b toward the point P.

Ω _in : solid angle range of light rays from the point P toward the pupil of the imaging optical system 15.

  h: Thickness of the observation object (colony 25a) at point P.

  f: The arrival rate of incident light that enters the observation object (colony 25a) and then enters the point P.

  s: Scattering efficiency of the observation object (colony 25a) at point P.

  g: Arrival rate of scattered light emitted from the point P after being emitted to the outside of the observation object (colony 25a).

Therefore, the luminance I _dark is expressed by the following equation (1).

なお、ここでは、被観察物（コロニー２５ａ）で発生した散乱光が再度散乱を受けること（多重散乱）は無いものと仮定した。

Here, it is assumed that the scattered light generated in the observation object (colony 25a) is not scattered again (multiple scattering).

  Here, scattering in the object to be observed (colony 25a) occurs at a portion where the refractive index is discontinuous, such as the cell membrane of each cell in the colony 25a or each organelle (nucleus, mitochondria, etc.) in the cell. To do. Therefore, in the expression (1), the scattering efficiency s is expressed as a function of the position (x, y, z).

  However, as described above, the object-side numerical aperture of the imaging optical system 15 is sufficiently small, and the pixel resolution of the image sensor 17 is larger than that of the organelle. Therefore, the scattering efficiency s is uniform in the observation object (colony 25a). (In other words, it does not depend on x, y, z), and there is no problem. Similarly, the arrival rates f and g can be regarded as uniform in the observation object (colony 25a).

Further, among the light rays incident on the point P and the light rays emitted from the point P, the light rays contributing to the luminance I _dark are sufficiently small in angle from the z-axis, so the product “f · g” in the equation (1) is , The transmittance when a light beam whose angle from the z-axis is zero passes through the observation object (colony 25a) having a thickness h, that is, the transmission of the observation object (colony 25a) at the point P (x, y). Can be considered equal to the rate. Here, the transmittance at the point P is expressed by exp (−hα), where α is the absorption coefficient (attenuation coefficient) of the observation object (colony 25a).

  Therefore, the above equation (1) is simplified as the following equation (2).

さらに、面光源の形状（ここではストライプ状）や結像光学系１５が不変であり、かつ被観察物（コロニー２５ａ）の種類も不変である限り、式（２）の積分記号以降は、定数Ｃで表現することができる。よって、式（２）は式（３）のとおり簡略化される。

Furthermore, as long as the shape of the surface light source (here stripes) and the imaging optical system 15 are invariable, and the type of the observation object (colony 25a) is also invariant, the integral symbols after the expression (2) are constants. C can be expressed. Therefore, Expression (2) is simplified as Expression (3).

図５は、ストライプ画像Ｉの明部を説明する図である。図５における点Ｐは、図４における点Ｐと同じである。但し、図５における点Ｐは、非発光部１ｄではなく発光部１ｂに正対する。なお、点Ｐに関する各量の定義は、前述したとおりであり、新たに以下の量を定義する。

FIG. 5 is a diagram illustrating a bright portion of the stripe image I. The point P in FIG. 5 is the same as the point P in FIG. However, the point P in FIG. 5 faces the light emitting portion 1b instead of the non-light emitting portion 1d. In addition, the definition of each quantity regarding the point P is as described above, and the following quantities are newly defined.

Ω _out : Solid angle range obtained by removing the solid angle range Ω _in from the solid angle range Ω.

Therefore, the luminance I _bright is expressed by the following equation (4).

なお、ここでも、被観察物（コロニー２５ａ）で発生した散乱光が再度散乱を受けること（多重散乱）は無いものと仮定した。

Here again, it is assumed that the scattered light generated in the observation object (colony 25a) is not scattered again (multiple scattering).

The first term of equation (4) is the amount of signal given to the luminance I _dark when the object to be observed (colony 25a) does not exist (a value that also takes into account light loss in the petri dish 25 and the imaging optical system 15). , {} Corresponds to the amount of light that has been removed from the imaging optical system 15 due to scattering caused by the observed object (colony 25a), and the latter half of {} represents the observed object (colony 25a). This corresponds to the amount of light that has entered the imaging optical system 15 due to the scattering generated in FIG.

