JP2010039438A - Confocal microscope system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a confocal microscope system which provides a high quality image by removing both of noise such as smearing noise due to an optical image incidence to an imaging apparatus during the term other than an exposure term and banding noise. <P>SOLUTION: An exposure operation of the imaging apparatus 11 and the quantity of light made incident to the imaging apparatus 11 are synchronously controlled with the scanning period of an optical scanning means having a mask pattern member 5 as a reference, so that the exposure time is made to be a multiple of integer of the minimum time necessary for scanning a specimen, and consequently the optical image incidence to the imaging apparatus during the term other than the exposure term is suppressed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention is configured such that a confocal image of the sample can be observed by using an optical scanning unit having a mask pattern member on which a predetermined pattern is formed and scanning the sample with illumination light from the illumination unit. The present invention relates to a confocal microscope system.

  A confocal microscope is a microscope used for observing and measuring the microstructure of a sample with excellent resolution on the image plane and in the optical axis direction by using a confocal optical system.

  Two confocal microscopes are generally known, a single beam scanning type and a multi-beam scanning type. The single-beam scanning confocal microscope needs to scan a single illumination light two-dimensionally, and thus has a drawback that the image forming speed is generally slow. On the other hand, the multi-beam scanning confocal microscope has an excellent feature that the image forming speed is generally high because a plurality of illumination lights are scanned simultaneously. In the multi-beam scanning type, a mask pattern member scanning type in which a mask pattern member on which a predetermined pattern is formed moves or rotates, and a light changing unit using a fixed mask pattern member and a light turning means such as a galvano scanner. A bidirectional scanning type is known. As the mask pattern member scanning type, there are known a pinhole disk type using a rotating disk in which a plurality of pinholes are arranged at predetermined positions, and a slit disk type using a rotating disk in which slits are arranged at predetermined positions. Among these, as a pinhole disk type, a Nipow disk type in which pinholes are spirally arranged from the inner periphery to the outer periphery of the disk is known. In the light turning scanning type, the illumination light that has passed through the mask pattern member in which a plurality of pinholes or slits are arranged at predetermined positions is scanned on the sample by turning the light with a galvano scanner. There is known a system in which a confocal image of a sample is formed on the image sensor by turning the return light again with a galvano scanner and scanning the image sensor.

  A multi-beam scanning confocal microscope is configured to obtain a confocal image of a sample by periodically scanning the sample with illumination light that has passed through a predetermined pattern. For this reason, in a confocal microscope system in which a multi-beam scanning confocal microscope and an imaging apparatus are combined, scanning unevenness occurs unless the scanning cycle and the exposure time of the imaging apparatus are in a suitable relationship. There is a problem that banding noise accompanied by uneven brightness occurs in the focus image. The preferred relationship means that the exposure time is an integral multiple of the minimum time for the illumination light to scan the sample uniformly. The banding noise not only deteriorates the texture of the image but also becomes an error factor when performing quantitative measurement or analysis.

  As a technique for solving such a problem, for example, there is a Nipkow disk type optical scanning device disclosed in Patent Document 1. This comprises a scanning start point detection pinhole corresponding to the start point of the scanning track of the Niipou disc, and means for photoelectrically converting the transmitted light of the scanning start point detection pinhole to generate a trigger signal for the imaging device, the trigger signal By controlling the image pickup device in synchronization with this, the synchronization between the scanning cycle and the image pickup cycle is suitably realized.

  In the confocal microscope motor control device disclosed in Patent Document 2, scanning is performed by driving the motor at a speed corresponding to a signal obtained by multiplying a vertical synchronizing signal obtained from the NTSC signal output from the CCD camera. The synchronization of the period and the imaging period is preferably realized.

  Further, in the confocal microscope disclosed in Patent Document 3, the rotational state of the rotating disk is controlled so that the phase of the vertical synchronizing signal and the rotating phase of the rotating disk are synchronized. Even when rotation unevenness occurs in the disk due to the frictional fluctuation of the shaft, the synchronization between the scanning period and the imaging period is suitably realized.

  Further, the confocal microscope disclosed in Patent Document 4 includes a control unit that controls the amount of illumination light in synchronization with the vertical synchronization signal of the imaging unit and the scanning cycle of the rotating disk. And the effective exposure period (the exposure step period of the image sensor and the period during which the light image is incident) are preferably realized, and the dynamic range of the light quantity can be easily changed. Is.

  Further, in the confocal image measurement system disclosed in Patent Document 5, the imaging cycle of the imaging device is set based on the scanning cycle of the rotating disk and the vertical transfer time of the frame transfer type CCD image sensor used as the imaging device. Thus, the synchronization between the scanning cycle and the imaging cycle is preferably realized.

  In the confocal microscope disclosed in Patent Document 6, the scanning period and the exposure time are suitably realized by controlling the exposure period of the imaging device so as to be synchronized with the scanning period of the rotating disk. In addition, by performing the exposure operation and the readout operation at the same time, it is possible to efficiently perform image acquisition. However, in the imaging method in the best embodiment described in Patent Document 6, it is assumed that the time required for the vertical transfer of the frame transfer type CCD image sensor is sufficiently shorter than the exposure period and the readout period. However, since vertical transfer requires a certain amount of time, it is sufficiently conceivable to shoot under conditions where the above assumption does not hold. Therefore, in the imaging method described in Patent Document 6, strictly speaking, there is a problem in that the synchronization between the scanning period and the exposure time cannot be suitably realized, and banding noise is generated.

  In a multi-beam scanning confocal microscope system, a two-dimensional image sensor is generally used as an imaging device. As the two-dimensional image sensor, a CCD image sensor, a CMOS image sensor, or the like is generally used. As a CCD image sensor, a frame transfer type CCD image sensor, a full frame transfer type CCD image sensor, an interline transfer type CCD image sensor, and the like are generally known.

