JP5957063B2 - Microscope system - Google Patents

Description

  The present invention relates to a microscope system.

  In the confocal microscope, laser light emitted from a laser light source is condensed on a measurement object by an objective lens. The reflected light from the measurement object is collected by the light receiving lens and enters the light receiving element through the pinhole (see, for example, Patent Document 1). The laser beam is scanned two-dimensionally on the surface of the measurement object. Further, the distribution of the amount of light received by the light receiving element is changed by changing the relative distance between the measurement object and the objective lens. A peak in the amount of received light appears when the surface of the measurement object is focused. An ultra-deep image having a very high depth of focus can be obtained based on the peak intensity of the received light amount distribution. Moreover, a height image showing the height distribution of the surface of the measurement object can be obtained based on the peak position of the received light amount distribution.

JP 2008-83601A

  According to the confocal microscope, a laser beam is scanned two-dimensionally on the surface of the measurement object while changing a relative distance between the measurement object and the objective lens, thereby allowing the measurement object and the objective lens to be scanned. A plurality of confocal image data corresponding to a plurality of relative distances are generated. Ultra-depth image data or height image data is generated based on the plurality of generated confocal image data. An ultra-deep image or a height image is displayed on the display unit based on the ultra-deep image data or the height image data. The user can specify the observation range of the measurement object.

  In the confocal microscope described in Patent Literature 1, the movement range (upper limit position and lower limit position) of the objective lens in the height direction with respect to the measurement target is set by the user. In this case, the user needs to set the movement range appropriately so that the peak of the received light amount appears in the movement range. If the movement range is set wide, the probability that the peak of the received light amount appears in the movement range increases, but the amount of confocal image data to be acquired increases. On the other hand, when the movement range is set to be narrow, the amount of confocal image data acquired is reduced, but the probability that the peak of the received light amount appears in the movement range is reduced. Therefore, the user needs to appropriately set the movement range of the objective lens so that the peak of the received light amount appears in the movement range and the amount of confocal image data acquired is as small as possible.

  When the user specifies a wider range than the observable area of the confocal microscope, the ultra-depth image data or height image data of multiple areas of the measurement object is moved by moving the stage of the confocal microscope. Each is generated. Thereafter, the ultra-depth image data or height image data of a plurality of regions are connected to display a wide-range ultra-depth image or height image of the measurement object on the display unit using the work memory.

  In such a case, the surface height may be different for each region of the measurement object. Therefore, the user needs to appropriately set the movement range in the height direction of the objective lens for each region.

  The objective of this invention is providing the microscope system which can set the range of the relative distance between a measurement object and an objective lens easily and appropriately about several unit area | regions.

[A] Present Invention (1) A microscope system according to the present invention is a microscope system for measuring the state of the surface of a measurement object, and an observation range in which a plurality of unit regions are set as an observation range on the surface of the measurement object. a setting unit, a light receiving element, for each unit region set by the observation range setting unit, irradiates the light while converging to the unit region through the objective lens, receives the light irradiated to the unit area an optical system for guiding the element, for each unit region set by the observation range setting unit, so that the irradiation of light is performed by optical science system at a plurality of positions along the direction perpendicular to the unit area, per unit area The moving unit moves the focal point of the optical system to a plurality of positions in the optical axis direction of the optical system by moving the objective lens relative to the measurement object in the optical axis direction of the optical system from the initial position determined by And the light receiving element A pixel data output unit that outputs a plurality of pixel data respectively corresponding to a plurality of pixels based on the output signal; and a control unit that controls the light receiving element and the moving unit . The control unit is set by the observation range setting unit A first process for adjusting the gain of the light receiving element based on the pixel data output from the pixel data output unit in a state where the objective lens is in the initial position determined for the unit area for each unit area; The objective lens is moved by the moving unit in the optical axis direction from the initial position determined for the unit area, and a predetermined number of pixels among a plurality of pixel data output from the pixel data output unit are supported at each position in the optical axis direction A second process of calculating an evaluation value using the pixel data value to be set, and automatically setting an upper limit position and a lower limit position in the optical axis direction based on the calculated evaluation value; Based on a plurality of pixel data output from the pixel data output unit at each position in the optical axis direction by moving the objective lens within the moving range determined by the upper limit position and lower limit position set by the And a third process for generating, as height image data, data representing peak positions in the optical axis direction of a plurality of pixel data for a plurality of pixels corresponding to the region, and a plurality of data set by the observation range setting unit A concatenation process for concatenating a plurality of height image data generated by repeatedly executing the first process, the second process, and the third process for the unit area is executed .

  (2) The control unit automatically executes processing for generating ultra-depth image data based on the plurality of pixel data for each of the plurality of unit regions set by the observation range setting unit, and generates each of the plurality of unit regions. The plurality of ultra-deep image data may be connected, and the ultra-depth image data may be data representing the peak intensity in the optical axis direction of the plurality of pixel data for the plurality of pixels corresponding to each unit region.

(3) When the control unit moves the objective lens to a plurality of positions in the optical axis direction in the second process, the value of the pixel data output from the pixel data output unit is greater than a predetermined output upper limit value. You may reduce the gain of a light receiving element so that it may become small.

(4) the second processing, the output from the pixel data output unit at each position of the objective lens while moving the objective lens in the direction and away from the direction toward the objective lens is the observation object from the initial position determined for each unit area In the second process for each unit region, the control unit moves the objective lens in a direction in which the objective lens and the observation target object approach each other from the determined initial position. When the level of the output signal of the light receiving element changes from a state higher than a predetermined level to a level equal to or lower than the predetermined level based on the acquired pixel data in detecting the position of the objective lens as the first relative position, the objective lens is moved from the determined initial position in a direction away the objective lens and the observation object If you are, based on the acquired pixel data, at the time the level of the output signal changes below a predetermined level at which the level of the light receiving element from the state higher than the predetermined level of the output signal of the light receiving element The position of the objective lens may be detected as the second relative position, and the movement range for the unit area may be set based on the detected first and second relative positions.

(5) the second processing, the output from the pixel data output unit at each position of the objective lens while moving the objective lens in the direction and away from the direction toward the objective lens is the observation object from the initial position determined for each unit area In the second process for each unit region, the control unit includes a process for acquiring a plurality of pixel data, and the relative distance between the objective lens and the observation object is determined from the determined initial position in the first process. The objective lens is moved relative to the object to be observed over a predetermined range from the distance to the second distance that is larger than the first distance, and based on the acquired pixel data, a plurality of objective lenses in a plurality of time varying from greater than the level that the level of the output signal reaches a predetermined state below the level of level predetermined output signal of the light receiving element Position detecting respectively, may be set movement range of the unit region so as to include the detected plurality of positions.

(6) In the second process for each unit region, the control unit determines whether the level of the output signal of the light receiving element is higher than a predetermined level based on the acquired pixel data, and receives light When the level of the output signal of the element is larger than a predetermined level, the objective lens is moved relative to the observation object by the first movement amount, and the level of the output signal of the light receiving element is predetermined. When the level is equal to or lower than the level, the objective lens may be moved relative to the observation object by the second movement amount, and the second movement amount may be larger than the first movement amount.

(7) In the second process for each unit region, the control unit moves the objective lens relative to the observation object over a predetermined range from the first distance to the second distance. If the moving range is not set even if the distance is set, the relative distance between the objective lens and the observation object from the determined initial position is larger than the second distance from the third distance smaller than the first distance. The objective lens is moved relative to the observation object over a predetermined range up to a distance of 4, and the level of the output signal of the light receiving element is determined based on the acquired pixel data. to include a plurality of positions respectively detected, it is detected a plurality of positions of the objective lens at a plurality of time varying below the level of level predetermined output signal of the light receiving element from higher than It may be set moving range of the unit region.

[B] Reference Form (1) A microscope system according to a first reference form is a microscope system for observing the state of the surface of an observation object, and includes a light receiving element and a unit region set on the surface of the observation object. The optical system irradiates the light while condensing the light on the unit area and guides the light irradiated to the unit area to the light receiving element, and the light by the optical system in the unit area at a plurality of positions along the direction perpendicular to the unit area A relative movement unit that relatively moves the optical system and the observation target so that irradiation is performed, an observation range setting unit that sets a plurality of unit areas as an observation range, and a plurality of sets set by the observation range setting unit For each of the unit areas, a movement range setting process for setting a relative movement range between the optical system and the observation object is performed, and the movement range setting process is set by controlling the optical system and the relative movement unit. Within moving range And a control unit that performs a measurement process of acquiring a plurality of pixel data for the unit region based on an output signal of the light receiving element while relatively moving the optical system and the observation target, and the observation range setting unit The plurality of unit areas are set so that overlapping portions are formed between adjacent unit areas, and the control unit sets the relative initial values of the optical system and the observation object in the movement range setting process for each unit area. The position is determined, and the optical system and the observation target are relatively moved in the direction in which the optical system and the observation target are approached and moved away from the determined initial position, and a plurality of pixel data are acquired and acquired. A moving range is set based on pixel data, and for each unit area other than the first unit area, an overlapping portion of the unit area of the pixel data acquired during measurement processing of other adjacent unit areas Based on the pixel data for those that determine the relative initial position of the optical system and the observation object such that the focal point of the optical system is aligned to at least a portion of the unit region.

  In this microscope system, a plurality of unit areas are set as the observation range. At this time, an overlapping portion is formed between adjacent unit regions. For each of the plurality of unit areas, a relative movement range between the optical system and the observation object is set by the movement range setting process, and the optical system and the observation object are within the movement range set in the unit area by the measurement process. Are moved relative to each other, and a plurality of pixel data for the unit area is acquired based on the output signal of the light receiving element.

  In the movement range setting process for each unit region, a relative initial position between the optical system and the observation object is determined. The optical system and the observation object are relatively moved in a direction in which the optical system and the observation object approach and move away from the determined initial position, and a plurality of pixel data are acquired. Based on the plurality of pixel data, a relative movement range between the optical system and the observation object is set for the unit region.

  For each unit area other than the first unit area, at least a part of the unit area based on the pixel data of the overlapping part of the unit area among the pixel data acquired during the measurement process of the other adjacent unit areas The relative initial position of the optical system and the observation object is determined so that the optical system is in focus.

  Accordingly, light is guided from at least a part of the unit region to the light receiving element in a state where the optical system and the observation target are at the initial positions. By moving the optical system and the observation object relative to each other in a direction in which the optical system and the observation object approach and move away from the determined initial position, the focus of the optical system is focused on at least a part of the unit region. It is possible to detect a relative position between the optical system and the observation object when the optical system is out of focus on the unit region from the state of being matched. Based on the detected position, the relative movement range between the optical system and the observation object can be appropriately set for the unit region.

  As described above, for each unit area other than the first unit area, the initial position can be determined based on the pixel data already acquired for the overlapping portion in the movement range setting process for the unit area. Therefore, it is not necessary to acquire pixel data in order to determine the initial position. Therefore, the initial position can be set in a short time. As a result, a plurality of pixel data for a plurality of unit regions can be efficiently acquired in a short time.

  (2) In the movement range setting process for each unit region, the control unit relatively moves the optical system and the observation object from the determined initial position in a direction in which the optical system and the observation object approach and away from each other. And a plurality of pixel data is acquired, and the level of the output signal of the light receiving element is lower than a predetermined level from a state where the level of the output signal of the light receiving element is higher than a predetermined level based on the acquired pixel data Detect the relative position between the optical system and the observation object at the time of changing to, set the movement range for the unit area based on the detected relative position, and each unit area except the first unit area In the case where the relative position is not detected, the pixel for the overlapping portion of the unit area among the pixel data acquired during the measurement processing of the other adjacent unit area Based on the data, the range of movement of the unit region so as to include a plurality of relative positions of the optical system and the observation object when the optical system is focused on each of the overlapping portions of the unit region. May be set.