  Again, as long as the shape of the surface light source (here stripes) and the imaging optical system 15 are unchanged, and the type of the object to be observed (colony 25a) is also unchanged, It can be expressed by a constant C ′.

  Therefore, the above equation (4) is simplified as the following equation (5).

そして、上述した式（３）と式（５）とに基づけば、式（６）が得られる。

And if it is based on Formula (3) and Formula (5) mentioned above, Formula (6) will be obtained.

この式（６）は、点Ｐにおける被観察物（コロニー２５ａ）の透過率ｅｘｐ（−ｈα）を導出するための式である。

This expression (6) is an expression for deriving the transmittance exp (−hα) of the observation object (colony 25a) at the point P.

Therefore, the measurement apparatus according to the present embodiment measures two types of luminance values I _dark and I _bright regarding the point P, and applies these values to the equation (6), thereby observing the object (colony 25a at the point P). ) Transmittance exp (−hα) can be calculated.

Furthermore, the measuring apparatus according to the present embodiment applies the value of the transmittance exp (−hα) calculated by the equation (6) and the value of the luminance I _dark to the equation (3), thereby observing the object at the point P. The value of the thickness h of (colony 25a) can be calculated.

However, the value and the transmittance exp (-hα), in order to calculate the value of thickness h is, B, C, C ', the value of Omega _in must be already known. Therefore, the measurement apparatus of the present embodiment performs preliminary measurement prior to the main measurement.

  Next, preliminary measurement and main measurement by the measuring apparatus will be described.

  The conditions of the preliminary measurement and the main measurement are common, and are set as follows, for example.

  -Stripe pitch of surface light source: 2 mm.

  Duty ratio between the light emitting unit 1b and the non-light emitting unit 1d is 1: 1.

  -Distance from transmissive liquid crystal panel 11 to petri dish 25: 25 mm.

Focal plane of imaging optical system 15: bottom surface of petri dish 25 Object side numerical aperture of imaging optical system 15: 0.01
-Pixel resolution of the image sensor 17: The object side distance is about 20 μm.

  In addition, in the preliminary measurement, a preliminary measurement sample that satisfies the following conditions is prepared.

  Α is 0.

  H is 0.

  -B is the same as that of this measurement sample.

  The preliminary measurement sample that satisfies the above conditions is, for example, the same type of petri dish as the main measurement petri dish 25, and is filled with the same type of culture liquid as the main measurement culture medium 25b, and no colonies exist. Is. Therefore, hereinafter, this preliminary measurement sample is referred to as a “colony-free sample”.

In addition, a preliminary measurement sample that satisfies the following conditions is also prepared for the preliminary measurement.
• exp (-hα) can be regarded as 1.
H is known
C and C ′ are the same as those of this measurement sample.

  The preliminary measurement sample that satisfies the above conditions is, for example, the same type of petri dish as the main measurement petri dish 25, filled with the same culture medium as the main measurement culture medium 25b, and a known thin thickness. A colony (for example, one layer of cells) is formed over the entire area of the sample. Therefore, hereinafter, this preliminary measurement sample is referred to as a “thin colony sample”.

  FIG. 6 is a flowchart of preliminary measurement by the CPU 37. This flow is started in accordance with a preliminary measurement start instruction from the user. Hereinafter, each step will be described in order.

Step S11: The CPU 37 instructs the user to place the non-colony sample on the stage 13. An instruction from the CPU 37 to the user is performed by displaying character information on the display 27. When receiving a notification from the user that the colony-free sample has been placed on the stage 13, the CPU 37 acquires one stripe image I of the colony-free sample. The notification from the user to the CPU 37 is made via the input device 28. Hereinafter, the stripe image I acquired in this step is referred to as “stripe image I ₁ ”.

Step S12: The CPU 37 instructs the user to place the thin colony sample on the stage 13 instead of the non-colony sample, and instructs the user to input the value of the thickness h of the thin colony. Upon receiving a notification from the user that the thin colony sample has been placed on the stage 13 and the value of the thickness h of the thin colony, the CPU 37 obtains one stripe image I of the thin colony sample and The value of the thickness h is written into the memory 41. Hereinafter, the stripe image I acquired in this step is referred to as “stripe image I ₂₁ ”.