  FIG. 10 is a schematic diagram showing an example of the configuration of a frame transfer type CCD image sensor. The frame transfer type CCD image sensor shown in FIG. 10 includes an imaging unit 31, a storage unit 32, a horizontal shift register 33, and a charge-voltage conversion circuit 34, each including a plurality of vertical shift registers. The vertical shift register and the horizontal shift register are composed of CCD elements and have a charge transfer function. In addition, the vertical shift register of the imaging unit 31 has a photoelectric conversion function of incident light in addition to the charge transfer function. By setting it as such a structure, the ratio of the area of the photoelectric conversion area which occupies for an imaging part can be made into 100%, and the outstanding photoelectric conversion efficiency is realizable. In addition, by vertically transferring the charge of the image pickup unit to the storage unit after the exposure is completed, it is possible to perform exposure in the image pickup unit at the same time as reading the charge from the storage unit, so that image acquisition is performed more efficiently. Can do. Further, the horizontal shift register unit can be configured to have an electron multiplication function. By adopting a configuration in which the horizontal shift register unit is equipped with an electron multiplication function, it is possible to amplify the charge at a predetermined magnification during horizontal transfer and realize a better S / N.

  The imaging operation in the frame transfer type CCD image sensor is performed through three steps of an exposure step, a vertical transfer step, and a horizontal transfer step. First, in the exposure step, a light image is incident on the image pickup unit 31, and charges generated according to the light intensity of the incident light are accumulated in the CCD elements constituting each pixel of the image pickup unit 31. In the subsequent vertical transfer step, charges are transferred from the imaging unit 31 to the storage unit 32 on the vertical shift register. In the subsequent horizontal transfer step, the charge is transferred from the storage unit 32 to the charge / voltage conversion circuit 34 through the horizontal shift register 33, and image information is output through the charge / voltage conversion circuit 34.

  In a frame transfer type CCD image sensor, it is known that when a light image is incident on an image pickup unit during a vertical transfer step period in an image pickup operation, vertical stripe noise called smear noise is generated in output image information. For example, Patent Document 7 refers to smear noise. The reason for the occurrence of smear noise is that the charge generated by the photoelectric conversion by the light image incident on the imaging unit 31 is mixed with the charge generated during the exposure step period during the vertical transfer step period. As a method for reducing smear noise in a frame transfer type CCD image sensor, there is a method for suppressing the incidence of a light image on the imaging unit 31 during the vertical transfer step period.

  FIG. 11 is a schematic diagram showing an example of the configuration of a full frame transfer type CCD image sensor. The full frame transfer type CCD image sensor shown in FIG. 11 includes an imaging unit 35 including a plurality of vertical shift registers, a horizontal shift register 36, and a charge-voltage conversion circuit 37. The vertical shift register and the horizontal shift register are composed of CCD elements and have a charge transfer function. The vertical shift register of the imaging unit 32 has a photoelectric conversion function of incident light in addition to the charge transfer function.

  The imaging operation in the full frame transfer type CCD image sensor is performed through two steps of an exposure step and a transfer step. First, in the exposure step, a light image is incident on the image pickup unit 35, and charges generated according to the light intensity of the incident light are accumulated in the CCD elements constituting each pixel of the image pickup unit 35. In the subsequent transfer step, the charge is transferred from the imaging unit to the charge / voltage conversion circuit 37 through the horizontal shift register 36, and image information is output through the charge / voltage conversion circuit 37.

  In a full-frame transfer type CCD image sensor, it is known that smear noise is generated in output image information when a light image is incident on the imaging unit 35 during a transfer step period in an imaging operation. The reason for the occurrence of smear noise is that charges generated by photoelectric conversion by the light image incident on the imaging unit 35 are mixed into charges generated during the exposure step period during the transfer step period. As a method for reducing smear noise in a full-frame transfer type CCD image sensor, there is a method for suppressing the incidence of a light image on the imaging unit 35 during the transfer step period.

  FIG. 12 is a schematic diagram showing an example of the configuration of an interline transfer type CCD image sensor. The interline transfer type CCD image sensor shown in FIG. 12 includes an imaging unit 40 including a plurality of photodiodes 38, a plurality of vertical shift registers 39, a horizontal shift register 41, and a charge-voltage conversion circuit 42. Details of the configuration of the interline transfer type CCD image sensor are described in, for example, Patent Document 8. The photodiode 38 has a photoelectric conversion function of incident light. The vertical shift register 39 and the horizontal shift register 41 are composed of CCD elements and have a charge transfer function. The vertical shift register 39 is configured to be masked with an aluminum light-shielding film or the like in order to prevent charge accumulation due to photoelectric conversion.

  The imaging operation in the interline transfer type CCD image sensor is performed through an exposure step, a charge transfer step from the photodiode 38 to the vertical shift register 39, and a transfer step. First, in the exposure step, a light image is incident on the imaging unit 40, and charges generated according to the light intensity of the incident light are accumulated in the photodiodes 38 constituting each pixel of the imaging unit. In the subsequent charge transfer step from the photodiode 38 to the vertical shift register 39, the charge accumulated in the photodiode 38 is moved to the CCD element on the adjacent vertical shift register. In the subsequent transfer step, the charge is transferred from the vertical shift register 39 to the charge voltage conversion circuit 42 through the horizontal shift register 41, and image information is output through the charge voltage conversion circuit 42.