  In the movement range setting process for each unit region, at the initial position, the optical system is focused on at least a part of the unit region. In this case, the level of the output signal of the light receiving element is high. On the other hand, when the optical system and the observation object are relatively moved in the direction in which the optical system and the observation object are approached and moved away from the determined initial position, the light is received as the focus of the optical system is separated from the unit area. The level of the output signal of the element is lowered.

  Therefore, by detecting the relative position between the optical system and the observation object at the time when the level of the output signal of the light receiving element changes from a state higher than the predetermined level to a predetermined level or less, the unit The moving range can be set to a minimum for the area. Thereby, the time for the measurement process is shortened.

  In addition, even when the relative position between the optical system and the observation target is not detected, the relative movement range between the optical system and the observation target is set, so that the measurement processing of the unit area can be performed. It becomes.

  (3) In the moving range setting process for each unit region, the control unit relatively moves the optical system and the observation object in a direction in which the optical system and the observation object approach from the determined initial position. In this case, the optical at the time when the level of the output signal of the light receiving element changes below a predetermined level from a state where the level of the output signal of the light receiving element is higher than a predetermined level based on the acquired pixel data. The relative position between the system and the observation object is detected as the first relative position, and the optical system and the observation object move relatively in a direction in which the optical system and the observation object move away from the determined initial position. In this case, based on the acquired pixel data, the level of the output signal of the light receiving element is changed from a level higher than a predetermined level to a level equal to or lower than the predetermined level. Detecting the relative position between the optical system and the observation object at the time of the second relative position, and setting a movement range for the unit region based on the detected first and second relative positions. Also good.

  In this case, the level of the output signal of the light receiving element is determined in advance when the optical system and the observation object are relatively moving in the direction in which the optical system and the observation object approach from the determined initial position. The first relative position is detected when the level of the output signal of the light receiving element changes below a predetermined level from a state higher than the level.

  In addition, when the optical system and the observation object are relatively moved in the direction in which the optical system and the observation object are moved away from the determined initial position, the level of the output signal of the light receiving element is determined in advance. The second relative position is detected when the level of the output signal of the light receiving element changes below a predetermined level from a higher state. Based on the detected first and second relative positions, a movement range for the unit area is set. Thereby, the time for the movement range setting process can be sufficiently shortened.

  (4) In the movement range setting process for each unit region, the control unit determines that the relative distance between the optical system and the observation object from the determined initial position is greater than the first distance from the first distance. The optical system and the observation object are relatively moved over a predetermined range up to a distance of 2, and based on the acquired pixel data, the level of the output signal of the light receiving element is higher than the predetermined level. Detecting a plurality of relative positions of the optical system and the observation object at a plurality of time points when the level of the output signal of the light receiving element changes from a high level to a predetermined level or less. The movement range for the unit region may be set so as to include

  In this case, in the movement range setting process for each unit area, the optical distance is set until the relative distance between the optical system and the observation object from the initial position becomes a second distance that is larger than the first distance from the first distance. The system and the observation object move relatively. A plurality of relative positions of the optical system and the observation object are detected at a plurality of time points when the level of the output signal of the light receiving element changes from a state higher than a predetermined level to a predetermined level or less. . The movement range for the unit area is set so as to include a plurality of detected positions.

  As a result, the optical system and the observation target at the time when the level of the output signal of the light receiving element becomes higher than a predetermined level while the optical system and the observation target move from the first distance to the second distance. The movement range can be set so that the relative distance to the object is included reliably. Therefore, measurement processing can be performed on the entire surface of the unit region regardless of the state of the surface of the observation object.

  (5) In the movement range setting process for each unit region, the control unit determines whether or not the level of the output signal of the light receiving element is higher than a predetermined level based on the acquired pixel data, and receives light When the level of the output signal of the element is larger than a predetermined level, the optical system and the observation object are relatively moved by the first movement amount, and the level of the output signal of the light receiving element is predetermined. When the following is true, the optical system and the observation object may be moved relative to each other by the second movement amount, and the second movement amount may be larger than the first movement amount.

  When the level of the output signal of the light receiving element is equal to or lower than the predetermined level, the level of the output signal of the light receiving element is lower than the predetermined level from the state where the level of the output signal of the light receiving element is higher than the predetermined level. The relative position between the optical system and the observation object at the time of changing to is not detected. Accordingly, when the level of the output signal of the light receiving element is equal to or lower than the predetermined level, the optical system and the object to be observed are relatively compared with the state in which the level of the output signal of the light receiving element is higher than the predetermined level. By increasing the typical movement amount, the time for the movement range setting process can be shortened.

  (6) In the movement range setting process for each unit region, the control unit relatively moves the optical system and the observation object over a predetermined range from the first distance to the second distance. However, if the movement range is not set, the relative distance between the optical system and the observation object from the determined initial position is a third distance that is smaller than the first distance and a fourth distance that is larger than the second distance. The optical system and the observation object are moved relative to each other over a predetermined range until the distance reaches the distance, and based on the acquired pixel data, the level of the output signal of the light receiving element is higher than the predetermined level. Detecting the relative positions of the optical system and the observation object at multiple points in time when the level of the output signal of the light receiving element changes below a predetermined level from a high state, and detecting the detected multiple positions Include It may be set the moving range of the urchin the unit area.

  In this case, when the movement range is not set even if the optical system and the observation object are relatively moved over a predetermined range from the first distance to the second distance, the optical system is moved from the initial position. The relative distance between the optical system and the observation object moves relatively from the third distance smaller than the first distance to the fourth distance larger than the second distance. . A plurality of relative positions of the optical system and the observation object are detected at a plurality of time points when the level of the output signal of the light receiving element changes from a state higher than a predetermined level to a predetermined level or less. .

  As described above, in the movement range setting process, the optical system for detecting a plurality of relative positions between the optical system and the observation object and the relative distance range between the observation object and the optical system are expanded. And the observation object move relatively, and a plurality of relative positions between the optical system and the observation object are detected. As a result, a plurality of relative positions between the optical system and the observation object can be detected over a wide range, so that it is possible to accurately set the movement range for each unit region over a wide range. It becomes.

  (7) The object observation method according to the second reference form is an object for setting a range in which the optical system included in the microscope system for observing the state of the surface of the observation object and the observation object are relatively moved. A method for observing an object, wherein the optical system irradiates light while condensing the unit area set on the surface of the observation target, guides the light irradiated to the unit area to the light receiving element, and A step of relatively moving the optical system and the observation target so that light is irradiated by the optical system in the unit region at a plurality of positions along a vertical direction, and a plurality of unit regions as an observation range. For each of the set unit areas, a setting step and a moving range setting process for setting a relative moving range between the optical system and the observation object are performed, and the optical system and the relative moving unit are controlled. Moved by A step of performing a measurement process of relatively moving the optical system and the observation object within the movement range set in the surrounding setting process and acquiring a plurality of pixel data for the unit area based on an output signal of the light receiving element And the step of setting the plurality of unit areas includes the step of setting the plurality of unit areas so that overlapping portions are formed between the adjacent unit areas, performing the movement range setting process, and performing the measurement process In the moving range setting process for each unit area, the relative initial position between the optical system and the observation target is determined, and the direction in which the optical system and the observation target are approached and away from the determined initial position. To move the optical system and the observation object relative to each other and acquire a plurality of pixel data, set a movement range based on the acquired pixel data, For each unit region excluding the unit region, an optical system is provided in at least a part of the unit region based on the pixel data for the overlapping portion of the unit region among the pixel data acquired during the measurement processing of the other adjacent unit regions. The method includes a step of determining an optical system and an initial position so as to be in focus.

  In this object observation method, a plurality of unit regions are set as the observation range. At this time, an overlapping portion is formed between adjacent unit regions. For each of the plurality of unit areas, a relative movement range between the optical system and the observation object is set by the movement range setting process, and the optical system and the observation object are within the movement range set in the unit area by the measurement process. Are moved relative to each other, and a plurality of pixel data for the unit area is acquired based on the output signal of the light receiving element.

  In the movement range setting process for each unit region, a relative initial position between the optical system and the observation object is determined. The optical system and the observation object are relatively moved in a direction in which the optical system and the observation object approach and move away from the determined initial position, and a plurality of pixel data are acquired. Based on the plurality of pixel data, a relative movement range between the optical system and the observation object is set for the unit region.

  For each unit area other than the first unit area, at least a part of the unit area based on the pixel data of the overlapping part of the unit area among the pixel data acquired during the measurement process of the other adjacent unit areas The relative initial position of the optical system and the observation object is determined so that the optical system is in focus.

  Accordingly, light is guided from at least a part of the unit region to the light receiving element in a state where the optical system and the observation target are at the initial positions. By moving the optical system and the observation object relative to each other in a direction in which the optical system and the observation object approach and move away from the determined initial position, the focus of the optical system is focused on at least a part of the unit region. It is possible to detect a relative position between the optical system and the observation object when the optical system is out of focus on the unit region from the state of being matched. Based on the detected position, the relative movement range between the optical system and the observation object can be appropriately set for the unit region.

  As described above, for each unit area other than the first unit area, the initial position can be determined based on the pixel data already acquired for the overlapping portion in the movement range setting process for the unit area. Therefore, it is not necessary to acquire pixel data in order to determine the initial position. Therefore, the initial position can be set in a short time. As a result, a plurality of pixel data for a plurality of unit regions can be efficiently acquired in a short time.

  (8) The object observation program according to the third reference form processes a process of setting a range in which the optical system included in the microscope system that observes the state of the surface of the observation object and the observation object are relatively moved. An object observation program to be executed by the apparatus, which irradiates light while condensing the unit area set on the surface of the observation object by the optical system and guides the light irradiated to the unit area to the light receiving element. And a process of relatively moving the optical system and the observation object so that light is irradiated by the optical system in the unit region at a plurality of positions along a direction perpendicular to the unit region, and as an observation range A process for setting a plurality of unit areas and a movement range setting process for setting a relative movement range between the optical system and the observation object for each of the set unit areas are performed. Control In this way, the optical system and the observation object are relatively moved within the movement range set in the movement range setting process, and a plurality of pixel data for the unit area are acquired based on the output signal of the light receiving element. The processing for causing the processing device to execute processing and setting a plurality of unit areas includes a process of setting a plurality of unit areas so that overlapping portions are formed between adjacent unit areas, and includes a movement range. The setting process is performed, and the measurement process is performed by determining a relative initial position between the optical system and the observation object in the movement range setting process for each unit area, and observing the optical system and the observation from the determined initial position. Move the optical system and the observation object relative to each other toward and away from the object, acquire multiple pixel data, and move based on the acquired pixel data For each unit area excluding the first unit area, the unit area is determined based on the pixel data for the overlapping portion of the unit area among the pixel data acquired during the measurement processing of the other adjacent unit areas. And a process of determining an optical system and an initial position so that at least a part of the optical system is in focus.

  In this object observation program, a plurality of unit areas are set as the observation range. At this time, an overlapping portion is formed between adjacent unit regions. For each of the plurality of unit areas, a relative movement range between the optical system and the observation object is set by the movement range setting process, and the optical system and the observation object are within the movement range set in the unit area by the measurement process. Are moved relative to each other, and a plurality of pixel data for the unit area is acquired based on the output signal of the light receiving element.

  In the movement range setting process for each unit region, a relative initial position between the optical system and the observation object is determined. The optical system and the observation object are relatively moved in a direction in which the optical system and the observation object approach and move away from the determined initial position, and a plurality of pixel data are acquired. Based on the plurality of pixel data, a relative movement range between the optical system and the observation object is set for the unit region.

  For each unit area other than the first unit area, at least a part of the unit area based on the pixel data of the overlapping part of the unit area among the pixel data acquired during the measurement process of the other adjacent unit areas The relative initial position of the optical system and the observation object is determined so that the optical system is in focus.

  Accordingly, light is guided from at least a part of the unit region to the light receiving element in a state where the optical system and the observation target are at the initial positions. By moving the optical system and the observation object relative to each other in a direction in which the optical system and the observation object approach and move away from the determined initial position, the focus of the optical system is focused on at least a part of the unit region. It is possible to detect a relative position between the optical system and the observation object when the optical system is out of focus on the unit region from the state of being matched. Based on the detected position, the relative movement range between the optical system and the observation object can be appropriately set for the unit region.