  Step S13: The CPU 37 shifts the light / dark phase of the surface light source by ½ pitch (ie, π).

Step S14: The CPU 37 acquires one stripe image I of the thin colony sample in this state. Hereinafter, the stripe image I acquired in this step is referred to as “stripe image I ₂₂ ”. Note that the light and dark positions are reversed between the stripe image I ₂₂ and the previously acquired stripe image I ₂₁ .

Step S15: The CPU 37 refers to the value of the brightness I _bright of an effective portion of the bright portion (portion outside the transition portion) located in the petri dish image in the stripe image I ₁ of the colony-free sample. Thereby, the signal amount given to the bright part of the stripe image I by an object other than the colony (the petri dish 25, the culture solution 25b, the imaging optical system 15, etc.) becomes known.

Then, the CPU 37 calculates the value of B by applying the referred luminance I _bright value and the above-described conditions (α = 0, h = 0) of the non-colony sample to the equation (5). It is assumed that the value of Ω _in in Equation (5) is known in advance from, for example, optical design data of the measuring device.

In addition, the CPU 37 refers to the value of the brightness I _bright of the effective portion of the bright portion (portion outside the transition portion) located in the petri dish image of the thin colony sample stripe image I ₂₁ , and In the sample stripe image I ₂₂ , reference is made to the value of the luminance I _dark of the effective portion (the portion outside the transition portion) of the _dark portion located at the same pixel coordinates as the bright portion. As a result, the signal amount given to the bright part of the stripe image I by the thin colony and the signal amount given to the dark part of the stripe image I by the thin colony become known.

Then, the CPU 37 refers to the value of the referenced luminance I _bright , the value of the referenced luminance I _dark , the above-described condition (e ^−hα = 1) of the thin colony sample, and the value of the thickness h of the thin colony (memory 41). Are stored in equations (3) and (5) to calculate each value of C and C ′.

  Thereafter, the CPU 37 writes each value of B, C, C ′ calculated in this step into the memory 41. If B, C, and C ′ have already been written in the memory 41 at that time, the values of B, C, and C ′ are updated by this step (step S15).

  Here, since the values of C and C ′ written in the memory 41 reflect the scattering efficiency of the observation object (colony 25a), unless the combination of the optical arrangement of the measuring apparatus and the sample type is changed. No need to update.

  On the other hand, since the value of B written in the memory 41 is a value indicating the luminance of the surface light source, it is necessary to update it appropriately if the variation of the luminance of the surface light source is taken into consideration. Therefore, in the following, it is assumed that the value of B is updated every predetermined period.

  FIG. 7 is a flowchart of the main measurement by the CPU 37. This flow is started in accordance with the start instruction of the main measurement from the user. Hereinafter, each step will be described in order.

  Step S20: The CPU 37 determines whether or not a predetermined period has elapsed since the previous update of the value of B. If it has elapsed, the process proceeds to step S21, and if not, step S23. Migrate to

Step S21: The CPU 37 instructs the user to place the non-colony sample on the stage 13. Thereafter, when receiving a notification from the user that the colony-free sample has been placed on the stage 13, the CPU 37 acquires the stripe image I (that is, the stripe image I ₁ ) of the colony-free sample. The operation in step S21 is the same as the operation in step S11 described above.

Step S22: The CPU 37 refers to the value of the brightness I _bright of the effective part of the arbitrary bright part (part deviated from the transition part) located in the petri dish image of the stripe image I ₁ of the colony-free sample, and the brightness The value of B is calculated by applying the value of I _bright and the condition of the colony-free sample (α = 0, h = 0) to the equation (5). Then, the CPU 37 updates the value of B by writing the value of B calculated in this step into the memory 41. Note that the B update process in step S22 is the same as the B update process in step S25 described above.