  In the interline transfer type CCD image sensor, smear noise may occur when a light image is incident on the image pickup unit 40 during the transfer step period in the image pickup operation. However, in the interline transfer type CCD image sensor, since the vertical shift register 39 is masked with an aluminum light shielding film or the like, the amount of smear noise generated is smaller than that of the frame transfer type CCD image sensor or the full frame transfer type CCD image sensor. Few. However, when the incident light to the imaging unit 40 has such a light intensity that it passes through the light shielding film, smear noise is also generated in the interline transfer type CCD image sensor. In the interline transfer type CCD image sensor, as a method of reducing smear noise, there is a method of suppressing light image incidence on the imaging unit 37 during the transfer step period.

Further, not only a CCD image sensor but also an image sensor using another image sensor, when a light image is incident on an image capturing unit during a period other than the exposure step in the image capturing operation, various image information is output. It is known that there are examples with different types of noise. For example, in a CMOS image sensor, since the charge of each pixel is read in a predetermined order after the exposure is completed, a difference occurs in the read timing of each pixel, and pattern noise accompanied by luminance unevenness corresponding to the read order occurs.
Japanese Patent No. 3019754 Japanese Patent No. 3319567 Japanese Patent No. 3670839 JP 2003-43363 A JP 2006-145634 A JP 2006-227315 A Japanese Patent No. 2517258 Japanese Patent No. 2506697 JP 2008-129172 A JP 2005-338136 A

  In a multi-beam scanning confocal microscope system, in order to acquire a high-quality image, it is caused by the incident of an optical image to the imaging unit other than the exposure period, along with the banding noise caused by the mismatch between the scanning period and the exposure time. It is necessary to reduce noise (including smear noise). However, in the conventional multi-beam scanning confocal microscope system, it is only possible to reduce banding noise, and it is not possible to reduce smear noise or noise caused by light image incidence on an imaging unit other than the exposure period. There was a problem. Due to this problem, in the conventional confocal microscope system, it is impossible to acquire a high-quality image due to smear noise and image quality deterioration due to noise caused by incidence of a light image on an imaging unit other than the exposure period.

  As a countermeasure against such a problem, for example, a method of suppressing the light amount of the light image incident on the imaging unit during a period in which these noises are generated, or a light amount of illumination light in the period in which these noises are generated. It is possible to suppress it. However, the configurations of the confocal microscopes disclosed in Patent Documents 3, 5, and 6 do not include means for suppressing light image incidence on the imaging unit or suppression for adjusting the amount of illumination light. It is not a configuration that solves the problem.

  In addition, the configuration of the confocal microscope disclosed in Patent Document 4 includes means for adjusting the amount of illumination light, which includes a scanning period and an effective exposure period (in the exposure step period of the image sensor, and the optical image is It is a configuration aimed at preferably realizing synchronization with the incident period) and easily changing the light quantity dynamic range. Therefore, the configuration is not intended to solve the above problems, and does not have an effect of solving the above problems. Actually, in the first and second embodiments described in Patent Document 4, the exposure operation and the transfer operation of the CCD image sensor are performed simultaneously, and the light source is turned on during this period. As a result, a light image is incident on the light source, resulting in a smear noise. In addition, if rotation unevenness occurs in the disk due to disk eccentricity or motor shaft friction fluctuation, the information cannot be fed back anywhere, so the timing of turning off the light source exceeds the exposure end timing of the CCD image sensor. Thus, there is a problem that both banding noise and smear noise are generated.

  An object of the present invention is to provide noise (including smear noise) caused by an optical image entering the image sensor imaging unit during a period other than the exposure period of the image sensor, and banding caused by a mismatch between the scanning period and the exposure time. An object of the present invention is to provide a multi-beam scanning confocal microscope system that eliminates both noises and enables high-quality image acquisition.

  In order to achieve the above object, the confocal microscope system according to claim 1 uses an optical scanning unit having a mask pattern member on which a predetermined pattern is formed, and scans illumination light from the illuminating unit onto the sample. In the confocal microscope system configured to be able to observe the confocal image of the sample, the imaging unit that captures the confocal image and the scanning period of the optical scanning unit are detected, and the scanning period corresponds to A scanning signal supply unit that outputs a scanning signal to be output, and a synchronization control unit that generates and outputs an imaging control signal and a light amount control signal in synchronization with the scanning signal. The imaging operation of the imaging unit is performed by the imaging control signal. And the amount of light incident on the imaging means is controlled by the light amount control signal.

  A confocal microscope system according to a second aspect of the present invention is the confocal microscope system according to the first aspect, further comprising illumination light adjusting means for adjusting the light intensity of the illumination light, and the light amount control signal includes the illumination light adjusting means. It is characterized by control.

  The confocal microscope system according to claim 3 is the confocal microscope system according to claim 1, further comprising return light adjusting means for adjusting the light intensity of the return light from the sample, wherein the light quantity control signal is the return light. It is characterized by controlling the adjusting means.

  The confocal microscope system according to claim 4 is the confocal microscope system according to any one of claims 1 to 3, wherein the optical scanning unit moves or rotates the mask pattern member to thereby form the pattern. The illumination light that has passed through is scanned.

  The confocal microscope system according to claim 5 is the confocal microscope system according to any one of claims 1 to 3, wherein the light scanning unit includes the fixed mask pattern member and a light turning unit. And the illumination light that has passed through the pattern is scanned by the light redirecting means.

  The confocal microscope system according to claim 6 is the confocal microscope system according to any one of claims 1 to 5, wherein the imaging means is an imaging means using a frame transfer type CCD image sensor, and the frame At least in the vertical transfer step period of the transfer type CCD image sensor, the light amount control signal for suppressing the amount of light incident on the image pickup means is generated and output by the synchronization control means.

  The confocal microscope system according to claim 7 is the confocal microscope system according to any one of claims 1 to 5, wherein the imaging unit is an imaging unit using a full frame transfer type CCD image sensor, In the full frame transfer type CCD image sensor, at least during the transfer step period, the light amount control signal for suppressing the amount of light incident on the image pickup means is generated and output by the synchronization control means.