  As described above, for each unit area other than the first unit area, the initial position can be determined based on the pixel data already acquired for the overlapping portion in the movement range setting process for the unit area. Therefore, it is not necessary to acquire pixel data in order to determine the initial position. Therefore, the initial position can be set in a short time. As a result, a plurality of pixel data for a plurality of unit regions can be efficiently acquired in a short time.

  According to the present invention, it is possible to easily and appropriately set the range of the relative distance between the measurement object and the objective lens for a plurality of unit regions.

It is a block diagram which shows the structure of the confocal microscope system which concerns on one embodiment of this invention. It is a figure for defining an X direction, a Y direction, and a Z direction. It is a figure which shows the example in which four unit area | regions are set to the acquisition range of pixel data. It is a figure which shows the relationship between the position of the Z direction of an observation target object, and the light reception intensity | strength of a light receiving element in one pixel. It is a figure which shows the example of a display of the display part before the setting of the acquisition range of pixel data. It is a figure for demonstrating the relationship between the position of the Z direction of the objective lens about one pixel, and the value of effective pixel data. It is a figure for demonstrating the peak position search process using an evaluation value. It is a figure for demonstrating the setting method of the upper limit position of the Z direction of the objective lens 3 at the time of the production | generation of height image data and ultra-depth image data. It is a schematic diagram which shows the positional relationship of the observation object which has a level | step difference in the surface, and an objective lens. It is a figure which shows the relationship between the position of the Z direction of the objective lens obtained by scanning a laser beam with respect to the observation target object of FIG. 9, and an evaluation value. It is a flowchart which shows the target object observation process performed in a confocal microscope system. It is a flowchart which shows the target object observation process performed in a confocal microscope system. It is a flowchart which shows the upper-lower limit automatic setting process of FIG. It is a flowchart which shows the upper-lower limit automatic setting process of FIG. It is a flowchart which shows the upper-lower limit automatic setting process of FIG. It is a flowchart which shows the boundary position search process of FIG. 14 and FIG. It is a flowchart which shows the boundary position search process of FIG. 14 and FIG. It is a flowchart which shows the boundary position search process of FIG. 14 and FIG. It is a flowchart which shows the other example of a boundary position search process.

  A microscope system according to an embodiment of the present invention will be described with reference to the drawings. Below, a confocal microscope system is demonstrated as an example of a microscope system.

(1) Basic Configuration of Confocal Microscope System FIG. 1 is a block diagram showing a configuration of a confocal microscope system 500 according to an embodiment of the present invention. As shown in FIG. 1, the confocal microscope system 500 includes a measurement unit 100, a PC (personal computer) 200, a control unit 300, and a display unit 400. The measurement unit 100 includes a laser light source 10, an XY scan optical system 20, a light receiving element 30, an illumination white light source 40, a color CCD (charge coupled device) camera 50, and a stage 60. An observation object S is placed on the stage 60.

  The laser light source 10 is a semiconductor laser, for example. The laser light emitted from the laser light source 10 is converted into parallel light by the lens 1, passes through the half mirror 4, and enters the XY scan optical system 20. Instead of the laser light source 10, another light source such as a mercury lamp may be used. In this case, a band pass filter is disposed between the light source such as a mercury lamp and the XY scan optical system 20. Light emitted from a light source such as a mercury lamp passes through a band-pass filter, becomes monochromatic light, and enters the XY scan optical system 20.

  The XY scan optical system 20 is, for example, a galvanometer mirror. The XY scan optical system 20 has a function of scanning a laser beam in the X direction and the Y direction on the surface of the observation object S on the stage 60. The definitions of the X direction, the Y direction, and the Z direction will be described later. The laser beam scanned by the XY scanning optical system 20 is reflected by the half mirror 5, then passes through the half mirror 6, and is focused on the observation object S on the stage 60 by the objective lens 3. Instead of the half mirrors 4 to 6, a polarization beam splitter may be used.

  The laser light reflected by the observation object S passes through the objective lens 3 and the half mirror 6, then is reflected by the half mirror 5, and passes through the XY scan optical system 20. The laser beam that has passed through the XY scan optical system 20 is reflected by the half mirror 4, collected by the lens 2, and transmitted through the pinhole of the pinhole member 7 and the ND (Neutral Density) filter 8 to receive the light receiving element 30. Is incident on. As described above, the reflection type confocal microscope system 500 is used in the present embodiment, but when the observation object S is a transparent body such as a cell, a transmission type confocal microscope system is used. Also good.

  The pinhole of the pinhole member 7 is disposed at the focal position of the lens 2. The ND filter 8 is used to attenuate the intensity of laser light incident on the light receiving element 30. Therefore, when the intensity of the laser beam is sufficiently attenuated, the ND filter 8 may not be provided.

  In the present embodiment, the light receiving element 30 is a photomultiplier tube. A photodiode and an amplifier may be used as the light receiving element 30. The light receiving element 30 outputs an analog electric signal (hereinafter referred to as a light receiving signal) corresponding to the amount of received light. The control unit 300 includes two A / D converters (analog / digital converter), a FIFO (First In First Out) memory, and a CPU (Central Processing Unit). The light reception signal output from the light receiving element 30 is sampled at a constant sampling period by one A / D converter of the control unit 300 and converted into a digital signal. Digital signals output from the A / D converter are sequentially stored in the FIFO memory. The digital signals stored in the FIFO memory are sequentially transferred to the PC 200 as pixel data.

  The illumination white light source 40 is, for example, a halogen lamp or a white LED (light emitting diode). White light generated by the illuminating white light source 40 is reflected by the half mirror 6 and then condensed by the objective lens 3 onto the observation object S on the stage 60.

  White light reflected by the observation object S passes through the objective lens 3, the half mirror 6 and the half mirror 5 and enters the color CCD camera 50. An imaging element such as a CMOS (complementary metal oxide semiconductor) image sensor may be used in place of the color CCD camera 50. The color CCD camera 50 outputs an electrical signal corresponding to the amount of received light. The output signal of the color CCD camera 50 is sampled at a constant sampling period by another A / D converter of the control unit 300 and converted into a digital signal. Digital signals output from the A / D converter are sequentially transferred to the PC 200 as camera data.

  The control unit 300 supplies pixel data and camera data to the PC 200 and controls the light receiving sensitivity (gain) of the light receiving element 30 and the color CCD camera 50 based on a command from the PC 200. Further, the control unit 300 controls the XY scanning optical system 20 based on a command from the PC 200 to scan the laser light on the observation object S in the X direction and the Y direction.

  The objective lens 3 is provided so as to be movable in the Z direction by the lens driving unit 63. The control unit 300 can move the objective lens 3 in the Z direction by controlling the lens driving unit 63 based on a command from the PC 200. Thereby, the position of the observation target S relative to the objective lens 3 in the Z direction can be changed.

  The PC 200 includes a CPU (Central Processing Unit) 210, a ROM (Read Only Memory) 220, a work memory 230, and a storage device 240. The ROM 220 stores a system program. The working memory 230 includes a RAM (Random Access Memory), and is used for processing various data. The storage device 240 is composed of a hard disk or the like. The storage device 240 stores an object observation program and is used for storing various data such as pixel data and camera data given from the control unit 300. Details of the object observation program will be described later.

  The CPU 210 generates image data based on the pixel data given from the control unit 300. Hereinafter, the image data generated based on the pixel data is referred to as confocal image data. An image displayed based on the confocal image data is referred to as a confocal image.

  The CPU 210 generates image data based on the camera data given from the control unit 300. Hereinafter, the image data generated based on the camera data is referred to as camera image data. An image displayed based on the camera image data is called a camera image.

  The CPU 210 performs various processes on the generated confocal image data and camera image data using the work memory 230, and displays a confocal image based on the confocal image data and a camera image based on the camera image data on the display unit 400. Let Further, the CPU 210 gives a driving pulse to the stage driving unit 62 described later.

  The display unit 400 is configured by, for example, a liquid crystal display panel or an organic EL (electroluminescence) panel.

  The stage 60 has an X direction moving mechanism, a Y direction moving mechanism, and a Z direction moving mechanism. Stepping motors are used for the X direction moving mechanism, the Y direction moving mechanism, and the Z direction moving mechanism.

  The X direction moving mechanism, the Y direction moving mechanism, and the Z direction moving mechanism of the stage 60 are driven by a stage operation unit 61 and a stage driving unit 62. The user can move the stage 60 in the X direction, the Y direction, and the Z direction relative to the objective lens 3 by manually operating the stage operation unit 61.

  The stage drive unit 62 moves the stage 60 relative to the objective lens 3 in the X direction, the Y direction, or the Z direction by supplying a current to the stepping motor of the stage 60 based on the drive pulse given from the PC 200. be able to.

(2) Confocal Image, Ultra-Deep Image, and Height Image FIG. 2 is a diagram for defining the X direction, the Y direction, and the Z direction. As shown in FIG. 2, the observation object S is irradiated with the laser light condensed by the objective lens 3. In the present embodiment, the direction of the optical axis of the objective lens 3 is defined as the Z direction. Further, two directions orthogonal to each other on the surface orthogonal to the Z direction are defined as an X direction and a Y direction, respectively. The X, Y, and Z directions are indicated by arrows X, Y, and Z, respectively.

  The relative position of the surface of the observation object S with respect to the objective lens 3 in the Z direction is referred to as the position of the observation object S in the Z direction. The confocal image data is generated for each unit area. The unit area is determined by the magnification of the objective lens 3.

  While the position of the observation object S in the Z direction is constant, the XY scanning optical system 20 scans the laser beam in the X direction at the end in the Y direction in the unit region. When the scanning in the X direction is completed, the laser beam is shifted by the XY scanning optical system 20 at a constant interval in the Y direction. In this state, the laser beam is scanned in the X direction. By repeating the scanning of the laser beam in the X direction and the shifting in the Y direction within the unit region, the scanning of the unit region in the X direction and the Y direction is completed. Next, the objective lens 3 is moved in the Z direction. Thereby, the scanning in the X direction and the Y direction of the unit area is performed in a constant state where the position of the objective lens 3 in the Z direction is different from the previous time. The unit region is scanned in the X and Y directions at a plurality of positions in the Z direction of the observation object S.

  Confocal image data is generated by scanning in the X and Y directions for each position of the observation object S in the Z direction. As a result, a plurality of confocal image data having different positions in the Z direction within the unit region is generated.

  Here, the number of pixels in the X direction of the confocal image data is determined by the scanning speed of the laser beam in the X direction by the XY scanning optical system 20 and the sampling period of the control unit 300. The number of samples in one X-direction scan (one scan line) is the number of pixels in the X direction. Further, the number of pixels in the Y direction of the confocal image data in the unit area is determined by the amount of shift in the Y direction of the laser beam by the XY scan optical system 20 at the end of scanning in the X direction. The number of scanning lines in the Y direction is the number of pixels in the Y direction. Furthermore, the number of confocal image data in the unit area is determined by the number of movements of the observation object S in the Z direction. Based on the plurality of confocal image data in the unit area, ultra-depth image data and height image data are generated by a method described later.

  In the example of FIG. 2, first, a plurality of confocal image data of the observation object S in the unit region s1 is generated at the initial position of the stage 60, and ultra-depth image data and height image data of the unit region s1 are generated. Is done. Subsequently, the stage 60 is sequentially moved to generate a plurality of confocal image data of the observation object S in the unit regions s2 to s4 and to generate ultra-depth image data and height image data of the unit regions s2 to s4. Is done. In this case, the unit regions s1 to s4 are set so that adjacent unit regions partially overlap each other. By performing pattern matching using a part of the super-depth image data and the height image data of the overlapping unit regions, the ultra-depth image data and the height image data of the plurality of unit regions s1 to s4 are connected with high accuracy. be able to. In the present embodiment, a portion where a plurality of adjacent unit regions overlap each other is called an overlapping portion.