Step S23: The CPU 37 instructs the user to place the main measurement sample on the stage 13. Thereafter, upon receiving a notification from the user that the main measurement sample has been placed on the stage 13, the CPU 37 acquires one stripe image I of the main measurement sample. Hereinafter, the stripe image I acquired in this step is referred to as “stripe image I ₃ ”.

Step S24: CPU 37, the number of acquisitions of the stripe image I ₃ it is determined whether or not reached the predetermined number of times in step S23, the process proceeds to step S25 if not been reached, when not reach Step S26 Migrate to Here, it is assumed that the predetermined number of times as a discrimination criterion in this step is “4”.

  Step S25: The CPU 37 shifts the phase of the surface light source by ¼ pitch (that is, π / 2) and then returns to step S23.

Step S26: CPU 37, the entire dark field image of the measurement sample by joining together from each of four stripe image _{I 3} acquired in the loop of steps S23~S25 cut only dark part _{I dark} (x, y) is created. Further, CPU 37 creates an overall bright field image _{I bright} of the measurement sample (x, y) by joining together from each of those four stripe image _{I 3} is cut out only the bright portion.

Since the phase shift amount in step S25 is ¼ pitch, the width of the cutout region in this step is also ¼ pitch. Therefore, only the effective part out of the bright / dark transition part of each stripe image I ₃ is reflected in the _bright field image I _bright (x, y) and the dark field image I _dark (x, y).

Step S27: The CPU 37 obtains a formula for calculating the transmittance exp (−hα) by applying the values of B, C, and C ′ currently stored in the memory 41 to the formula (6). It is assumed that the value of Ω _in in Equation (6) is known in advance from, for example, optical design data of the measuring device.

Then, the CPU 37 determines the luminance I _dark of a certain pixel of the _dark field image I _dark (x, y) created in step S26 and the luminance of the same pixel of the _bright field image I _bright (x, y) created in step S26. By applying I _dark to the calculation formula, the value of the transmittance exp (−hα) of the portion corresponding to the pixel in the observed object (colony 25a) is calculated. Then, the CPU 37 calculates the transmittance distribution e ^−hα (x, y) of the observation object (colony 25a) in the measurement sample by performing this calculation for at least all the pixels located in the petri image. .

Step S28: The CPU 37 ^applies the value of the transmittance e ^−hα of a certain pixel in the transmittance distribution e ^−hα (x, y) calculated in Step S27 to the expression (3), thereby The thickness h of the part corresponding to the pixel in the colony 25a) is calculated. Then, the CPU 37 calculates the thickness distribution h (x, y) of the observation object (colony 25a) in the main measurement sample by performing this calculation for all the pixels.

Step S29: The CPU 37 displays the dark field image I _dark (x, y) created in step S26 and the thickness distribution h (x, y) calculated in step S28 on the display 27, and ends the flow. .

As described above, the measurement apparatus of the present embodiment uses the transmittance distribution e ^−hα (x, y) of the observation object (colony 25a) in the entire area of the measurement sample as the dark field image I _dark (x Y), the _bright field image I _bright (x, y) of the sample for measurement, and the information (C, C ′) of the scattering efficiency of the observation object (colony 25a) stored in the memory 41 in advance. Calculate based on

In addition, the measuring apparatus of the present embodiment acquires four stripe images I ₃ necessary for creating the _dark field image I _dark (x, y) and the bright field image I _bright (x, y) by the image sensor 17. Therefore, there is no need to scan the measurement sample with a beam.

Therefore, the measurement apparatus of the present embodiment can measure the transmittance distribution e ^−hα (x, y) at a higher speed than when the measurement sample is scanned with a beam.

In addition, the measurement apparatus of the present embodiment uses both a dark field image I _dark (x, y) and a bright field image I _bright (x, y) as a common optical system (striped surface light source, imaging optical system, Since it is acquired by the imaging device), it is efficient.

In addition, the measurement apparatus of the present embodiment acquires three or more stripe images I _{3 of the} sample for main measurement, and only the effective portion of each stripe image I ₃ is obtained with the _dark field image I _dark (x, y) and the bright field. Since it is used to create the image I _bright (x, y), it is hardly affected by noise.
[Supplement of embodiment]
In the above-described embodiment, the value of B is updated every predetermined period. However, the brightness of the surface light source may be monitored, and may be performed every time the brightness changes more than a certain value.

