JP2004264048A - Object motion measuring apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple apparatus for measuring the motion of an object with high sensitivity even if the object is moving in any direction. <P>SOLUTION: The measuring apparatus of the motion of the object comprises: an objective lens for condensing light from the object; a lattice-like filter, where a part for projecting the objective image of the object for transmitting light transmitted through the objective lens and a part for shielding light are alternately arranged crosswise; a condensing lens for condensing light transmitted through the lattice-like filter; and a photodetector for measuring the intensity of light transmitted through the condensing lens. The motion of the object is measured by measuring a change in the quantity of light when the objective image of the object moves at the boundary section between the part for transmitting light in the lattice-like filter and a part for shielding light. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an apparatus for measuring the movement of an object by an optical method.
[0002]
[Prior art]
Conventionally, the following two methods have been used to measure the movement speed and vibration period of an object by an optical method.
[0003]
One method is to perform image processing using a video camera. After capturing and recording a moving image of the object, the position change of each frame of the object to be measured is obtained from the recorded image, and the speed and vibration period of the motion are obtained therefrom.
[0004]
Although this method is superior to the method described below in that it can handle various types of exercise, the time accuracy is limited by the video recording speed (1/60 second for general video recording), and real-time measurement is not possible. There is a point that it is impossible and that the analysis is complicated.
[0005]
Another method is to use an optical sensor. The image of the object to be measured is projected onto the light receiving surface of the optical sensor, and the successive changes in the light intensity of the image are measured by the optical sensor to determine the movement speed and displacement.
[0006]
In this method, the change of the optical signal in which the movement speed and the displacement are measured differs depending on the arrangement of the object and the optical sensor, the direction in which the shadow or the reflected light travels, and the like. For example, when there is a dark object on a light background, the image must be placed in such a manner that the image of the object edge portion is just projected on the light receiving surface. Alternatively, in the case of measuring light reflected by an object or the like as a clue, it is necessary to dispose an apparatus such that the light beam efficiently enters the light receiving surface. In any situation, similar measurement sensitivity and accuracy cannot be expected, and it is not a general-purpose method.
[0007]
Other methods for measuring the movement speed and vibration period of an object using an optical sensor have been devised (for example, see Non-Patent Document 1). When measuring the movement velocity v of an object moving in a fixed direction as shown in FIG. 15A, a light transmitting part and a light shielding part are separated by a fixed distance d as shown in FIG. 15B. / 2, a filter having a lattice pattern alternately arranged in the fixed direction is used.
[0008]
When the object is observed through the above filter, the observed image looks as shown in FIG. When the entire observation image is measured by the optical sensor, as shown in FIG. 15D, the output voltage of the optical sensor has a period t according to the light intensity. ₁ It can be seen that it changes in Period t ₁ The movement velocity v of the object can be measured from the distance d of the grid.
Non-Patent Documents 1 to 3 describe such a method of measuring the movement speed.
[0009]
The following methods are available for measuring the reciprocating motion of an object. As shown in FIG. 16A, when measuring the frequency f of an object that reciprocates in a certain direction, a filter is used that increases or decreases the light transmittance along the certain direction.
[0010]
For example, when an object is observed through a filter whose light transmittance decreases from left to right as shown in FIG. 16B, the observed image becomes as shown in FIG. 16C when the object is on the left side. It looks bright and dark on the right. When the entire observation image is measured by the optical sensor, as shown in FIG. 16D, the output voltage of the optical sensor has a period t according to the light intensity. ₂ And the frequency f can be measured.
[0011]
[Non-patent document 1]
The Secretariat of the Science Council of Japan, The Zoological Society of Japan, Kanto Chapter, "Micro-Exploration of Cells-Seeing Invisible Things", Nikkei Selection B, Japan Science Cooperation Foundation, 1996, p. 57-72
[0012]
[Problems to be solved by the invention]
However, in the above method, it is necessary to always match the gradient direction of the light transmittance of the filter with the vibration direction of the object, and the vibration period of an object whose vibration direction changes over time, for example, a microorganism or sperm that performs flagellar movement, is required. It was difficult to measure.
[0013]
Further, in order to improve the measurement sensitivity, the gradient of the light transmittance of the filter must be steep. However, if the gradient of the light transmittance is steep, there is a disadvantage that the measurement region is narrowed.