  FIG. 3 is a diagram illustrating an example in which four unit regions s1 to s4 are set in the pixel data acquisition range. In the example of FIG. 3, the unit areas s1 to s4 are set in 2 rows and 2 columns. Unit areas s1 and s2 are arranged in the first line, and unit areas s3 and s4 are arranged in the second line.

  In this case, an overlapping portion ov is set between the unit region s1 and the unit region s2 that are adjacent to each other in the horizontal direction (row direction). In addition, an overlapping portion ov is set between the unit region s1 and the unit region s3 that are adjacent to each other in the vertical direction (column direction). Furthermore, an overlapping portion ov is set between the unit region s1 and the unit region s4 that are adjacent to each other in the oblique direction. Similarly, an overlapping portion ov is set between each two adjacent unit regions. In FIG. 3, the overlapping portion ov is hatched.

  FIG. 4 is a diagram illustrating the relationship between the position of the observation object S in the Z direction and the light receiving intensity of the light receiving element 30 in one pixel. As shown in FIG. 1, the pinhole of the pinhole member 7 is disposed at the focal position of the lens 2. Therefore, when the surface of the observation object S is at the focal position of the objective lens 3, the laser light reflected by the observation object S is condensed at the pinhole position of the pinhole member 7. Thereby, most of the laser beam reflected by the observation object S passes through the pinhole of the pinhole member 7 and enters the light receiving element 30. In this case, the light receiving intensity of the light receiving element 30 is maximized. As a result, the voltage value of the light reception signal output from the light receiving element 30 is maximized.

  On the other hand, when the observation object S is at a position out of the focal position of the objective lens 3, the laser light reflected by the observation object S is condensed at a position before or after the pinhole of the pinhole member 7. . Thereby, most of the laser light reflected by the observation object S is blocked by the portion around the pinhole of the pinhole member 7, and the light receiving intensity of the light receiving element 30 is reduced. As a result, the voltage value of the light reception signal output from the light receiving element 30 decreases.

  Thus, a peak appears in the light reception intensity distribution of the light receiving element 30 in a state where the surface of the observation object S is at the focal position of the objective lens 3. The received light intensity distribution in the Z direction is obtained for each pixel from a plurality of confocal image data of each unit region. Thereby, the peak position and peak intensity (peak received light intensity) of the received light intensity distribution are obtained for each pixel.

  Data representing peak positions in the Z direction for a plurality of pixels corresponding to each unit area is referred to as height image data, and an image displayed based on the height image data is referred to as a height image. The height image represents the surface shape of the observation object S. In addition, data representing the peak intensity for a plurality of pixels corresponding to each unit region is referred to as ultra-deep image data, and an image represented based on the ultra-depth image data is referred to as an ultra-deep image. The ultra-deep image is an image obtained in a state where all parts of the surface of the observation object S are in focus. The PC 200 generates a plurality of confocal image data of the unit region based on the plurality of pixel data of the unit region given from the control unit 300, and the height image data and the super image of the unit region based on the plurality of confocal image data. Generate depth image data. Hereinafter, the height image data and the ultradeep image data are collectively referred to as surface image data, and the height image and the ultradeep image are collectively referred to as a surface image.

(3) Setting of Pixel Data Acquisition Range (Observation Range) FIG. 5 is a diagram illustrating a display example of the display unit 400 before setting of the pixel data acquisition range. As shown in FIG. 5, an image display area 410 and a condition setting area 420 are displayed on the screen of the display unit 400. In the image display area 410, a confocal image based on the confocal image data or a camera image based on the camera image data is displayed. In the condition setting area 420, a range setting button 421 and an acquisition start button 422 are displayed.

  The user places the observation object S on the stage 60 of the confocal microscope system 500 of FIG. The control unit 300 sequentially provides camera data to the PC 200. The CPU 210 of the PC 200 generates camera image data based on the camera data given by the control unit 300 and displays the camera image of the observation object S in the image display area 410 of the display unit 400. In this case, the user can change the range of the camera image of the observation object S displayed in the image display area 410 by moving the stage 60 in the X direction or the Y direction.

  The user operates the range setting button 421 in the condition setting area 420 using a pointing device such as a mouse connected to the PC 200. As a result, the CPU 210 of the PC 200 causes the display unit 400 to display a pixel data acquisition range setting screen (not shown). The user sets the pixel data acquisition range using a pointing device while the acquisition range setting screen is displayed.

  When the set acquisition range is larger than the unit area, the CPU 210 of the PC 200 sets n (n is a natural number of 2 or more) unit areas in the set acquisition range. In this case, the CPU 210 sets an overlapping portion ov between each two unit areas adjacent to each other. Hereinafter, the plurality of unit areas set when the acquisition range is larger than the unit area are referred to as first to nth unit areas, respectively.

  After the pixel data acquisition range is set, the user operates the acquisition start button 422 in the condition setting area 420. As a result, the CPU 210 of the PC 200 executes the upper / lower limit automatic setting process as a process corresponding to the movement range setting process of the present invention for the first unit area.

  The upper / lower limit automatic setting process is a process of setting an upper limit position and a lower limit position in the Z direction of the objective lens 3 that moves when generating height image data and ultra-depth image data. Details of the upper / lower limit automatic setting process will be described later.

  After the upper / lower limit automatic setting process is executed, the CPU 210 of the PC 200 scans the laser beam in the X direction and the Y direction at a plurality of positions in the Z direction between the set upper limit position and lower limit position, and the first unit area A plurality of pixel data in is acquired. Thereby, a plurality of confocal image data corresponding to a plurality of positions different in the Z direction in the first unit region are generated, and surface image data (height image data) is generated based on the generated plurality of confocal image data. And ultra-deep image data).

  Subsequently, the CPU 210 of the PC 200 executes upper / lower limit automatic setting processing described later for the second unit area. Further, the CPU 210 of the PC 200 scans the laser beam in the X direction and the Y direction at a plurality of positions in the Z direction between the set upper limit position and lower limit position, and acquires a plurality of pixel data in the second unit area. To do. Thereby, in the second unit region, a plurality of confocal image data corresponding to a plurality of positions different in the Z direction are generated, and surface image data is generated based on the generated plurality of confocal image data.

  Thereafter, similarly, the CPU 210 of the PC 200 executes the upper / lower limit automatic setting process for each of the third to nth unit areas and acquires a plurality of pixel data. In this way, surface image data is generated for all unit regions.

  Finally, the CPU 210 of the PC 200 connects the generated surface image data of all (1st to nth) unit regions, and displays the image of the surface of the observation object S based on the connected surface image data on the display unit 400. To display.

  Hereinafter, the upper and lower limit automatic setting process executed for the first unit area is referred to as the first upper and lower limit automatic setting process, and is executed for the second and subsequent m (m is a natural number of 2 to n) unit areas. This upper / lower limit automatic setting process is called the mth upper / lower limit automatic setting process.

  In the present embodiment, the first upper / lower limit automatic setting process is different from the mth upper / lower limit automatic setting process. In the m-th upper / lower limit automatic setting process, a plurality of pieces of pixel data already acquired in the overlapping portion ov between the m-th unit region and the unit region adjacent to the m-th unit region are used. Thereby, the upper and lower limit automatic setting process after the mth is simplified as compared with the first upper and lower limit automatic setting process. Details of the first upper / lower limit automatic setting process and the mth upper / lower limit automatic setting process will be described.

(4) First Upper / Lower Limit Automatic Setting Process Details of the first upper / lower limit automatic setting process will be described. FIG. 6 is a diagram for explaining the relationship between the position in the Z direction of the objective lens 3 for one pixel and the value of effective pixel data. In FIG. 6, the vertical axis represents the pixel data value, and the horizontal axis represents the position of the objective lens 3 in the Z direction. In the horizontal axis of FIG. 6, the position of the objective lens 3 in the Z direction increases from left to right.

  Hereinafter, the value of the pixel data is used as the level of the output signal of the light receiving element 30. Further, the noise level nl is used as a predetermined level of the output signal of the light receiving element 30.

  The pixel data is a digital signal corresponding to the light reception signal output from the light receiving element 30. For this reason, the value of the pixel data increases as the gain of the light receiving element 30 increases and decreases as the gain of the light receiving element 30 decreases. Pixel data is output from the A / D converter. Therefore, the upper limit value of the pixel data is the upper limit value of the output range of the A / D converter (hereinafter referred to as the output upper limit value max).

  When the pixel data value is saturated at the output upper limit value max, the pixel data value corresponding to the light receiving intensity of the light receiving element 30 cannot be obtained. If the pixel data value is less than or equal to the noise level nl, the peak of the pixel data cannot be clearly identified from the noise of the light receiving element 30. Hereinafter, pixel data values that are smaller than the output upper limit value max and larger than the noise level nl are referred to as effective pixel data values.

  In FIG. 6, the range of valid pixel data values is indicated by an arrow HL. In this case, as indicated by the curve l1, when an arbitrary first gain is set in the light receiving element 30, the pixel data value for one pixel is a position where the objective lens 3 is lower than the peak position z0. It is effective in a state where it is in a range from ma1 to a position mb1 higher than the peak position z0.

  Therefore, the upper limit position and the lower limit position in the Z direction of the objective lens 3 for obtaining a valid pixel data value for one pixel are determined according to the output upper limit value max and the noise level nl.

  As indicated by a curve l2 in FIG. 6, when a second gain smaller than the first gain is set in the light receiving element 30, the pixel data value is set to the first gain in the light receiving element 30. Compared to the case, it becomes smaller overall. In this case, the value of the pixel data for one pixel is effective when the objective lens 3 is in the range from the position ma2 higher than the position ma1 to the position mb2 lower than the position mb1. As described above, when the gain set in the light receiving element 30 is changed, the upper limit position and the lower limit position in the Z direction of the objective lens 3 capable of obtaining effective pixel data values are also changed.

  When setting the upper limit position and the lower limit position for one pixel, the gain is set so that the value of the pixel data at the peak position z0 is as close as possible to the output upper limit value max. In this case, the peak of the pixel data is sufficiently larger than the noise level nl. Therefore, the peak of the pixel data can be clearly identified from the noise of the light receiving element 30.

  Here, when the upper limit position and the lower limit position for one pixel are set, first, the objective lens 3 is moved in the Z direction so that the focus is on the surface of the observation object S. That is, the objective lens 3 is moved to the peak position z0 as the initial position. When the objective lens 3 is at the peak position z0, the value of the pixel data is reliably valid. In this case, when the focus of the objective lens 3 is sufficiently deviated from the surface of the observation object S by moving the objective lens 3 in the upward direction and the downward direction from the peak position z0, that is, the value of the pixel data is It is possible to detect the position of the objective lens 3 when the noise level is nl or less. Based on the detected position, the upper limit position and the lower limit position of the objective lens 3 in the Z direction can be appropriately set.

  Thus, by setting the upper limit position and the lower limit position of the objective lens 3 in the Z direction, it is not necessary to move the objective lens 3 in the Z direction within a range where effective pixel data cannot be obtained. As a result, it is possible to shorten the processing time for setting the upper limit position and the lower limit position of the objective lens 3 in the Z direction.

  Further, in this case, the lower limit position of the objective lens 3 in the Z direction is such that the focal position of the objective lens 3 moves below the surface of the observation object S, and the pixel data value is from a valid value to a noise level nl or less. It is set to the position at the moment. For this reason, when the lower limit position in the Z direction of the objective lens 3 is set, the objective lens 3 is prevented from colliding with the surface of the observation object S due to excessive movement downward.

  In the present embodiment, the position in the Z direction of the objective lens 3 corresponding to the peak position z0 (evaluation peak position Ez0 described later) is detected using the evaluation value described later during the first upper / lower limit automatic setting process. At the same time, a peak position search process for adjusting the gain of the light receiving element 30 is performed.