  The confocal microscope system according to claim 8 is the confocal microscope system according to any one of claims 1 to 5, wherein the imaging unit is an imaging unit using an interline transfer type CCD image sensor, At least in the transfer step period of the interline transfer type CCD image sensor, the light amount control signal for suppressing the amount of light incident on the image pickup means is generated and outputted by the synchronization control means.

  The confocal microscope system according to claim 9 is the confocal microscope system according to any one of claims 1 to 5, wherein the imaging unit is an imaging unit using a two-dimensional image sensor, and the two-dimensional image In the period other than the exposure step period of the sensor, the synchronization control unit generates and outputs the light amount control signal for suppressing the amount of incident light on the imaging unit.

  According to the confocal microscope system of the present invention, noise (including smear noise) caused by the light image entering the image sensor imaging unit during a period other than the exposure period of the image sensor, and the scanning period and the exposure time. It is possible to acquire a high-quality image that eliminates both the banding noise caused by the mismatch.

  Below, with reference to FIGS. 1-9, embodiment of the confocal microscope system which concerns on this invention is described. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. Further, the dimensional ratios in the drawings do not necessarily match those described.

  Hereinafter, Example 1 of the confocal microscope system according to the present invention will be described in detail.

  FIG. 1 is a diagram showing a schematic configuration of a confocal microscope system according to the present embodiment. FIG. 2 is a schematic diagram showing a configuration of a mask pattern formed on the Niipou disc 5 used as an optical scanning unit according to the present embodiment.

  The confocal microscope system shown in FIG. 1 captures a confocal image of the sample 9 by using a confocal optical system. The light source device 1 functions as an illuminating unit, and the light control functions as an illuminating light adjusting unit. Device 2, collimating lens 3, half mirror 4, Nipkow disk 5, motor 6, motor control device 7, objective lens 8, imaging lens 10, imaging device 11 that functions as imaging means, and photosensor that functions as scanning signal supply means 12, a synchronization control device 13 functioning as a motivation control means, and a computer 14 are provided.

  The light source device 1 includes a solid-state laser light source 1a that emits illumination light. However, the illumination means is not limited to the above configuration. As an example for implementing the illumination means, for example, a gas laser, a semiconductor laser, a discharge tube, or a light emitting diode can be used as a light source.

  The light control device 2 includes an acousto-optic modulator 2a, and adjusts the amount of illumination light introduced from the light source device 1 according to the light amount control signal 13b from the synchronization control device 13 and emits it. However, the illumination light adjusting means is not limited to the above configuration. As an example of implementing the illumination light adjusting means, an electric shutter device or a mechanical shutter device can be used. An example of the electrical shutter device is a liquid crystal shutter device.

  As optical scanning means, a Nipkow disk 5, a motor 6 for rotating the Nipkow disk 5, and a motor control device 7 for controlling the rotational speed of the motor 6 to a predetermined value are provided. The motor control device 7 receives the rotational speed command signal 14b from the computer 14 and controls the rotational speed of the motor 6 to a predetermined value. The rotation speed command value can arbitrarily specify a range from 1500 revolutions per minute to 10,000 revolutions per minute, but is not particularly limited to this range.

  The nipou disk 5 is produced by vapor-depositing chromium on a glass substrate to form a predetermined mask pattern. In the configuration of the mask pattern formed on the Nipkow disk 5 schematically shown in FIG. 2, a large number of pinhole patterns that transmit light are predetermined on the eight spirals on the inner periphery of the mask pattern. The pinhole part 51 arrange | positioned by this arrangement | sequence is provided. However, the number of spirals is not limited to this, and may be one, for example. On the outer periphery of the mask pattern, there are provided slit portions 52 in which a slit pattern equal to the number of the helical arrays is formed with reference to the position corresponding to the start point of each helical array of the pinhole portion 51.

  However, the optical scanning unit is not limited to the above-described configuration, and any optical scanning unit may be used as long as it includes a mask pattern member and a scanning unit for periodically scanning a sample with light that has passed through a predetermined pattern. As the mask pattern member, for example, a glass substrate member in which a pinhole pattern or a slit pattern is formed by mask vapor deposition at a predetermined position can be used. Moreover, the structure provided with the micro lens member which formed the micro lens in the position corresponding to a pin hole with the mask pattern member in which the pin hole pattern was formed can be used. The micro lens member and the mask pattern member in which the pinhole is formed are configured such that the corresponding pinhole is located at the focal position of the microlens where the illumination light passing through each microlens is collected. As the scanning means, for example, a motor or a galvano scanner can be used.

  The photosensor 12 functioning as a scanning signal supply means includes an infrared light emitting diode (not shown), an infrared phototransistor (not shown), and a scanning signal generation circuit (not shown), and is arranged at a position sandwiching the slit portion 52 of the Nipkow disk 5. Yes. The photosensor 12 outputs the scanning signal 12a generated by the scanning signal generation circuit. However, the configuration and arrangement of the scanning signal supply means are not limited to the above as long as the scanning signal 12a corresponding to the scanning period of the optical scanning means is output. Other implementation methods include, for example, a method in which a rotary encoder is attached to the rotating shaft of the optical scanning means or the rotating shaft of the motor to convert the encoder signal into a scanning signal, or a magnetic sensor such as a Hall sensor is provided. A method of converting a rotation angle signal into a scanning signal using a motor capable of outputting a rotation angle signal of a rotor, or a method of obtaining a motor driving cycle signal from a motor driving circuit and converting the motor driving cycle signal into a scanning signal. Can be used.