[0014]
An object of the present invention is to provide a simple device that can measure the motion of an object with high sensitivity regardless of the direction in which the object is moving.
[0015]
[Means for Solving the Problems]
In order to solve the above problems, an invention according to claim 1 of the present invention is an object motion measuring device, wherein an objective lens for condensing light from an object, and an objective image of the object is projected and A lattice filter in which a portion that transmits light transmitted through the objective lens and a portion that blocks light are alternately arranged vertically and horizontally, a condenser lens that collects light transmitted through the lattice filter, and a light that passes through the condenser lens. A light detector for measuring light intensity.
[0016]
According to the first aspect of the present invention, by measuring the change in the amount of light when the objective image of the object moves at the boundary between the light-transmitting part and the light-shielding part of the lattice filter, It is possible to easily measure the motion of the object even if it is moving in any direction. In addition, the present invention can be applied to general imaging devices such as an optical microscope, a magnifying glass, a microlens, a telescope, etc., fluorescence correlation spectroscopy, and the like to measure the motion of various objects of various sizes.
[0017]
The invention according to claim 2 is an apparatus for measuring the motion of an object, comprising: an objective lens for condensing light from the object; and a portion for transmitting an object image projected on the object and transmitted through the objective lens. A lattice-shaped half mirror in which the reflecting portions are alternately arranged vertically and horizontally, two condenser lenses for condensing light transmitted through the lattice-shaped half mirror and light reflected by the lattice-shaped half mirror, respectively, It has two photodetectors for measuring the intensity of light transmitted through each condenser lens, and an operational amplifier for amplifying an output voltage difference between the two photodetectors.
[0018]
According to the second aspect of the present invention, when the object image of the object moves at the boundary between the light transmitting portion and the light reflecting portion of the lattice-shaped half mirror, the transmitted light and the reflected light of the lattice-shaped half mirror are moved. By amplifying and measuring the difference between the light amount changes, the motion of the object can be measured simply and accurately regardless of the direction in which the object moves. In addition, the present invention can be applied to general imaging devices such as an optical microscope, a magnifying glass, a microlens, a telescope, etc., fluorescence correlation spectroscopy, and the like to measure the motion of various objects of various sizes.
[0019]
The invention according to claim 3 is an apparatus for measuring the motion of an object, wherein an objective lens for condensing light from the object, and a light on which an objective image of the object is projected and transmitted through the objective lens are provided at two different angles. A grid-like mirror in which two types of reflecting surfaces that reflect light are alternately arranged vertically and horizontally, two condenser lenses that respectively collect light reflected in two directions by the lattice-like mirror, and a light that passes through each of the condenser lenses. It has two photodetectors for measuring the intensities of the light thus obtained, and an operational amplifier for amplifying an output voltage difference between the two photodetectors.
[0020]
According to the third aspect of the present invention, when the object image of the object moves at the boundary between the two types of reflection surfaces of the lattice-shaped mirror, the difference between the light amount changes of the two types of reflected light of the lattice-like mirror is amplified. By measuring the movement of the object in any direction, the movement of the object can be measured simply and accurately. In addition, the present invention can be applied to general imaging devices such as an optical microscope, a magnifying glass, a microlens, a telescope, etc., fluorescence correlation spectroscopy, and the like to measure the motion of various objects of various sizes.
[0021]
The invention according to claim 4 is an apparatus for measuring the movement of an object, wherein an objective lens for condensing light from the object and light that is projected on an objective image of the object and transmitted through the objective lens are alternately vertically and horizontally. It is characterized by having a photodetector matrix for receiving light on the light receiving surfaces of the two types of photodetectors and an operating amplifier for amplifying the output voltage difference between the two types of photodetectors.
[0022]
According to the fourth aspect of the present invention, when the object image of the object moves at the boundary between the light receiving surfaces of the two types of photodetectors, the difference between the outputs of the two types of photodetectors is amplified and measured. Regardless of the direction in which the object is moving, the motion of the object can be measured simply and accurately. In addition, the present invention can be applied to general imaging devices such as an optical microscope, a magnifying glass, a microlens, a telescope, etc., fluorescence correlation spectroscopy, and the like to measure the motion of various objects of various sizes.
[0023]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, an example applied to an optical microscope as an embodiment of the present invention will be described in detail.