  In the above-described embodiment, the measurement is performed on the premise that B is common over the entire area of the sample. However, the measurement may be performed on the premise that B is different depending on the position on the sample.

  Further, in steps S23 to S25 of the above-described embodiment, the number of stripe images acquired from the main measurement sample is four, but may be another number of three or more. When the number of acquired images is n, the phase shift amount for each round is 1 / n pitch (that is, 2π / n), and the width of the cutout area in step S26 is also 1 / n pitch.

Also, in the preliminary measurement of the above-described embodiment, although two the number of stripe image I ₂ acquired from the thin colonies sample may be 1. However, in that case, the amount of signal thin colonies give a bright portion of the stripe image, required to obtain both the amount of signal thin colonies give the dark portion of the stripe image from one different pixel coordinates of the stripe image I ₂ Therefore, the thickness of the thin colony needs to be completely uniform.

  In the preliminary measurement of the above-described embodiment, two preliminary measurement samples, a non-colony sample and a thin colony sample, were used, but the measurement should be performed on the premise that B is common over the entire area of the sample. For example, one preliminary measurement sample having a non-colony region and a thin colony region may be used. In that case, both the signal amount given to the bright part of the stripe image by the object other than the colony and the signal amount given to the bright part and the dark part of the stripe image by the thin colony are acquired from different regions of the same stripe image. become.

  17 ... Imaging device, 15 ... Imaging optical system, 11 ... Transmission type liquid crystal panel

Claims (8)

An observation means for acquiring both a dark field image and a bright field image from the object to be observed;
Storage means for storing in advance information indicating the scattering efficiency of the object to be observed;
An arithmetic means for calculating a transmittance distribution of the object to be observed based on a dark field image and a bright field image acquired by the observation means, and information stored in advance by the storage means;
A measuring apparatus comprising: The measuring apparatus according to claim 1,
The observation means includes
An illumination optical system that illuminates the object to be observed with a surface light source in which bright portions and dark portions are alternately arranged;
Imaging means for obtaining an image of the object illuminated by the surface light source;
Control means for repeatedly driving the imaging means while changing the light / dark phase of the surface light source;
Image processing means for acquiring a dark field image of the object to be observed based on dark portions of the plurality of images acquired by the imaging means, and acquiring a bright field image of the object to be observed based on bright parts of the plurality of images; ,
A measuring apparatus comprising: The measuring apparatus according to claim 2,
The computing means is
The measurement apparatus characterized by converting the transmittance distribution of the observation object into the thickness distribution of the observation object. The measuring apparatus according to claim 2,
The control means includes
The measuring apparatus, wherein the imaging unit is driven at least three times during a period in which the light / dark phase of the surface light source changes by one period. An observation procedure for obtaining both a dark field image and a bright field image from the object to be observed;
A storage procedure for storing in advance information indicating the scattering efficiency of the object to be observed;
A calculation procedure for calculating a transmittance distribution of the object to be observed based on the dark field image and the bright field image acquired in the observation procedure, and information stored in advance by the storage unit,
A measurement method comprising: The measurement method according to claim 5,
The observation procedure is as follows:
An illumination procedure for illuminating the object to be observed with a surface light source in which bright and dark portions are alternately arranged,
An imaging procedure for obtaining an image of the object illuminated by the surface light source;
A control procedure for repeatedly executing the imaging procedure while changing the light / dark phase of the surface light source;
An image processing procedure for acquiring a dark field image of the object to be observed based on the dark part of the plurality of images acquired in the imaging procedure, and acquiring a bright field image of the object to be observed based on the bright part of the plurality of images; ,
A measurement method comprising: The measurement method according to claim 6,
In the calculation procedure,
A measurement method, wherein the transmittance distribution of the observation object is converted into the thickness distribution of the observation object. The measurement method according to claim 6,
In the control procedure,
The measurement method, wherein the imaging procedure is executed at least three times during a period in which the light / dark phase of the surface light source changes by one period.

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