  The imaging device 11 includes an imaging sensor 11a using a frame transfer type CCD image sensor and an imaging control unit 11b. The imaging control unit 11b is based on an imaging control signal 13a input from the synchronization control device 13, and the imaging sensor The imaging operation of 11a is controlled. The imaging control unit 11b performs exposure while the imaging control signal 13a is at a high level. That is, exposure starts when the imaging control signal 13a becomes high level, and exposure ends when it becomes low level. After the exposure is completed, the image sensor 11a is controlled so that the vertical transfer operation and the horizontal transfer operation are sequentially performed. The captured image information is transmitted to the computer 14, recorded in a storage device (not shown), and / or displayed on a monitor device (not shown).

  The synchronization control device 13 includes a micro controller (not shown). The synchronization operation control signal 14a from the computer 14 and the scanning signal 12a from the photosensor 12 are input signals, and the imaging control signal 13a and the light amount control signal 13b are output. To do. The imaging control signal 13a is input to the imaging device 11 and controls the imaging operation of the imaging device 11. The light amount control signal 13b is input to the light control device 2 to adjust the amount of light emitted from the light control device 2.

  Next, the operation of the confocal microscope system configured as described above will be described.

  3 to 4 are flowcharts for explaining the operation of the synchronization control device 13. 5 shows a scanning signal 12a, an imaging control signal 13a, a light amount control signal 13b, an exposure operation of the frame transfer type CCD image sensor 11a, a vertical transfer operation of the image sensor 11a, a horizontal transfer operation of the image sensor 11a, and an adjustment. 3 is a time chart showing the amount of illumination light emitted from the optical device 2; However, in the flowcharts of FIGS. 3 to 4 and the time chart of FIG. 5, only the part related to the imaging operation, which is the main operation of the confocal microscope system, is described, and the parts related to other operations are omitted.

  When the motor control device 7 receives the rotational speed command signal 14b from the computer 14, the motor control apparatus 7 drives the motor 6 to rotate at the designated rotational speed, and the Nipkow disk 5 rotates accordingly. The spiral array of the Nipkow disk 5 is an array that can scan the sample uniformly by a pinhole group on one spiral. Therefore, the sample scanning is performed a number of times equal to the number of spirals (eight) by rotating the Nipkow disk 5 once.

  A scanning signal 12 a is output from the photosensor 12 along with the rotation of the Nipkow disk 5. Since the slit pattern 52 equal to the number of spiral arrangements is formed in the slit portion 52 of the nipou disk 5, the cycle of the scanning signal 12a coincides with the scanning cycle of the nipou disk 5.

  When the synchronous control device 13 receives the synchronous operation control signal 14a from the computer 14 (step S1 in FIG. 3; Yes), it reads the scanning frequency parameter from the synchronous operation control signal 24, sets internal variables, and performs the synchronous imaging operation. It is validated (steps S2, S3).

  When the synchronous control device 13 detects the rising edge of the scanning signal 12a immediately after the synchronous imaging operation is validated (step S11; No, step S12; Yes), it performs an exposure start process (step S13). In the exposure start process, the imaging control signal 13a and the light amount control signal 13b are simultaneously set to the high level, and the exposure operation and illumination light irradiation are started simultaneously (steps S21 and S22 in FIG. 4).

  Each time the synchronization control device 13 detects the rising edge of the scanning signal 12a, the scanning number counter is incremented (step S14), and the exposure is continued until the count number reaches the designated number (step S15; Yes).

  The synchronization control device 13 performs an exposure end process (step S16) after detecting the rising edge of the scanning signal 12a for the number of times indicated by the scanning number parameter (step S15; Yes). In the exposure end process, the imaging control signal 13a and the light amount control signal 13b are simultaneously set to the low level, and the exposure operation and illumination light irradiation are simultaneously ended (steps S31 to S34 in FIG. 4).

  In the frame transfer type CCD image sensor 11a of the imaging device 11, a vertical transfer operation and a horizontal transfer operation are sequentially performed immediately after the exposure operation is completed. The synchronous control device 13 waits for a predetermined time after executing the exposure end processing so that the next exposure operation is not started during the period during which these transfers are performed (step S41 in FIG. 4). When the standby time has elapsed, the immediately following rise of the scanning signal 12a is detected (step S11; No, step S12; Yes), and the exposure start process (step S13) is performed again.

  By repeating the above operation, the continuous synchronous imaging operation shown in the time chart of FIG. 5 is performed.

  As described above, in this embodiment, illumination light irradiation is performed only during the exposure step period of the imaging device 11, thereby avoiding the incidence of a light image on the imaging unit during the vertical transfer step period of the frame transfer type CCD image sensor 11a. Therefore, it is possible to acquire an image without image quality deterioration due to smear noise.

  In the present embodiment, the exposure period of the imaging device 11 is an integral multiple of the period of the scanning signal 12a. Therefore, since the exposure period is an integral multiple of the minimum time during which the illumination light scans the sample 9 uniformly, an image without image quality degradation due to banding noise can be acquired.

  Therefore, this embodiment can acquire a high-quality image that eliminates both image quality deterioration due to banding noise and smear noise, and achieves the object of the present invention.

  In addition, this embodiment has a feature that the dimming device 2 uses the acousto-optic modulator 2a, thereby realizing the effect of completely preventing the occurrence of smear noise. In general, in a frame transfer type CCD image sensor, the time required for the vertical transfer step is about 0.1 milliseconds to several milliseconds, and in order to reliably prevent the incidence of a light image during this period, a microsecond is required. It is necessary to adjust the amount of incident light in order. In general, an acousto-optic modulator has a characteristic that it has a very high-speed response characteristic on the order of several tens of nanoseconds, and has sufficient performance to completely prevent smear noise in this embodiment. Therefore, this embodiment realizes an effect of completely preventing the occurrence of smear noise.