[First Embodiment]
In FIG. 1, the motion measuring apparatus according to the present invention includes an optical microscope 10, a lattice filter 21, an optical sensor 30, and analysis means (not shown).
[0024]
The optical microscope 10 is configured by combining a light source 11, an illumination lens 12, a condenser lens 13, a sample stage 14, an objective lens 15, and an eyepiece 16 (condensing lens). Alternatively, any commercially available optical microscope may be used as the optical microscope 10. The optical microscope 10 is preferably a bright-contrast phase-contrast microscope or a dark-field microscope, and particularly preferably a dark-field microscope, in order to increase the difference in luminance between the object whose motion is to be measured and the background.
[0025]
To make the optical microscope 10 a phase contrast microscope, a combination of the condenser lens 13 and the objective lens 15 is used for the phase contrast microscope. In order to use a dark field microscope, a condenser lens 13 for dark field is used.
[0026]
As the light source 11, any illumination such as a halogen / tungsten lamp or the like can be used, but it is preferable that there is no temporal change in the amount of light, and the voltage applied to the illumination is preferably a DC voltage.
[0027]
The lattice filter 21 is configured such that transmissive portions that transmit light and light-shielding portions S that block light are alternately arranged vertically and horizontally.
As a pattern of such a lattice filter, for example, as shown in FIG. 2A, there is a so-called Ichimatsu pattern in which square transmissive portions T and light-shielding portions S are alternately arranged vertically and horizontally. Alternatively, as shown in FIG. 2B, the pattern may be a pattern in which transparent portions T and light-shielding portions S of an equilateral triangle are alternately arranged vertically and horizontally.
[0028]
As a material for the lattice filter, a material known as an ND filter can be used. Alternatively, the pattern may be perforated on a light-shielding paper, or the pattern may be photographed on a photographic film and developed.
[0029]
The lattice filter 21 is provided between the objective lens 15 and the eyepiece 16 of the optical microscope 10. When a commercially available optical microscope is used, it can be easily installed from a position where a normal eyepiece micrometer, scale plate, reticle, or the like is inserted.
[0030]
In addition, instead of being provided between the objective lens 15 and the eyepiece 16, the lattice filter 21 is substantially optically common with other observation sample surfaces, for example, between the light source 11 and the condenser lens. It may be installed or it may be installed on a sample stage.
[0031]
In addition, the optical microscope 10 may be provided with a beam splitter between the objective lens 15 and the lattice filter 21 so that the reflected image can be measured by transmitted light while observing the reflected image on a monitor with a video camera. . Further, a video camera may be attached to a C mount portion for attaching and detaching a camera of a commercially available optical microscope.
[0032]
As the optical sensor 30 (photodetector), a known photoelectric conversion device such as a photodiode or a photomultiplier tube can be used, but it is preferable to use an inexpensive photodiode from the viewpoint of cost. In addition, a well-known amplifier that amplifies a signal such as an operational amplifier can be used as needed.
When light of different wavelengths is radiated between the object and the background, the motion of the object may be measured using the optical sensor 30 having different light receiving sensitivity depending on the wavelength.
[0033]
The analysis unit is a device that performs frequency analysis, correlation analysis, and the like on the input signal, and receives a signal from the optical sensor 30. As the analysis means, a dedicated machine for FFT analysis (Ono Sokki, CF-5220, etc.) or a personal computer having an AD converter and analysis software can be used.
[0034]
As the analysis software, an FFT algorithm described in a general language such as Basic or C, dedicated software, spreadsheet software, or the like can be used. Specifically, it is described in "Shigeo Minami's Waveform Data Processing for Scientific Measurement", by CQ Publishing Co., Ltd., pp. 154-165.
[0035]
Further, a recording unit for recording a signal from the optical sensor 30 can be arbitrarily provided. As the recording means, any recording means for recording the time change of the voltage can be used, and specifically, a recording device for recording on a magnetic tape such as an audio tape, a video tape, and a DAT can be used.
[0036]
Furthermore, when the signal from the optical sensor 30 is converted into digital data, various analyzes such as frequency analysis, autocorrelation analysis, and cross-correlation analysis can be performed using well-known spreadsheet software. The digitalized signal can be recorded on an arbitrary digital data recording medium.
[0037]
Next, a measuring method using the motion measuring device according to the first embodiment of the present invention will be described.