  FIG. 7 is a diagram for explaining the peak position search process using the evaluation value. Here, the evaluation value is a value calculated based on the values of a plurality of pixel data for a predetermined region of the surface of the observation object S. In the present embodiment, the evaluation value is the sum of the values of all the pixel data for the unit area. The position in the Z direction of the objective lens 3 when the evaluation value indicates the peak value is referred to as an evaluation peak position Ez0. Further, a product value of the output upper limit max of the A / D converter and the number of pieces of pixel data for calculating the evaluation value (the number of all pixel data in the unit area in this example) is set as the output upper limit Emax. Call. Further, a multiplication value of the noise level nl and the number of pixel data for calculating the evaluation value (in this example, the number of all pixel data in the unit area) is referred to as a noise evaluation level Enl.

  Hereinafter, an evaluation value described later is used as the level of the output signal of the light receiving element 30. Further, a noise evaluation level Enl described later is used as a predetermined level of the output signal of the light receiving element 30.

  FIG. 7A shows the relationship between the position of the objective lens 3 in the Z direction and the evaluation value. In FIG. 7A, the vertical axis represents the evaluation value, and the horizontal axis represents the position of the objective lens 3 in the Z direction. On the horizontal axis in FIG. 7A, the position of the objective lens 3 in the Z direction increases from left to right.

  At the start of the peak position search process, the gain of the light receiving element 30 is set so that the calculated evaluation value is smaller than the evaluation upper limit value Emax and sufficiently larger than the noise evaluation level Enl. In this example, the gain of the light receiving element 30 is set so that the calculated evaluation value is ½ of the evaluation upper limit value Emax.

  Thereafter, as shown by a thick solid arrow in FIG. 7A, the objective lens 3 is set while being gradually moved upward from the current position zs1 in the Z direction (position lower than the evaluation peak position Ez0). All the pixel data for the unit area is acquired with the gain obtained and the evaluation value is calculated.

  In this case, the evaluation value increases exponentially until the objective lens 3 reaches the evaluation peak position Ez0. Therefore, in this peak position search process, the gain is decreased by a certain amount every time the evaluation value reaches the evaluation upper limit value Emax.

  This prevents the evaluation value that increases exponentially from being saturated at the evaluation upper limit value Emax until the objective lens 3 moves from the position zs1 in the Z direction to the evaluation peak position Ez0. As a result, in the vicinity of the evaluation peak position Ez0, the gain of the light receiving element 30 is finally set to an appropriate value.

  The evaluation value decreases as the gain of the light receiving element 30 decreases. Therefore, in the peak position search process, the calculated evaluation value is corrected based on the number of times the gain of the light receiving element 30 is decreased.

  In the present embodiment, the evaluation value is corrected by adding the multiplication value of the gain reduction count of the light receiving element 30 after the start of the peak position search process and the evaluation upper limit value Emax to the calculated evaluation value. Thereby, it is possible to approximately associate the corrected evaluation value with the light receiving intensity of the light receiving element 30 over the entire range of the objective lens 3 in the Z direction.

  FIG. 7B shows the relationship between the position of the objective lens 3 in the Z direction and the corrected evaluation value. In FIG. 7B, the vertical axis represents the evaluation value after correction, and the horizontal axis represents the position of the objective lens 3 in the Z direction. On the horizontal axis of FIG. 7B, the position of the objective lens 3 in the Z direction increases from left to right.

  As shown in FIG. 7B, the corrected evaluation value exponentially increases to the peak value and then decreases exponentially from the peak value, similarly to the light reception intensity of the light receiving element 30. Thereby, it is determined whether the increase / decrease in the evaluation value after correction is switched by moving the objective lens 3 in one direction (upward direction) in the Z direction. When the increase / decrease in the evaluation value after correction is switched, the evaluation value after correction is a predetermined value from the peak value by further moving the objective lens 3 in one direction (upward direction) in the Z direction. It is determined whether or not it has decreased by pi2.

  When the corrected evaluation value decreases by the value pi2 from the peak value, it is determined whether the corrected evaluation value has increased by a predetermined value pi1 before the increase / decrease in the corrected evaluation value is switched. . When the corrected evaluation value increases by a predetermined value pi1, the position of the objective lens 3 when the evaluation value indicates the peak value can be detected as the evaluation peak position Ez0.

  The range in which the objective lens 3 can move in the Z direction is determined in advance by the magnification of the objective lens 3. The lower position of the objective lens 3 allowed in advance is called a lower end position zt1, and the upper position of the objective lens 3 allowed in advance is called an upper end position zt2.

  When the objective lens 3 is initially at a position zs2 higher than the evaluation peak position Ez0, the evaluation peak position Ez0 cannot be detected even if the objective lens 3 is moved upward. In this case, as indicated by a thick dotted line arrow in FIG. 7B, after the objective lens 3 is moved to the upper end position zt2, the objective lens 3 is moved in the reverse direction (downward direction). Thereby, the evaluation peak position Ez0 can be detected as described above.

  The value of the pixel data changes due to the influence of noise of the light receiving element 30. Therefore, the values pi1 and pi2 are higher than the noise evaluation level Enl, for example, the noise level nl and the number of pieces of pixel data for calculating the evaluation value (in this example, the number of all pixel data in the unit area). Set to a large value. Thereby, even when the corrected evaluation value changes due to the influence of noise of the light receiving element 30, it is possible to prevent the evaluation peak position Ez0 from being erroneously detected. The values pi1 and pi2 may be the same value or different values.

  In the first upper / lower limit automatic setting process, as described above, the evaluation peak position Ez0 is searched while the gain of the light receiving element 30 is adjusted by the peak position search process.

  In this example, the evaluation peak position Ez0 is approximately used as the position where the objective lens 3 is focused on the first unit region. The objective lens 3 is moved to the evaluation peak position Ez0 as an initial position, and the upper limit position and the lower limit position in the Z direction of the objective lens 3 are set. In this case, the surface of the observation object S exists at the focal position of the objective lens 3. Therefore, as described later, by calculating the evaluation value while changing the position of the objective lens 3 in the Z direction upward from the evaluation peak position Ez0, the position where the evaluation value is equal to or lower than the noise evaluation level Enl is obtained. The upper limit position of the objective lens 3 in the Z direction can be set appropriately. On the other hand, by calculating the evaluation value while changing the position of the objective lens 3 in the Z direction downward from the evaluation peak position Ez0, the position where the evaluation value is equal to or lower than the noise evaluation level Enl The lower limit position of the direction can be set appropriately.

  FIG. 8 is a diagram for explaining a method of setting the upper limit position and the lower limit position in the Z direction of the objective lens 3 when generating height image data and ultra-depth image data.

  In this setting method, first, in the state where the objective lens 3 is held at the evaluation peak position Ez0 detected by the peak position search process, the unit area is scanned in the X direction and the Y direction, and all the pixels in the unit area are scanned. Get the data.

  When the value of any pixel data is the output upper limit value max (FIG. 6), the value of the pixel data is not valid. Therefore, the gain of the light receiving element 30 is decreased by a certain amount. After that, by scanning the unit area in the X direction and the Y direction again, all the pixel data for the unit area are acquired.

  Whether the evaluation value is equal to or less than a predetermined threshold when all the pixel data values are smaller than the output upper limit value max (FIG. 6) by repeating the above gain adjustment and pixel data acquisition. Determine whether. In the present embodiment, the predetermined threshold is the noise evaluation level Enl (FIGS. 7 and 10). If the evaluation value is not less than or equal to the noise evaluation level Enl (FIGS. 7 and 10), the objective lens 3 is moved in a direction upward by a certain amount, as indicated by a thick solid arrow na in FIG.

  Subsequently, the above gain adjustment, pixel data acquisition, pixel data determination operation, and movement in the upward direction of the objective lens 3 are repeated. Thereby, the position in the Z direction of the objective lens 3 when the evaluation value is determined to be equal to or less than the noise evaluation level Enl is set as the upper limit position UP.

  When the gain of the light receiving element 30 changes, the value of the acquired pixel data also changes. Therefore, after the upper limit position UP is set, the objective lens 3 is moved in the Z direction to the position Ez1 when the gain of the light receiving element 30 is finally decreased, as indicated by a thick solid arrow nb in FIG. If the gain of the light receiving element 30 does not change until the upper limit position UP is set after the peak position search process, the objective lens 3 is moved in the Z direction to the evaluation peak position Ez0. In this state, by scanning the unit area in the X direction and the Y direction, all pixel data for the unit area is acquired.

  Also in this case, as described above, when the value of any pixel data is the output upper limit value max (FIG. 6), the gain of the light receiving element 30 is decreased by a certain amount. After that, by scanning the unit area in the X direction and the Y direction again, all the pixel data for the unit area are acquired.

  If the values of all the pixel data become smaller than the output upper limit max (FIG. 6) by repeating the above gain adjustment and pixel data acquisition, whether or not the evaluation value is equal to or less than the noise evaluation level Enl. Determine whether.

  If the evaluation value is not less than or equal to the noise evaluation level Enl, the objective lens 3 is moved in a direction downward by a certain amount as indicated by a thick solid arrow nc in FIG. Subsequently, the above gain adjustment, pixel data acquisition, pixel data determination operation, and movement downward in the objective lens 3 are repeated. Thereby, the position in the Z direction of the objective lens 3 when the evaluation value is determined to be equal to or less than the noise evaluation level Enl is set as the lower limit position BP.

  As described above, when the gain of the light receiving element 30 changes, the value of the acquired pixel data also changes, and the range of the objective lens 3 in which the effective pixel data value can be obtained also changes. That is, if the gain of the light receiving element 30 changes after the upper limit position UP is set and before the lower limit position BP is set, the upper limit position UP also changes according to the amount of gain change. Therefore, when the gain of the light receiving element 30 is reduced when the lower limit position BP is set after the setting of the upper limit position UP, the gain of the light receiving element 30 is finally reduced as shown by a thick solid line arrow nd in FIG. The objective lens 3 is moved in the Z direction to the current position Ez2.

  Thereafter, as shown by a thick solid line arrow ne in FIG. 8, the upper limit position UP is searched again. As described above, once the upper limit position UP and the lower limit position BP are set, it is considered that the gain of the light receiving element 30 is sufficiently reduced. Therefore, when searching for the upper limit position UP again, the gain of the light receiving element 30 hardly decreases. Therefore, the upper limit position UP is reset with the light receiving element 30 adjusted to a gain that is substantially the same as the gain when the lower limit position BP is set.

(5) mth Upper / Lower Limit Automatic Setting Process As described above, in the mth upper / lower limit automatic setting process, an overlapping portion between the mth unit area and a unit area adjacent to the mth unit area. A plurality of pixel data already acquired for ov is used. Details of the upper and lower limit automatic setting processing after the mth will be described below.

  When the mth and subsequent upper / lower limit automatic setting processing is started, a plurality of pixel data is already acquired for the overlapping portion ov between the mth unit region and the unit region adjacent to the mth unit region. Has been.

  For example, when the second upper / lower limit automatic setting process is started for the second unit region s2 of FIG. 3, the overlapping portion ov between the first unit region s1 and the second unit region s2 A plurality of pixel data has already been acquired. Similarly, when the fourth upper / lower limit automatic setting process is started for the fourth unit region s4 in FIG. 3, the overlapping portion ov between the second unit region s2 and the fourth unit region s4 and A plurality of pixel data has already been acquired for the overlapping portion ov between the third unit region s3 and the fourth unit region s4.

  Therefore, in the m-th upper / lower limit automatic setting process, the m-th unit area and the m-th unit area are adjacent to each other instead of the peak position search process executed in the first upper / lower-limit automatic setting process. The initial position of the objective lens 3 in the Z direction for searching for the upper limit position UP and the lower limit position BP is determined based on a plurality of pixel data already acquired for the overlapping portion ov with the unit area.

  The plurality of pixel data already acquired for the overlapping portion ov is valid pixel data. Therefore, the initial position of the objective lens 3 can be determined so that the evaluation value becomes larger than the noise evaluation level Enl by using the plurality of effective pixel data. As a result, in the m-th upper / lower limit automatic setting process, the upper limit position and the lower limit position of the objective lens 3 in the Z direction can be set in a short time.