  Further, in observation / measurement using a confocal microscope, unintentional alteration of the sample 9 due to illumination light may be a problem. For example, when a living cell in which a fluorescent protein is expressed, a fixed cell, or a living tissue is used as a sample, and illumination light is irradiated to obtain a confocal image of fluorescence emitted from the sample, fluorescence is emitted by the illumination light. There is a problem that the protein is faded and it is difficult to obtain an image, and a cell is damaged and killed due to phototoxicity of illumination light. Since such alteration depends on the illumination time of illumination light, a countermeasure against this problem is a method of avoiding unnecessary illumination light irradiation other than the exposure period. In the present embodiment, illumination light irradiation is performed only during a period equal to the exposure period, and illumination light irradiation is not performed during other periods, thereby realizing the effect of minimizing the deterioration of the sample 9 due to illumination light irradiation.

  Hereinafter, the configuration of the confocal microscope system according to the second embodiment will be described. FIG. 6 is a diagram illustrating a schematic configuration of the confocal microscope system according to the present embodiment. The same parts as those in FIG. 1 are denoted by the same reference numerals, and detailed description thereof is omitted.

  As shown in FIG. 6, in the confocal microscope system of the present embodiment, the illumination unit and the illumination light adjustment are performed by directly controlling the output of the light source device 1 without using the light control device as the illumination light adjustment unit. A function as a means is realized in the light source device 1. Moreover, 11 A of imaging devices provided with a two-dimensional image sensor are used.

  The light source device 1 includes a solid-state laser light source 1a that emits illumination light. However, the illumination means is not limited to the above configuration. As an example for implementing the illumination means, for example, a gas laser, a semiconductor laser, a discharge tube, or a light emitting diode can be used as a light source.

  The imaging device 11A includes an imaging sensor 11d using a two-dimensional image sensor and an imaging control unit 11e, and controls the imaging operation of the imaging sensor 11d by an imaging control signal 13a input to the imaging device 11A. The imaging control unit 11e controls the imaging sensor 11d to perform exposure while the imaging control signal 13a is at a high level, end exposure at the same time as the level becomes low, and start a transfer operation. However, the imaging unit is not limited to the above configuration, and any imaging unit and imaging control unit may be used. As the imaging sensor, for example, an interline transfer type CCD image sensor, a full frame transfer type CCD image sensor, a CMOS image sensor, or the like can be used.

  The synchronization control device 13 is the same as that in the first embodiment. However, the light amount control signal 13b output from the synchronization control device 13 is input to the light source device 1 to adjust the amount of light emitted from the light source device 1.

  3 to 4 are flowcharts for explaining the operation of the synchronization control device 13. FIG. 7 is a time chart of the scanning signal 12a, the imaging control signal 13a, the light amount control signal 13b, the exposure operation of the two-dimensional image sensor, the transfer operation of the image sensor, and the amount of light emitted from the light source device 1 according to the present embodiment. It is. In the time chart of FIG. 7, only the part related to the imaging operation, which is the main operation of the confocal microscope system, is shown, and the part related to the other operations is omitted.

  In the two-dimensional image sensor 11d of the imaging device 11A, a transfer operation is performed immediately after the exposure operation is completed. In order to prevent the next exposure operation from starting during the transfer period, the synchronization control device 13 waits for a predetermined time after executing the exposure end process. When the standby time elapses, the immediately following rise of the scanning signal 12a is detected, and the exposure start process is performed again.

  In the confocal microscope system of the present embodiment, by directly controlling the light source device 1, illumination light irradiation is performed only during the exposure period of the imaging device 11 </ b> A, so that the two-dimensional image sensor is transferred to the imaging unit during the transfer step period. Avoid light image incidence. For this reason, it is possible to acquire an image without image quality deterioration due to noise caused by the incidence of the light image on the imaging unit other than during exposure.

  In the present embodiment, the exposure period of the imaging device 11A is an integral multiple of the scanning signal cycle. Therefore, since the exposure period is an integral multiple of the minimum time during which the illumination light scans the sample 9 uniformly, an image without image quality degradation due to banding noise can be acquired. Therefore, this embodiment can acquire a high-quality image that eliminates both image quality deterioration due to banding noise and smear noise, and achieves the object of the present invention.

  Further, in this embodiment, illumination light irradiation is performed only during a period equal to the exposure period, and illumination light irradiation is not performed during other periods, thereby realizing an effect of minimizing sample deterioration due to illumination light irradiation.

  In addition, since the light source device 1 controls the output of the light source and adjusts the amount of illumination light in the light source device 1, the present embodiment does not require illumination light adjusting means, so that the present invention is inexpensive and simple. To achieve this goal.

  Hereinafter, the configuration of the confocal microscope system according to the third embodiment will be described.

  FIG. 8 is a diagram illustrating a schematic configuration of the confocal microscope system according to the present embodiment. The same parts as those in FIG. 1 are denoted by the same reference numerals, and detailed description thereof is omitted.

  The light source device 1 includes a solid-state laser light source 1a and emits illumination light. However, the illumination means is not limited to the above configuration. As an example of implementing the illumination means, for example, a gas laser, a semiconductor laser, a discharge tube, or a light emitting diode can be used.

  The imaging device 11B includes an imaging sensor 11f using a frame transfer CCD image sensor and an imaging control unit 11g, and controls an imaging operation of the imaging sensor 11f by an imaging control signal 13a input to the imaging device 11B. .

  In this embodiment, a liquid crystal shutter 15 is provided as return light adjusting means for controlling the amount of light incident on the imaging device 11B by adjusting the return light, and the light amount of the return light from the sample 9 according to the light amount control signal 13b. Is adjusted and introduced into the image pickup apparatus 11B. However, the return light adjusting means is not limited to the above configuration. For example, an electric shutter device or a mechanical shutter device can be used. An example of the electrical shutter device is an acousto-optic modulator.