[0038]
An observed image of an object with a bright contrast phase contrast microscope or a dark field microscope has high luminance against a dark background. Here, when the lattice-shaped filter 21 is installed between the objective lens 15 and the eyepiece 16 and an object that vibrates and moves from the eyepiece 16 is observed, the object becomes a transparent part T that transmits light of the lattice-shaped filter 21 and a light-shielding part. It appears to vibrate at the boundary with the part.
[0039]
At this time, when the luminance of the light transmitted through the lattice filter 21 and condensed by the eyepiece is measured by the optical sensor, when the image O of the object is biased toward the light shielding portion S as shown in FIG. The output voltage is high when the output voltage is low and the image O of the object is biased toward the transmission portion T as shown in FIG. When measured over time, the output voltage changes according to the vibration period of the object. By analyzing the output voltage of the optical sensor, the vibration period of the object can be measured.
[0040]
Note that the image O of the object may not be in focus. Further, the measurement can be performed without depending on the magnification of the image.
When there are a plurality of objects, the average vibration period can be measured.
[0041]
Further, since the transmissive portions T and the light-shielding portions S are alternately arranged vertically and horizontally, when measuring an object vibrating while moving in a certain direction, it is possible to measure a motion speed and a vibration period.
[0042]
Note that, even when the lattice filter 21 is installed on the illumination side of the object to be measured, the object vibrates at the boundary between the illuminated part and the shadow part on the sample table, so that the measurement can be performed similarly. .
[0043]
When light of different wavelengths is emitted between the object and the background, the light from the object such as a high-pass filter, a low-pass filter, a band-pass filter, etc. is better transmitted and the background light is A well-known light-shielding filter may be appropriately used.
[0044]
As described above, according to the first embodiment of the present invention, a signal for measuring the motion of an object can be obtained only by attaching the lattice filter 21 and the optical sensor 30 to the optical microscope 10. Further, since the image of the object does not have to be in focus, it is possible to measure the motion of the object accompanied by three-dimensional displacement. When there are a plurality of objects, the average vibration period can be measured.
[0045]
[Second embodiment]
Next, a second embodiment of the present invention will be described. As shown in FIG. 4, in the motion measuring apparatus according to the second embodiment, the light source 11, the illumination lens 12, the condenser lens 13, the sample stage 14, the objective lens 15, and the analyzing means are the same as those in the first embodiment. However, instead of the lattice filter 21, a lattice half mirror 22 including a light transmitting part and a light reflecting part is provided. Further, the eyepieces 16 and 16 for condensing the light transmitted through the lattice half mirror 22 and the light reflected by the lattice half mirror 22, respectively, and the intensity of the light transmitted through the eyepieces 16 and 16 are measured. Two optical sensors 30 and 30 are provided. Further, an output of the two optical sensors 30, 30 is input, and an operation amplifier 40 for amplifying the potential difference is provided.
[0046]
The lattice-shaped half mirror 22 has a configuration in which transmissive portions T that transmit light and reflective portions R that reflect light are alternately arranged vertically and horizontally, and a transmissive portion T that transmits light and a reflective portion that reflects light. Are equal in area.
[0047]
As such a lattice half mirror 22, for example, as shown in FIGS. 5A and 5B, a mirror or a prism having a reflection surface in which transmission portions T and reflection portions R are alternately arranged vertically and horizontally are used. Conceivable. Further, in the figure, the shape of the transmitting portion T and the reflecting portion R is a square or a regular triangle, but may be an arbitrary shape such as a circle or a polygon.
[0048]
Next, the measurement principle of the motion measurement device according to the second embodiment of the present invention will be described. FIG. 6 is a schematic diagram of a phase contrast microscope image or a dark field microscope image of the bright contrast of the vibrating object projected on the boundary between the transmission part T and the reflection part R on the lattice half mirror 22.
[0049]
At this time, when the image O of the object moves in the direction of the transmission portion T as shown in FIG. 6A, the amount of light transmitted through the transmission portion T increases and the amount of light reflected by the reflection portion R decreases. When the image O of the object moves in the direction of the reflection portion R as shown in FIG. 6B, the amount of light transmitted through the transmission portion T decreases and the amount of light reflected by the reflection portion R increases.
At this time, the absolute values of the amounts of change in the transmitted light and the reflected light are equal.