  Specifically, the initial position can be determined as follows. By obtaining a plurality of pixel data for the unit region, height image data and ultra-depth image data of the unit region are generated. Therefore, during the m-th upper / lower limit automatic setting process, height image data and ultra-depth image data are already generated for the overlapping portion ov between the m-th unit region and the unit region adjacent to the m-th unit region. Has been. Therefore, in the m-th upper / lower limit automatic setting process, the lowest height (or the highest height) of the surfaces of the overlapping portion ov in the Z direction is based on the height image data about the already generated overlapping portion ov. Ask for. The position in the Z direction of the objective lens 3 when the focal position matches the obtained height is determined as the initial position.

  Alternatively, the initial position can be determined as follows. Based on the height image data of the already generated overlapping portion ov, the average height of the surface of the overlapping portion ov in the Z direction is calculated. The position in the Z direction of the objective lens 3 when the focal position matches the calculated average height is determined as the initial position.

  Alternatively, the initial position can be determined as follows. Based on the height image data for the overlapped portion ov that has already been generated, a histogram (frequency distribution) for the height of the surface of the overlapped portion ov in the Z direction is obtained. The position of the objective lens 3 when the focal position coincides with the height indicating the peak in the obtained histogram is determined as the initial position.

  When the pixel data value is less than or equal to the noise level nl, it is difficult to clearly distinguish the peak of the pixel data value from the noise of the light receiving element 30. Therefore, when the initial position is determined, the noise level nl among the plurality of pieces of pixel data already acquired for the overlapping portion ov between the mth unit region and the unit region adjacent to the mth unit region is set as the noise level nl. Exceeding pixel data may be extracted. In this case, by determining the initial position based on the extracted pixel data, the initial position can be appropriately determined based on the effective pixel data.

  When the mth unit region is adjacent to a plurality of unit regions, the initial position of the objective lens 3 may be determined based on a plurality of pixel data for a plurality of overlapping portions ov.

  Subsequently, in a state where the objective lens 3 is held at the initial position in the Z direction, scanning of the m-th unit area in the X direction and the Y direction is performed, thereby acquiring all pixel data for the unit area. In this state, when the values of all the pixel data are not equal to or greater than a predetermined value, the gain of the light receiving element 30 is increased by a certain amount. When the acquisition of the pixel data and the adjustment of the gain of the light receiving element 30 are repeated and the values of all the pixel data are equal to or greater than a predetermined value, the search for the upper limit position UP and the lower limit position BP of the objective lens 3 with the initial position as a reference To start.

  The search operation for the upper limit position UP and the lower limit position BP in the m-th upper / lower limit automatic setting process is the same as the search operation for the upper limit position UP and the lower limit position BP in the first upper / lower limit automatic setting process.

  As described above, in the m-th upper / lower limit automatic setting process, the initial position for searching for the upper limit position UP and the lower limit position BP of the objective lens 3 based on the already acquired pixel data for the overlapping portion ov is set. It is determined. Therefore, it is not necessary to execute the peak position search process. As a result, the processing time of the upper / lower limit automatic setting process is sufficiently shortened.

(6) Another Example of Upper / Lower Limit Automatic Setting Process FIG. 9 is a schematic diagram showing the positional relationship between the observation object S having a step on the surface and the objective lens 3. In the example of FIG. 9, a step including the first, second, and third surfaces Sa1, Sa2, Sa3 is formed on the surface of the observation object S. The first and third surfaces Sa1 and Sa3 are parallel to the XY direction, and the second surface Sa2 is parallel to the YZ direction. In the Z direction, the first surface Sa1 is higher than the third surface Sa3. In the X direction, the second surface Sa2 is located between the first and third surfaces Sa1 and Sa3.

  As shown in FIG. 9A, the position of the objective lens 3 in the Z direction when the focal position fp of the objective lens 3 is located at the intersection of the first surface Sa1 and the second surface Sa2 in the Z direction. Called the first Z position zp1. As shown in FIG. 9B, when the focal position fp of the objective lens 3 is located on the second surface Sa2 and between the first surface Sa1 and the third surface Sa3 in the Z direction in the Z direction. The position of the objective lens 3 in the Z direction is referred to as a second Z position zp2. As shown in FIG. 9C, the position of the objective lens 3 in the Z direction when the focal position fp of the objective lens 3 is located at the intersection of the second surface Sa2 and the third surface Sa3 in the Z direction. This is called the third Z position zp3.

  The laser beam is scanned in the X direction and the Y direction at a plurality of positions in the Z direction of the observation object S in FIG. FIG. 10 is a diagram showing the relationship between the evaluation value and the position in the Z direction of the objective lens 3 obtained by scanning the observation target S of FIG. 9 with laser light. The vertical axis in FIG. 10 represents the evaluation value, and the horizontal axis represents the position of the objective lens 3 in the Z direction. On the horizontal axis, the position of the objective lens 3 in the Z direction increases from left to right. Further, the above-described lower end position zt1, upper end position zt2, and first to third Z positions zp1 to zp3 are shown on the horizontal axis of FIG.

  As shown in FIG. 10, when the position of the objective lens 3 in the Z direction is at the first Z position zp1, the focal position fp of the objective lens 3 is located on the first surface Sa1 (FIG. 9A )reference). In this case, a part of the laser light is reflected by the first surface Sa1 at the focal position fp of the objective lens 3, so that the evaluation value is increased. Further, when the position of the objective lens 3 in the Z direction is at the third Z position zp3, the focal position fp of the objective lens 3 is located on the third surface Sa3 (see FIG. 9C). In this case, a part of the laser light is reflected by the third surface Sa3 at the focal position fp of the objective lens 3, so that the evaluation value is increased. On the other hand, when the objective lens 3 is at the second Z position zp2, the focal position fp of the objective lens 3 does not exist on a plane orthogonal to the optical axis of the objective lens 3 (a plane parallel to the XY direction). In this case, there is no surface that reflects the laser beam in the Z direction at the focal position fp of the objective lens 3. Therefore, the evaluation value becomes small.

  In this example, when the objective lens 3 is in the range AZ1 from the lower end position zt1 to the position zn1, the range AZ2 from the position zn2 to the position zn3, and the range AZ3 from the position zn4 to the upper end position zt2. The evaluation value is equal to or less than the above-described noise evaluation level Enl. The first Z position zp1 is located between the position zn1 and the position zn2, the second Z position zp2 is located between the position zn2 and the position zn3, and the third Z position zp3 is located at the position zn3. And the position zn4.

  According to the above upper / lower limit automatic setting process, after the objective lens 3 is moved to the first position zp1, the gain adjustment, the acquisition of pixel data, and the movement in the upward direction of the objective lens 3 are repeated. Thereby, the position zn2 in the Z direction of the objective lens 3 when the evaluation value first becomes equal to or lower than the noise evaluation level Enl is set as the upper limit position UP. In addition, after setting the upper limit position UP, the objective lens 3 is finally moved to the position when the gain of the light receiving element 30 is decreased, and then gain adjustment, acquisition of pixel data, and downward direction of the objective lens 3 are performed. Repeat the move. Thereby, the position zn1 in the Z direction of the objective lens 3 when the evaluation value first becomes equal to or lower than the noise evaluation level Enl is set as the lower limit position BP. When the gain of the light receiving element 30 changes, the upper limit position UP is searched again and reset. Thus, when the upper limit position UP and the lower limit position BP are set, the third Z position zp3 deviates from the range between the upper limit position UP and the lower limit position BP. Therefore, the user cannot observe the third surface Sa3.

  Therefore, in the confocal microscope system 500 according to the present embodiment, the following upper / lower limit automatic setting process may be performed in addition to the upper / lower limit automatic setting process described with reference to FIG.

  In the upper and lower limit automatic setting processing of this example, after the objective lens 3 is moved to the first position zp1, the gain adjustment, the acquisition of pixel data, and the top of the objective lens 3 are performed until the objective lens 3 reaches the upper end position zt2. The movement in the direction toward is repeated (see the thick arrow in FIG. 10). The position in the Z direction (position zn4 in this example) of the objective lens 3 when the evaluation value is finally lower than the noise evaluation level Enl from a value larger than the noise evaluation level Enl is set as the upper limit position UP.

  After the upper limit position UP is set, the objective lens 3 is moved to the position at the time when the gain of the light receiving element 30 is finally decreased, and then the gain adjustment and pixel data are changed until the objective lens 3 reaches the lower end position zt1. The acquisition and movement in the downward direction of the objective lens 3 are repeated. The position in the Z direction (position zn1 in this example) of the objective lens 3 when the evaluation value is finally lower than the noise evaluation level Enl from a value larger than the noise evaluation level Enl is set as the lower limit position BP.

  When the gain of the light receiving element 30 changes, the objective lens 3 is finally moved to a position where the gain of the light receiving element 30 is decreased, and then gain adjustment and pixel data are performed until the objective lens 3 reaches the upper end position zt2. And the movement toward the top of the objective lens 3 are repeated. The position in the Z direction of the objective lens 3 when the evaluation value is finally lower than the noise evaluation level Enl from a value larger than the noise evaluation level Enl is reset as the upper limit position UP.

  According to this method, even when the surface of the observation object S has a large step, the user observes all the surfaces (the first and third surfaces Sa1 and Sa3 in FIG. 9) constituting the step. Can do.

  As described above, the range in which the objective lens 3 can move in the Z direction is determined in advance by the magnification of the objective lens 3. However, the lower limit position BP and the upper limit position UP are not necessarily located between the lower end position zt1 and the upper end position zt2 that are predetermined according to the magnification of the objective lens 3.

  Accordingly, in the upper / lower limit automatic setting process, when the lower limit position BP and the upper limit position UP cannot be set, a position that is a certain distance higher than the upper end position zt2 (FIGS. 7, 9, and 10) is set as a new upper end position. After setting a position lower than the position zt1 (FIGS. 7, 9, and 10) by a certain distance as a new lower end position, the upper / lower limit automatic setting process is performed again based on the newly set upper end position and lower end position. Also good.

  As described above, when the upper limit position UP and the lower limit position BP are not set within a predetermined range in which the objective lens 3 can move in the Z direction, new upper limit positions and lower limit positions are set. Thus, the upper / lower limit automatic setting process is performed again in a state where the range in which the objective lens 3 can move in the Z direction is expanded, so that the upper limit position UP and the lower limit position BP are accurately set over a wide range in the Z direction. It becomes possible to do.

  When searching for the upper limit position UP and the lower limit position BP, gain adjustment and pixel data acquisition are performed substantially in the range from the lower end position zt1 to the upper end position zt2. Therefore, the upper and lower limit automatic setting process takes longer time. Therefore, in the upper / lower limit automatic setting process of this example, the upper limit position UP and the lower limit position BP may be searched as follows.

  As described above, when searching for the upper limit position UP or the lower limit position BP, the position in the Z direction of the objective lens 3 when the evaluation value is finally lower than the noise evaluation level Enl from a value larger than the noise evaluation level Enl is the upper limit position. It is set as UP or lower limit position BP. Therefore, when searching for the upper limit position UP or the lower limit position BP, if the evaluation value is larger than the noise evaluation level Enl, the movement pitch in the Z direction of the objective lens 3 is set small. This reason will be described together with a specific setting example of the movement pitch.

  In the Z direction, the intensity of the laser light emitted from the objective lens 3 to the observation object S is highest at the focal position fp of the objective lens 3 and decreases as the distance from the focal position fp of the objective lens 3 increases. If the intensity of the laser light applied to the surface of the observation object S is too small, the surface of the observation object S cannot be observed accurately. Therefore, the movement pitch in the Z direction of the objective lens 3 when searching for the upper limit position UP or the lower limit position BP is set so that the entire surface of the unit region is irradiated with laser light having a certain intensity or higher.