  FIG. 9 shows a scanning signal 12a, an imaging control signal 13a, a light amount control signal 13b, an exposure operation of a frame transfer type CCD image sensor, a vertical transfer operation of the image sensor, a horizontal transfer operation of the image sensor, and 4 is a time chart of the amount of light transmitted through the liquid crystal shutter 15 that is transmitted back. In the time chart of FIG. 9, only the part related to the imaging operation, which is the main operation of the confocal microscope system, is shown, and the part related to the other operations is omitted.

  When receiving the synchronous operation control signal 14a from the computer 14, the synchronous control device 13 reads the scanning frequency parameter from the synchronous operation control signal 24, sets internal variables, and validates the synchronous imaging operation. When the synchronous control device 13 detects the rising edge of the scanning signal 12a immediately after the synchronous imaging operation is validated, the synchronous control device 13 performs an exposure start process. In the exposure start process, the imaging control signal 13a and the light amount control signal 13b are simultaneously set to the high level, and at the same time as the exposure operation is started, the transmitted light amount of the liquid crystal shutter 15 is maximized.

  Subsequently, the synchronization control device 13 performs an exposure end process after detecting the rising edge of the scanning signal 12a for the number of times indicated by the scanning number parameter. In the exposure end process, the imaging control signal 13a and the light amount control signal 13b are simultaneously set to the low level, and the transmitted light amount of the liquid crystal shutter 15 is set to 0 simultaneously with the end of the exposure operation.

  As described above, in this embodiment, the light image is incident on the image capturing unit only during the exposure period of the image capturing apparatus 11B, so that the light image is incident on the image capturing unit in the vertical transfer step period of the frame transfer type CCD image sensor. Since this can be avoided, it is possible to acquire an image without image quality deterioration due to smear noise.

  In the present embodiment, the exposure period of the imaging device 11B is an integral multiple of the period of the scanning signal. Therefore, since the exposure period is an integral multiple of the minimum time during which the illumination light scans the sample 9 uniformly, an image without image quality degradation due to banding noise can be acquired. Therefore, this embodiment can acquire a high-quality image that eliminates both image quality degradation due to banding noise and smear noise, and achieves the object of the present invention.

  In the first to third embodiments, the number of scans of the sample can be arbitrarily designated by the scan number parameter indicated in the synchronous operation control signal 14a. Furthermore, the rotational speed command signal 14b can arbitrarily specify the rotational speed of the motor 6 within a predetermined range, and can thereby arbitrarily specify the scanning period of the Nipo disk 5. With the above two actions, according to the first to third embodiments, any exposure time can be set, and imaging is performed under the most suitable conditions depending on the type or state of the sample 9. The effect of being able to be realized. It is also conceivable to realize an effect of performing imaging under more favorable conditions by making it possible to arbitrarily adjust the illumination light intensity during illumination light irradiation by the light quantity control signal 13b.

  In the first to third embodiments, even if rotation unevenness occurs in the Nipkow disk 5 due to eccentricity of the Nipkow disk 5 or frictional fluctuation of the shaft of the motor 6, it is synchronized with the scan cycle of the Nipkow disk 5. Thus, an imaging operation and a light image incident on the imaging unit are performed. Therefore, even if rotation unevenness occurs, smear noise and banding noise do not occur, so that an effect of minimizing image degradation is realized.

  In the first to third embodiments, it is also possible to use the encoder pattern of the pinhole disk of the confocal scanner unit disclosed in Patent Document 9 in the slit portion 52 of the nipou disk 5. This encoder pattern is characterized in that one encoder pattern has a circumferential width different from other encoder patterns. In this case, in the synchronous control device 13, it is possible to obtain rotation cycle information indicating a rotation reference point that exists at one location for one rotation of the Niipou disc together with the scanning cycle from the scanning signal 12a. In a confocal microscope system using a pinhole disk, a brightness fluctuation synchronized with the rotation of the disk mentioned in Patent Document 10 may rarely occur. This is because the amount of transmitted light of illumination light and return light differs depending on the position on the pinhole disk due to pinhole pattern formation error, eccentricity of the pinhole disk, and the like. In the conventional confocal microscope system, there is a problem that even if the images are captured under the same imaging conditions, the brightness varies from image to image due to this problem. This problem becomes an error factor when performing quantitative analysis of images. According to the configuration of the slit portion 52 and the synchronization control device 13 described above, it is possible to realize setting the start timing and the end timing of all exposure operations with the rotation reference point as a reference. Become. According to this, all exposures can be started at a fixed position of the rotating disk and can be ended at a fixed position. Therefore, it is possible to perform imaging and analysis in a state in which the error factor due to the luminance fluctuation is suppressed to a minimum.

  In the first to third embodiments, the objective lens moving means (not shown) and / or the sample 9 that moves the focal position of the objective lens 8 in the optical axis direction is perpendicular to the optical axis direction and / or the optical axis. A configuration including a sample moving means (not shown) that realizes movement in a smooth surface direction can be considered. In this configuration, the synchronization control device 13 drives the objective lens moving means and / or the sample moving means in synchronization with the scanning signal 12a. In this case, the objective lens moving means and / or the sample moving means are driven during a period when the exposure operation is not performed in the imaging devices 11, 11A, 11B, and the focal position of the objective lens 8 and / or the imaging position of the sample 9 is driven. It is preferable to move.