[0050]
The light transmitted through the transmitting portion T and the light reflected by the reflecting portion R are detected by separate optical sensors 30 and 30, respectively, and the difference between their output voltages is amplified by an operational amplifier 40. Is output as a signal. When the output voltage of the operational amplifier 40 is analyzed, the vibration period of the object can be measured as in the first embodiment.
[0051]
According to the motion measuring device of the second embodiment, the same measurement as that of the first embodiment can be performed, and the reflected light and the transmitted light of the lattice-shaped half mirror 22 are measured by the two optical sensors 30, 30. Since the difference between the output voltages is amplified by the operational amplifier 40, the DC component of the output of the optical sensors 30 and 30 due to the background light can be cut to perform measurement with higher sensitivity. Further, since there is no light shielding portion S, light from the object to be measured can be effectively used. Also, as in the first embodiment, the image of the object does not have to be in focus, so that the motion of the object accompanied by three-dimensional displacement can be measured. When there are a plurality of objects, the average vibration period can be measured.
[0052]
[Third Embodiment]
Next, a third embodiment of the present invention will be described. The difference between the third embodiment of the present invention and the second embodiment is that, as shown in FIG. 7, instead of splitting light from an object into transmitted light and reflected light by a lattice half mirror 22, as shown in FIG. This is a point measured by dividing the reflected light into two by the lattice mirror 23.
[0053]
As shown in FIGS. 8A and 8B, the grating mirror 23 has a reflection surface R that reflects light at two different angles with respect to the optical axis from the object. ₁ , R ₂ Are alternately arranged vertically and horizontally.
For example, a DMD (Digital Micromirror Device) can be used as such a grid-like mirror 23. When a DMD is used as the lattice-like mirror 23, for example, as shown in FIG. ₁ And R ₂ And R ₁ , R ₂ May be detected by separate optical sensors 30 and 30 and amplified by the operational amplifier 40. When the output voltage of the operational amplifier 40 is analyzed, the vibration period of the object can be measured.
[0054]
As described above, according to the motion measuring device of the third embodiment of the present invention, R ₁ , R ₂ Is detected by the separate optical sensors 30 and 30 and the difference between the output voltages is amplified by the operational amplifier 40. Therefore, the optical sensors 30 and 30 are operated according to the same principle as that described in the second embodiment. The measurement can be performed with higher sensitivity by cutting the DC component of the output voltage due to the background light. Further, since there is no light shielding portion S, light from the object to be measured can be effectively used. Further, as in the first and second embodiments, the image of the object does not have to be in focus, so that the motion of the object accompanied by three-dimensional displacement can be measured. When there are a plurality of objects, the average vibration period can be measured.
[0055]
When a DMD is used as the grating mirror 23, the reflection surface R ₁ And R ₂ In addition to alternately arranging the patterns in the vertical and horizontal directions, arbitrary patterns such as alternately arranging two in the vertical and horizontal directions can be used.
[0056]
[Fourth Embodiment]
Next, a fourth embodiment of the present invention will be described. As shown in FIG. 9, in the motion measuring apparatus according to the third embodiment of the present invention, the light source 11, the condenser lens 13, the sample stage 14, the objective lens 15, and the analyzing means are the same as those in the first to third embodiments. However, a two-dimensional photodiode array 24 in which photodiodes are arranged vertically and horizontally is provided at a position where an objective image is formed. The two-dimensional photodiode array 24 has two outputs, which are input to the operational amplifier 40.
[0057]
Although only a single lens is shown as the objective lens 15 in the figure, one or more lenses are provided between the objective lens 15 and the two-dimensional photodiode array 24 in order to increase the magnification of the objective lens. An eyepiece image may be formed on the light receiving surface of the two-dimensional photodiode array. That is, a two-dimensional photodiode array may be attached to a position where an eyepiece image of an eyepiece of a commercially available optical microscope is formed. Attachment to a commercially available optical microscope can be easily performed by using, for example, a C-mount portion of the optical microscope.
[0058]
As the two-dimensional photodiode array 24, one in which photodiodes having the same performance are arranged vertically and horizontally can be used. Alternatively, a commercially available two-dimensional photodiode array may be used. For example, a 5 × 5 element SiPIN photodiode array (Hamamatsu Photonics, S7585), a 16 × 16 element SiPIN photodiode array (Hamamatsu Photonics, S3805), or the like is used. be able to.