  As described above, when setting the upper limit position and the lower limit position for one pixel, the gain is set so that the value of the pixel data at the peak position z0 is as close as possible to the output upper limit value max. Therefore, even when the surface of the observation object S is irradiated with laser light with a half intensity of the peak intensity, sufficient reflected light can be obtained for observing the surface of the observation object S.

  Here, the distance between the position in the Z direction and the focal position fp when the intensity of the laser light is ½ of the peak intensity in a state where the objective lens 3 is fixed is called an intensity half distance. In this example, when the evaluation value is larger than the noise evaluation level Enl, the movement pitch in the Z direction of the objective lens 3 is set to the intensity half distance. In this case, when searching for the upper limit position UP or the lower limit position BP, it is possible to irradiate the entire surface of the unit region with laser light with an intensity of ½ or more of the peak intensity. Thereby, the upper limit position UP or the lower limit position BP can be set with high accuracy.

  When the evaluation value is equal to or less than the noise evaluation level Enl (for example, when the position of the objective lens 3 is in the range AZ1, AZ2, AZ3 in FIG. 10), the movement pitch in the Z direction is set large. This reason will be described together with a specific setting example of the movement pitch.

  In a state where the evaluation value is equal to or lower than the noise evaluation level Enl, the upper limit position UP and the lower limit position BP are not set. When the evaluation value is equal to or lower than the noise evaluation level Enl, it is only necessary to determine whether or not the evaluation value has changed to a value larger than the noise evaluation level Enl.

  Therefore, in this example, when the evaluation value is equal to or less than the noise evaluation level Enl, the gain of the light receiving element 30 is increased by a certain amount. In this case, the value of the acquired plurality of pixel data is increased, and the evaluation value is increased. Thereby, even when the intensity of the laser beam irradiated on the surface of the unit region is low, the evaluation value and the noise evaluation level Enl can be compared. Thereby, the movement pitch of the objective lens 3 in the Z direction can be set larger than the above-mentioned intensity half-distance.

  In this way, the movement pitch in the Z direction of the objective lens 3 is set larger than in the case where the evaluation value is larger than the noise evaluation level Enl. As a result, the number of pixel data acquisition times can be reduced, so that the upper and lower limit automatic setting processing time is shortened.

  When the evaluation value is equal to or less than the noise evaluation level Enl, the amount of laser light emitted from the laser light source 10 may be increased by a certain amount instead of increasing the gain of the light receiving element 30 by a certain amount. In this case, the above-mentioned strength half distance is increased. Thereby, the movement pitch in the Z direction of the objective lens 3 can be set larger than when the evaluation value is larger than the noise evaluation level Enl.

  When the evaluation value is equal to or lower than the noise evaluation level Enl, the attenuation factor of the ND filter 8 may be decreased by a certain amount instead of increasing the gain of the light receiving element 30 by a certain amount. In this case, the above-mentioned strength half distance is increased. Thereby, the movement pitch in the Z direction of the objective lens 3 can be set larger than when the evaluation value is larger than the noise evaluation level Enl.

  In the above example, after the upper limit position UP is set, the objective lens 3 is finally moved to the position at the time when the gain of the light receiving element 30 is decreased, and then the gain adjustment is performed until the objective lens 3 reaches the lower end position zt1. The acquisition of pixel data and the movement in the downward direction of the objective lens 3 are repeated, and the objective lens 3 in the Z direction when the evaluation value is finally lower than the noise evaluation level Enl from a value larger than the noise evaluation level Enl. The position is set as the lower limit position BP.

(7) Object Observation Process In the confocal microscope system 500, the object observation process performed by the CPU 210 of the PC 200 in FIG. 1 when observing the observation object S will be described.

  FIG. 11 and FIG. 12 are flowcharts showing the object observation process executed in the confocal microscope system 500. The CPU 210 of the PC 200 in FIG. 1 operates according to the object observation program stored in the storage device 240.

  In the object observation process described below, an upper / lower limit automatic setting process (step S4 to be described later) is executed as a movement range setting process of the present invention, and a process of acquiring pixel data for a unit region as a measurement process of the present invention. (Step S7 described later) is executed. In steps S8 and S9 of the flowchart, the confocal image data and the surface image data are abbreviated as image data. The object observation process includes the above upper / lower limit automatic setting process. As described above, in the m-th upper / lower limit automatic setting process, a plurality of pieces of pixel data already acquired in the overlapping portion ov between the m-th unit area and the unit area adjacent to the m-th unit area are used. It is done. This simplifies the m-th and subsequent upper / lower limit automatic setting processes compared to the first upper / lower limit automatic setting process, so that the time of the mth upper / lower limit automatic setting process is sufficiently short.

  First, the CPU 210 generates camera image data based on the camera data given by the control unit 300, and displays the camera image of the observation object S in the image display area 410 of the display unit 400 of FIG. 5 based on the camera image data. (Step S1).

  Next, the user instructs the acquisition range of the pixel data using the pointing device. CPU210 sets the acquisition range of pixel data based on a user's instruction (step S2). In the present embodiment, when the pixel data acquisition range is larger than the unit region, the position of each unit region and the number of unit regions in the acquisition range are determined so that adjacent unit regions partially overlap each other. The The position of each unit area and the number of unit areas within the determined acquisition range are stored in the work memory 230.

  Subsequently, the CPU 210 moves the stage 60 by applying a driving pulse to the stage driving unit 62 of FIG. 1 based on the position of the unit area stored in the work memory 230 (step S3).

  Subsequently, the CPU 210 performs the above upper / lower limit automatic setting process (step S4). The movement range in the Z direction of the objective lens 3 is set by the upper / lower limit automatic setting process. Details of the upper and lower limit automatic setting processing will be described later (see FIGS. 13 to 16). Thereafter, the CPU 210 determines whether or not the upper / lower limit automatic setting process has succeeded (step S5). When the upper / lower limit automatic setting process is successful, the CPU 210 notifies the control unit 300 of the number of samplings in the X-direction scanning, the number of scanning lines, and the movement range in the Z-direction, and commands acquisition of pixel data for the unit area ( Step S6).

  The control unit 300 controls the XY scan optical system 20 of FIG. 1 and the Z of the objective lens 3 based on the number of samplings in the X direction, the number of scanning lines, and the movement range in the Z direction notified from the CPU 210. The position (position in the Z direction of the observation object S) is moved, and pixel data is given to the CPU 210 of the PC 200 based on the light reception signal output from the light receiving element 30. CPU210 acquires the pixel data about a unit area from the control part 300 (step S7). The CPU 210 generates a plurality of confocal image data of the unit area based on the acquired pixel data and also generates surface image data (step S8) and stores it in the storage device 240.

  Here, the CPU 210 determines whether or not the confocal image data and the surface image data of all the unit areas have been generated (step S9). When the confocal image data and the surface image data of all the unit areas have not been generated, the CPU 210 returns to the process of step S3. The CPU 210 moves the stage 60 to a position where the confocal image data of the next unit area can be generated, and repeats the processes of steps S4 to S9.

  On the other hand, when the confocal image data and the surface image data of all the unit areas are generated in step S <b> 9, the CPU 210 connects the surface image data of the plurality of unit areas stored in the storage device 240 using the storage device 240. (Step S10). Thereby, surface image data of the designated acquisition range is generated. When only one unit region is included in the pixel data acquisition range, the surface image data is not connected in step S10.

  Thereafter, the CPU 210 causes the display unit 400 to display an image of the surface of the observation object S based on the connected surface image data using the work memory 230 (step S11). Finally, the CPU 210 stores the connected surface image data in the storage device 240 and ends the object observation process.

  If the upper / lower limit automatic setting process fails in step S5, the CPU 210 displays an image indicating that the image process has failed on the display unit 400 (step S12), and ends the object observation process.

  Thus, in the object observation process of FIGS. 11 and 12, the object observation process ends when the upper and lower limit automatic setting process fails in steps S5 and S12. Not limited to this, when the upper / lower limit automatic setting process fails in step S5, the CPU 210 sets the upper end position zt2 (FIGS. 7, 9, and 10) as the upper limit position UP, and sets the lower end position zt1 (FIG. 7, 9 and 10) may be set as the lower limit position BP. In this case, the CPU 210 may proceed to step S6 after setting the lower limit position BP and the upper limit position UP. Thereby, even when the upper / lower limit automatic setting process fails, the display unit 400 can display an image of the surface of the observation object S.

  Not limited to the above example, when the upper / lower limit automatic setting process fails in step S5, the CPU 210 sets a position a certain distance higher than the upper end position zt2 (FIGS. 7, 9, and 10) as a new upper end position, After a position lower than the lower end position zt1 (FIGS. 7, 9, and 10) by a certain distance is set as a new lower end position, an upper / lower limit automatic setting process in step S4 based on the newly set upper end position and lower end position. May be performed again.

  A constant distance between the upper end position zt2 (FIGS. 7, 9, and 10) and the new upper end position, and a constant distance between the lower end position zt1 (FIGS. 7, 9, and 10) and the new lower end position. May be equal or different from each other. These fixed distances may be predetermined distances depending on the magnification of the objective lens 3 or may be distances set by a user using a pointing device such as a mouse connected to the PC 200 (FIG. 1). There may be.

  As described above, when the upper limit position UP and the lower limit position BP are not set within a predetermined range in which the objective lens 3 can move in the Z direction, new upper limit positions and lower limit positions are set. Thus, the upper / lower limit automatic setting process is performed again in a state where the range in which the objective lens 3 can move in the Z direction is expanded, so that the upper limit position UP and the lower limit position BP are accurately set over a wide range in the Z direction. It becomes possible to do.

  When the upper limit position UP is set and the lower limit position BP is not set, the CPU 210 sets only the new upper end position and sets the newly set upper end position and lower end position zt1 (FIGS. 7, 9, and 10). ), The upper / lower limit automatic setting process of step S4 may be performed again. Similarly, when the lower limit position BP is set and the upper limit position UP is not set, the CPU 210 sets only the new lower end position, and sets the newly set lower end position and upper end position zt2 (FIG. 7, FIG. 9 and FIG. The upper and lower limit automatic setting process of step S4 may be performed again based on 10).

(8) Upper / Lower Limit Automatic Setting Process FIGS. 13 to 15 are flowcharts showing the upper / lower limit automatic setting process of FIG. As shown in FIG. 13, the CPU 210 first determines whether or not the current upper / lower limit automatic setting process is the first upper / lower limit automatic setting process (step S20). When the current upper / lower limit automatic setting process is the first upper / lower limit automatic setting process, the CPU 210 detects the evaluation peak position Ez0 by performing the peak position search process (see FIG. 7) using the evaluation value. (Step S21). Subsequently, the CPU 210 sets the detected evaluation peak position Ez0 as the initial position of the first upper / lower limit automatic setting process, and moves the objective lens 3 to the initial position (step S22).

  At the time when the m-th upper / lower limit automatic setting process is started, a plurality of pixel data is already acquired for the overlapping portion ov between the m-th unit region and the unit region adjacent to the m-th unit region. Yes. Therefore, when the current upper / lower limit automatic setting process is the mth upper / lower limit automatic setting process other than the first in step S20, the CPU 210 determines the initial position based on the plurality of pixel data for the overlapping portion ov. The determined initial position is stored in the work memory 230 (step S23). Thereafter, the CPU 210 moves the objective lens 3 to the initial position (step S24). Further, the CPU 210 adjusts the gain of the light receiving element 30 in a state where the objective lens 3 is at the initial position (step S25). Specifically, the CPU 210 acquires all the pixel data values for the unit area in a state where the objective lens 3 is at the initial position, and all the acquired pixel data values are equal to or greater than a predetermined value. The gain of the light receiving element 30 is adjusted so that Thereby, the value of the acquired several pixel data can be made sufficiently larger than the noise level nl.