  Moreover, in the said Examples 1-3, the structure provided with the optical filter switching means which switches a plurality of optical filters and the optical filter arrange | positioned on an optical path can be considered. As the optical filter switching means, for example, an optical filter disk capable of arranging a plurality of the optical filters on the disk, a motor for rotationally driving the optical filter disk, and a motor drive circuit for driving the motor There is a filter wheel device provided. In this configuration, the synchronization control device 13 drives the optical filter switching means in synchronization with the scanning signal 12a. In this case, it is preferable that the optical filter switching unit is driven to switch the optical filter during a period when the exposure operation is not performed in the imaging apparatuses 11, 11A, and 11B.

  As described above, according to the confocal microscope system of the present invention, the imaging operation and the amount of light incident on the imaging device are controlled based on the scanning period of the optical scanning unit, so that other than the banding noise and the exposure period. Both noises (including smear noise) caused by the light image incident on the imaging device during the period can be eliminated, and a high-quality image can be acquired.

  The scope of application of the present invention is not limited to the above embodiment. The present invention is widely applied to a confocal microscope system having a predetermined mask pattern member and configured to scan a sample with illumination light through the mask pattern member and obtain a confocal image of the sample. Can be applied.

1 is a diagram illustrating a schematic configuration of a confocal microscope system according to Embodiment 1. FIG. The schematic diagram which shows an example of a structure of the mask pattern formed in the nipou disk. The flowchart explaining operation | movement of a synchronous control apparatus. The flowchart explaining operation | movement of a synchronous control apparatus. 3 is a time chart showing the operation timing of the first embodiment. FIG. 3 is a diagram illustrating a schematic configuration of a confocal microscope system according to a second embodiment. 6 is a time chart showing the operation timing of the second embodiment. FIG. 5 is a diagram illustrating a schematic configuration of a confocal microscope system according to a third embodiment. 10 is a time chart showing the operation timing of the third embodiment. FIG. 3 is a schematic diagram showing an example of the configuration of a frame transfer type CCD image sensor. FIG. 3 is a schematic diagram illustrating an example of a configuration of a full frame transfer type CCD image sensor. The schematic diagram which shows an example of a structure of an interline transfer type CCD image sensor.

Explanation of symbols

1 Light source device (illumination means)
2 Light control device (light quantity control means)
3 Collimating lens 4 Half mirror 5 Nipou disk (light scanning means)
6 Motor 7 Motor control device 8 Objective lens 9 Sample 10 Imaging lens 11 Imaging device (imaging means)
12 Photosensor (scanning signal supply means)
13 Synchronous control device (synchronous control means)
14 Computer 15 Liquid crystal shutter (return light adjusting means)
31 Frame transfer type CCD image sensor Image pickup unit 32 Frame transfer type CCD image sensor Accumulation unit 33 Frame transfer type CCD image sensor Horizontal transfer register 34 Frame transfer type CCD image sensor Charge voltage conversion circuit 35 Full frame transfer type CCD image sensor Image pickup unit 36 Full frame transfer type CCD image sensor Horizontal transfer register 37 Full frame transfer type CCD image sensor Charge voltage conversion circuit 38 Interline transfer type CCD image sensor Photodiode 38 Interline transfer type CCD image sensor Vertical transfer register 38 Interline transfer type CCD image Sensor Image pickup unit 38 Interline transfer type CCD image sensor Horizontal transfer register 38 Interline transfer type CCD image sensor Charge voltage conversion circuit 51 Nipow disc Pinhole unit 52 Nipou circle Panel Slit part

Claims (9)

A confocal microscope system configured so that a confocal image of the sample can be observed by using an optical scanning unit having a mask pattern member on which a predetermined pattern is formed and scanning the sample with illumination light from the illumination unit Because
An imaging means for imaging the confocal image;
A scanning signal supply means for detecting a scanning period of the optical scanning means and outputting a scanning signal corresponding to the scanning period;
Synchronous control means for generating and outputting an imaging control signal and a light amount control signal in synchronization with the scanning signal,
A confocal microscope system, wherein an imaging operation of the imaging unit is controlled by the imaging control signal, and an incident light amount to the imaging unit is controlled by the light amount control signal. Comprising illumination light adjusting means for adjusting the light intensity of the illumination light;
The confocal microscope system according to claim 1, wherein the light amount control signal controls the illumination light adjusting unit. A return light adjusting means for adjusting the light intensity of the return light from the sample;
The confocal microscope system according to claim 1, wherein the light amount control signal controls the return light adjusting unit. 4. The confocal microscope according to claim 1, wherein the light scanning unit scans the illumination light that has passed through the pattern by moving or rotating the mask pattern member. system. The light scanning means includes the fixed mask pattern member and light turning means,
The confocal microscope system according to any one of claims 1 to 3, wherein the illumination light that has passed through the pattern is scanned by the light redirecting means. The imaging means is an imaging means using a frame transfer type CCD image sensor,
6. The synchronization control unit generates and outputs the light amount control signal for suppressing the amount of light incident on the imaging unit at least during a vertical transfer step period of the frame transfer type CCD image sensor. The confocal microscope system according to any one of the above. The imaging means is an imaging means using a full frame transfer type CCD image sensor,
6. The synchronization control unit generates and outputs the light amount control signal for suppressing the amount of incident light to the imaging unit at least during a transfer step period of the full frame transfer type CCD image sensor. The confocal microscope system according to any one of the above. The imaging means is an imaging means using an interline transfer type CCD image sensor,
6. The synchronization control unit generates and outputs the light amount control signal for suppressing the amount of light incident on the imaging unit at least during a transfer step period of the interline transfer type CCD image sensor. The confocal microscope system according to any one of the above. The imaging means is an imaging means using a two-dimensional image sensor,
6. The light amount control signal for suppressing the amount of light incident on the imaging unit is generated and output by the synchronization control unit during a period other than the exposure step period of the two-dimensional image sensor. The confocal microscope system according to any one of the preceding claims.

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