[0059]
Next, how to use the two-dimensional photodiode array in the present invention will be described. First, as shown in FIG. 10, each element of the two-dimensional photodiode array 24 (6 × 6 elements in the figure) is classified every other row and column (in the figure, A and B). Next, the output terminal of A and the output terminal of B are connected to each other to obtain two output terminals of an output terminal a and an output terminal b.
[0060]
Although the figure shows a two-dimensional photodiode array consisting of an even number of elements, in the case of a two-dimensional photodiode array consisting of an odd number of elements, for example, the central element is not used, and A The number of elements and the number of elements of B are set to be the same.
The two output terminals a and b are connected to the operational amplifier 40, and the output voltage difference is activated and amplified and output to the analysis means.
[0061]
Next, the measurement principle of the motion measuring device according to the fourth embodiment of the present invention will be described.
Similarly to the second and third embodiments, when the image of the vibrating object projected on the boundary between the light receiving surface A and the light receiving surface B on the two-dimensional photodiode array 24 moves in the direction of the light receiving surface A, , The output voltage at terminal a increases and the output voltage at terminal b decreases. When the image of the object moves toward the light receiving surface B, the output voltage at the terminal a decreases and the output voltage at the terminal b increases. When the difference between the output voltages of a and b is amplified by the operational amplifier 40, a voltage that changes according to the vibration cycle of the object is output as a signal. When the output voltage of the operational amplifier 40 is analyzed, the vibration period of the object can be measured in the same manner as in the second and third embodiments.
[0062]
According to the fourth embodiment of the present invention, the same measurement as that of the second and third embodiments can be easily performed simply by attaching the two-dimensional photodiode array to the optical microscope, and fine adjustment of the mirror angle. Becomes unnecessary and the design becomes easy. Further, the pattern of the light receiving surface can be easily changed by changing the connection between the elements. Also, as in the first to fourth embodiments, the image of the object does not have to be in focus, so that the motion of the object accompanied by three-dimensional displacement can be measured. When there are a plurality of objects, the average vibration period can be measured.
[0063]
According to the above embodiment, the motion of the object under the optical microscope can be easily measured. Thus, for example, by measuring the self-diffusion coefficient D of a molecule in a solution having an absolute temperature T and a viscosity η, the radius r of the molecule can be calculated by Einstein's diffusion formula (D = kT / 6πηr, where k is Boltzmann's constant). It can be considered that it can be used to measure the structural change and association state of the molecules in the aqueous solution. In this way, it can be applied to fluorescence correlation spectroscopy and the like.
[0064]
The present invention is not limited to the above embodiments. For example, by excluding the light source 11, the condenser lens 13, and the sample stage 14 from the above-described four embodiments, changing the magnification of the objective lens 15 and the eyepiece 16 allows a distant telescope. The vibration motion of the object can also be measured.
[0065]
<Example 1 Measurement of sea urchin sperm movement>
The movement of sea urchin sperm (Bahun sea urchin) was measured. Sea urchin spermatozoa were diluted in artificial seawater (aquamarine) and placed in a chamber. The chamber was set on a sample table, and measurement was performed while observing the movement of sea urchin sperm.
[0066]
A phase contrast condenser lens (Olympus U-UCDB-2) and a phase contrast objective lens (Olympus Uplan 20NH) are attached to an inverted microscope (Olympus IX-70), and a two-dimensional photodiode array with an operation amplifier is attached to the inverted microscope C. It was attached to the mount.
[0067]
As a two-dimensional photodiode array, a 5 × 5 element SiPIN photodiode array (Hamamatsu Photonics, S7585) was used. Each element was alternately distributed vertically and horizontally and connected to obtain two output terminals. The element at the center was not used.
[0068]
Each output of the two-dimensional photodiode array was amplified using two operational amplifiers (Burr-Brown, OPA111BM) and operation-amplified using operation amplifiers (Burr-Brown, INA106KP). The output of the operational amplifier was passed through a voltage follower (National Instruments, LF356N), digitized by an AD converter (ONKYO, USB digital audio processor SE-U55X), input to a personal computer, saved, and analyzed by spreadsheet software (EXCEL). .
[0069]
<Result 1>
The obtained signal is shown in FIG. FIG. 11B is an enlarged view of a portion between 0.5 and 1.0 seconds in FIG. It can be seen that the light amount changes with the movement of sea urchin sperm.
[0070]
FIG. 11C shows the relationship between the frequency of vibration and the power spectrum obtained by Fourier transforming the signal shown in FIG. 11A. In this example, it can be seen from the graph that the flagellation frequency of sea urchin sperm was about 47 Hz. This value was close to the frequency of flagellation of sea urchin sperm reported so far.
[0071]
<Example 2 Measurement of rainbow trout sperm movement>
Rainbow trout spermatozoa are inactive in male testes, but are activated when they are inseminated in freshwater, start exercising, swim for several tens of seconds, and stand still. The movement of rainbow trout sperm immediately after being spermized in freshwater was observed and measured.
[0072]
Illumination was performed by applying a DC voltage of 11 V (LEADER LPS-160-5) to a halogen / tungsten lamp (PHILIPS 12V-50W). For dark field condenser lens (Nikon Dark Field Condenser Dry 0.95-0.80), objective lens (OLYMPUS, A10NH 0.25 160 / 0.1), eyepiece lens (Sigma Kogaku, DLB-20-30PM) Was.
[0073]
The above optical elements were assembled horizontally on an optical bench (Newport, BREADBOARD) to form an optical microscope, and mounted on an air spring type anti-vibration table (Meiratsu Seiki, VISOLATOR).
[0074]
A lattice filter was installed between the objective lens and the eyepiece, and an optical sensor was installed at the focal point of light transmitted through the eyepiece. The sample stage was set between the condenser lens and the objective lens. A half mirror was set between the objective lens and the lattice filter, and the half mirror was set so that reflected light was projected on a CCD camera (SONY XC-75).
[0075]
The filter was prepared by printing a checkerboard pattern in which black squares and white squares were alternately arranged vertically and horizontally on a positive slide film (FUJICHROME RDP II 135) with a laser beam (LASERGRAPHICS, Inc. LFR Mark II).
[0076]
As the optical sensor, a photodiode (Hamamatsu Photonics, S5821-01) and an operational amplifier (BURR-BROWN, OPA9544) were used. The output of the optical sensor was amplified with a lock-in amplifier (NF ELECTRONIC INSTRUMENTS 5610B), measured with an FFT analyzer (Ono Sokki, CF-5220), and recorded on a DAT recorder (PIONER, D-05).
[0077]
In addition, an experiment to analyze the movement using high-speed video was also performed in parallel, and it was confirmed that the movement cycle was measured. Remove the lattice half mirror, eyepiece, and optical sensor behind the objective lens, and set up so that the light receiving surface of the high-speed CCD camera (HAS200R, Detect) is located at the same position where the lattice half mirror was installed. did. Images were input and recorded on a personal computer equipped with an image processing board (DIG-ATII, Detect) and image processing software (DIP95, Detect).
[0078]
Rainbow trout sperm were diluted 1000 times with seminal plasma (centrifugal supernatant of rainbow trout semen) and placed in a syringe (NARISIGE SYR-6) equipped with an injection needle (HAMILTON 90031).
In the chamber, artificial fresh water (120 mM NaCl, 10 μM CaCl ₂ , 10 mM HEPES-Cl (pH 8.5)).
[0079]
The injection needle attached to the syringe containing the sperm was inserted into the chamber provided on the sample stage, and the observation was performed with a CCD camera so that the tip of the injection needle was positioned at the center of the visual field.
Rainbow sperm were sperm into freshwater by pushing the piston of the syringe, and the movement was observed and measured.
[0080]
<Result 2>
FIG. 12 shows the obtained signal. FIGS. 13A to 13D are diagrams showing the relationship between the average power spectrum and the frequency for 6 seconds, 16 seconds, 19 seconds, and 3 seconds after 22 seconds from the start of the measurement of the signal in FIG. 12, respectively. The rainbow trout sperm started to move and a peak at about 100 Hz was observed in the power spectrum. This peak moved to a lower frequency side while attenuating with time. This can be seen from a frequency-time chart (FIG. 14) showing the power spectrum in black and white gradations and showing the relationship between frequency and time.
[0081]
【The invention's effect】
According to the present invention, it is possible to easily measure the motion of an object regardless of the direction in which the object is moving. Further, the present invention can be applied to general imaging devices such as an optical microscope, a magnifying glass, a microlens, a telescope, and the like, and can measure the motion of various large and small objects. Further, since the image of the object does not have to be in focus, it is possible to measure the motion of the object accompanied by three-dimensional displacement. When there are a plurality of objects, the average vibration period can be measured.
[Brief description of the drawings]
FIG. 1 is a diagram showing a first embodiment of the present invention.
FIGS. 2A and 2B are schematic diagrams of a lattice filter used in the first embodiment of the present invention.
FIG. 3 is a diagram illustrating a measurement principle according to the first embodiment of the present invention.
FIG. 4 is a diagram showing a second embodiment of the present invention.
FIGS. 5A and 5B are schematic diagrams of a lattice-shaped half mirror used in a second embodiment of the present invention.
FIG. 6 is a diagram illustrating a measurement principle according to a second embodiment of the present invention.
FIG. 7 is a diagram showing a third embodiment of the present invention.
FIG. 8A is a schematic view of a lattice-like mirror used in a third embodiment of the present invention, and FIG. 8B is a cross-sectional view taken along the line II of FIG.
FIG. 9 is a diagram showing a fourth embodiment of the present invention.
FIG. 10 is a schematic diagram of a light receiving surface of a two-dimensional photodiode array used in a fourth embodiment of the present invention.
FIG. 11 (a) is a signal obtained by measuring the movement of sea urchin spermatozoa as Example 1 of the present invention, and FIG. 11 (b) is an enlarged view of a portion of 0.5 to 1.0 seconds of (a). (C) is a diagram illustrating a relationship between a power spectrum obtained by Fourier-transforming the signal of (a) and a frequency.
FIG. 12 shows a signal obtained by measuring the movement of rainbow trout sperm as Example 2 of the present invention.
13A and 13B are diagrams showing the relationship between the power spectrum and the frequency of the signal in FIG. 12, wherein FIG. 13A shows about 6 seconds after the start of measurement, FIG. 13B shows about 16 seconds, and FIG. 13C shows about 19 seconds. , (D) is the average spectrum of the signal for 3 seconds after about 22 seconds.
FIG. 14 is a diagram showing the power spectrum of the signal of FIG. 12 in black and white gradation and showing the relationship between frequency and time.
FIG. 15 is a diagram showing a conventional method for measuring the speed of an object.
FIG. 16 is a diagram showing a conventional method of measuring vibration of an object.
[Explanation of symbols]
10 Optical microscope
11 Lighting
12 Condenser lens
13 Sample table
14 Objective lens
15 Eyepiece
21 Grid filter
22 Lattice half mirror
23 lattice mirror
24 Two-dimensional photodiode array
30 Optical Sensor
40 operational amplifier
O Object image
R reflection part
R ₁ , R ₂ Reflective surface
S Shading part
T transmission part

Claims (4)

An objective lens for condensing light from an object, a lattice filter in which an object image of the object is projected and a portion for transmitting light transmitted through the objective lens and a portion for shielding light are alternately arranged vertically and horizontally, An object motion measuring device, comprising: a condenser lens for condensing light transmitted through a filter; and a photodetector for measuring the intensity of light transmitted through the condenser lens. An objective lens for condensing light from an object, a lattice-shaped half mirror in which an object image of the object is projected and a portion for transmitting light and a portion for reflecting light transmitted through the objective lens are alternately arranged vertically and horizontally, and the grating Condenser lenses for condensing the light transmitted through the conical half mirror and the light reflected on the lattice-shaped half mirror, and two photodetectors for measuring the intensity of the light transmitted through each of the condensing lenses And an operation amplifier for amplifying a difference between output voltages of the two photodetectors. An objective lens for condensing light from an object, and a lattice-shaped mirror in which two types of reflecting surfaces for projecting an object image of the object and reflecting light transmitted through the objective lens at two different angles are alternately arranged vertically and horizontally. Two condensing lenses for condensing light reflected in two directions by the lattice mirror, two photodetectors for measuring the intensity of light transmitted through each condensing lens, and the two light beams A motion measuring device for an object, comprising: an operational amplifier for amplifying an output voltage difference of a detector. An objective lens for condensing light from an object, and a photodetector matrix for receiving light received by light receiving surfaces of two types of photodetectors in which an objective image of the object is projected and light transmitted through the objective lens is alternately arranged vertically and horizontally. A motion amplifier for amplifying the difference between the output voltages of the two types of photodetectors.

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