  As described above, in the m-th upper / lower limit automatic setting process, a plurality of pieces of pixel data already acquired in the overlapping portion ov between the m-th unit area and the unit area adjacent to the m-th unit area are used. It is done. Thereby, it is not necessary to detect the evaluation peak position Ez0 in the m-th and later upper and lower limit automatic setting processes. Accordingly, since the m-th and lower upper / lower limit automatic setting processes are simplified compared to the first upper / lower limit automatic setting process, the time of the mth upper / lower limit automatic setting process is sufficiently shortened.

  After the process of step S22 or step S25, the CPU 210 starts the boundary position search process of FIGS. 16 to 18 described later in one direction of the Z direction (for example, the upward direction) with the objective lens 3 in the initial position. (Step S26). The boundary position search process is a process of searching for the upper limit position UP or the lower limit position BP while adjusting the gain of the light receiving element 30. Details of the boundary position search processing will be described later.

  Subsequently, the CPU 210 determines whether or not the upper limit position UP is set in step S28, that is, whether or not the boundary position search process is successful (step S27). When the boundary position search process fails, the CPU 210 proceeds to the process of step S149 described later.

  In the boundary position search process, the gain of the light receiving element 30 may be decreased by a certain amount in step S81 of FIG. Therefore, when the boundary position search process is successful in step S27, the CPU 210 determines whether or not the gain of the light receiving element 30 has decreased in the boundary position search process of step S26 (step S28). If the gain of the light receiving element 30 has not decreased, the CPU 210 moves the objective lens 3 to the initial position (step S29). On the other hand, when the gain of the light receiving element 30 is decreased, the CPU 210 stores the objective lens 3 last stored in step S71 (FIG. 16) or step S82 (FIG. 16) of the boundary position search process described later. To the Z position (step S30). Thereafter, the CPU 210 starts the boundary position search process of FIGS. 16 to 18 described later in the reverse direction of the Z direction (for example, the downward direction) (step S31).

  Subsequently, the CPU 210 determines whether or not the lower limit position BP is set in step S31, that is, whether or not the boundary position search process is successful (step S32). When the boundary position search process fails, the CPU 210 proceeds to the process of step S149 described later.

  When the boundary position search process is successful, the CPU 210 determines whether or not the gain of the light receiving element 30 has decreased in the boundary position search process of step S31 (step S33), similarly to step S28.

  In the boundary position search process described later, the boundary position of the Z position of the objective lens 3 is stored or updated in the work memory 230 in the process of step S76 in FIG. If the gain of the light receiving element 30 has not decreased in step S33, the CPU 210 sets the upper limit position UP of the objective lens 3 based on the boundary position stored by the boundary position search process in step S26, and the boundary position in step S31. The lower limit position BP of the objective lens 3 is set based on the boundary position stored by the search process (step S34), and the upper / lower limit automatic setting process is terminated.

  When the gain of the light receiving element 30 is decreased in step S34, the CPU 210 stores the objective lens 3 last in step S71 (FIG. 16) or step S82 (FIG. 16) of the boundary position search process described later. The objective lens 3 is moved to the Z position (step S146). Thereafter, the CPU 210 starts again the boundary position search process of FIGS. 16 to 18 described later with respect to one direction (eg, upward direction) in the Z direction (step S147).

  Subsequently, the CPU 210 determines whether or not the upper limit position UP is set in step S147, that is, whether or not the boundary position search process is successful (step S148). When the boundary position search process fails, the CPU 210 proceeds to the process of step S149 described later.

  When the boundary position search process is successful, the CPU 210 proceeds to the process of step S34. In step S34, the CPU 210 sets the upper limit position UP of the objective lens 3 based on the boundary position stored in step S147, and sets the lower limit position BP of the objective lens 3 based on the boundary position stored in step S31. . Accordingly, the upper limit position UP and the lower limit position BP of the objective lens 3 are set in a state where the gain of the light receiving element 30 is adjusted to an optimum value.

  When the boundary position search process fails in step S148, the CPU 210 determines whether or not the current upper / lower limit automatic setting process is the first upper / lower limit automatic setting process (step S149). When the current upper / lower limit automatic setting process is the first upper / lower limit automatic setting process, the CPU 210 proceeds to the process of step S12 in FIG. 11 and causes the display unit 400 to display an image indicating that the image process has failed.

  At the time when the m-th upper / lower limit automatic setting process is started, a plurality of pixel data is already acquired for the overlapping portion ov between the m-th unit region and the unit region adjacent to the m-th unit region. Yes. Therefore, in step S149, when the current upper / lower limit automatic setting process is the mth upper / lower limit automatic setting process other than the first, the CPU 210 determines the upper limit position UP and the lower limit based on the plurality of pixel data for the overlapping portion ov. The position BP is set (step S150), and the upper / lower limit automatic setting process is terminated. Thus, in the m-th upper / lower limit automatic setting process, even when the boundary position search process described later fails, the upper limit position UP and the lower limit position BP are based on a plurality of pieces of pixel data for the overlapped portion ov already stored. Is set.

  In step S150, the CPU 210 obtains the lowest height of the surfaces of the overlapping portion ov in the Z direction based on the plurality of pixel data of the overlapping portion ov, and the objective when the focal position matches the obtained height. The position of the lens 3 in the Z direction may be set as the lower limit position BP. The size of the objective lens 3 in the XY directions is sufficiently larger than the size of each unit area. Therefore, when the position in the Z direction of the objective lens 3 when the focal position coincides with the lowest height among the surfaces of the overlapped portion ov is set as the lower limit position BP, the objective lens 3 is used when acquiring a plurality of pixel data. Colliding with the surface of the observation object S is prevented.

  In step S150, the CPU 210 obtains the highest height among the surfaces of the overlapping portion ov in the Z direction based on the plurality of pixel data of the overlapping portion ov, and the objective when the focal position matches the determined height. The position in the Z direction of the lens 3 may be set as the upper limit position BP.

  In step S150, the CPU 210 may set the upper limit position UP and the lower limit position BP already set in the adjacent unit area as the upper limit position UP and the lower limit position BP of the current unit area, respectively.

  Further, in step S150, the CPU 210 may set the upper limit position UP of the current unit area at a position higher than the upper limit position UP already set in the adjacent unit area. Further, the CPU 210 may set the lower limit position BP of the current unit area at a position lower than the lower limit position BP that has already been set in the adjacent unit area.

(9) Boundary Position Search Processing Method FIGS. 16 to 18 are flowcharts showing the boundary position search process of FIGS. 14 and 15. In the confocal microscope system 500 according to the present embodiment, the boundary position search processing method can be switched between two modes. One of the two modes is the upper / lower limit automatic setting process described with reference to FIG. 8, and the other of the two modes is the upper / lower limit automatic setting process described with reference to FIG. This flowchart corresponds to the upper and lower limit automatic setting processing described with reference to FIG.

  As shown in FIG. 16, the CPU 210 stores the current Z position of the objective lens 3 in the work memory 230 (step S70).

  Subsequently, the CPU 210 acquires all pixel data values for the unit area at the current Z position of the objective lens 3, and stores the acquired pixel data values in the work memory 230 of FIG. S71).

  Next, the CPU 210 determines whether or not any value of the stored plurality of pixel data is the output upper limit value max (FIG. 6) (step S72). When the values of all the pixel data are not the output upper limit value max (FIG. 6), the CPU 210 calculates the evaluation value by acquiring the values of all the pixel data for the unit area (step S73), and the calculated evaluation It is determined whether or not the value is equal to or less than a predetermined threshold value (step S74). Here, the threshold value may be set to a value equal to the noise evaluation level Enl (FIGS. 7 and 10), or set to a value larger than the noise evaluation level Enl (FIGS. 7 and 10). May be. Furthermore, a plurality of threshold values may be set so as to correspond to a plurality of gains of light receiving elements 30 different from each other. In this case, in step S74, the CPU 210 determines whether or not the calculated evaluation value is equal to or less than a threshold value corresponding to the gain of the light receiving element 30 that is currently set.

  If the Z position of the objective lens 3 does not reach the lower end position zt1 (FIGS. 7, 9, and 10) or the upper end position zt2 (FIGS. 7, 9, and 10) in the process of step S77 described later, The processes of steps S71 to S74 are repeated. When the calculated evaluation value is equal to or less than the threshold value, the CPU 210 determines whether or not the evaluation value was larger than the threshold value during the previous processing of step S74 (step S75). If the evaluation value is equal to or less than the threshold value during the previous processing in step S74, the CPU 210 proceeds to processing in step S77 described later.

  On the other hand, if the evaluation value is larger than the threshold value in the previous processing of step S74, the CPU 210 stores the current Z position of the objective lens 3 in the work memory 230 as a boundary position in one direction of the Z direction. Alternatively, when the boundary position is already stored in the work memory 230, the CPU 210 updates the boundary position with the current Z position of the objective lens 3 (step S76).

  Subsequently, the CPU 210 determines whether or not the Z position of the objective lens 3 has reached the lower end position zt1 or the upper end position zt2 (step S77). When the Z position of the objective lens 3 reaches the lower end position zt1 or the upper end position zt2, the boundary position search process ends. On the other hand, when the Z position of the objective lens 3 does not reach the lower end position zt1 or the upper end position zt2, the CPU 210 moves the objective lens 3 in the Z direction in the second pitch (step S87), and performs the process of step S71. Return. The second pitch is set to be larger than the first pitch described later. By setting the second pitch to be larger than the first pitch, the time for the boundary position search process is shortened.

  In step S72, when the value of any pixel data is the output upper limit value max, the CPU 210 reduces the gain of the light receiving element 30 by a certain amount and stores the fact that the gain has been reduced in the work memory 230 (step S81). ). Subsequently, the CPU 210 updates the Z position of the objective lens 3 stored in the work memory 230 in step S70 with the current Z position of the objective lens 3 (step S82), and returns to the process of step S71.

  If the calculated evaluation value is larger than the threshold value in step S74, the CPU 210 moves the objective lens 3 in the Z direction in the first pitch (step S83), and the Z position of the objective lens 3 is the lower end position. It is determined whether or not zt1 or upper end position zt2 has been reached (step S84). The first pitch is set to a size equal to the above-described intensity half distance. Thereby, the upper limit position UP and the lower limit position BP of the objective lens 3 can be set with high accuracy.

  In step S84, when the Z position has not reached the lower end position zt1 or the upper end position zt2, the CPU 210 returns to the process of step S71. On the other hand, when the Z position reaches the lower end position zt1 or the upper end position zt2, as shown in FIG. 18, the CPU 210 determines whether or not the boundary position is stored in the work memory 230 by the process of step S76 ( Step S85).

  If the boundary position is stored in the work memory 230, the boundary position search process ends. On the other hand, when the boundary position is not stored in the work memory 230, the CPU 210 determines that the boundary position search process has failed (step S86).

  As described above, in the boundary position search process of FIGS. 16 to 18, the CPU 210 moves the objective lens 3 in the Z direction until the Z position of the objective lens 3 reaches the lower end position zt1 or the upper end position zt2.

  In contrast, the CPU 210 does not perform steps S75, S77, S84, and S87 in the upper and lower limit automatic setting process described with reference to FIG. That is, the CPU 210 ends the boundary position search process when the boundary position is stored in the work memory 230 in the process of step S76 of FIG. In this case, it is not necessary to move the objective lens 3 in the Z direction over the entire range from the lower end position zt1 to the upper end position zt2 during the boundary position search process. As a result, the time for the boundary position search process is shortened.

(10) Another Example of Boundary Position Search Processing Method FIG. 19 is a flowchart illustrating another example of the boundary position search process. FIG. 19 illustrates only processing different from the boundary position search processing of FIGS.

  As shown in FIG. 19, after the process of step S <b> 87 in FIG. 17, the CPU 210 may perform the following process in addition to the boundary position search process in FIGS. 16 to 18.

  After step S87, the CPU 210 increases the gain of the light receiving element 30 by a certain amount (step S91). Subsequently, the CPU 210 acquires all pixel data values for the unit area at the current Z position of the objective lens 3, and stores the acquired pixel data values in the work memory 230 of FIG. S92).

  Next, the CPU 210 calculates an evaluation value based on the value of the pixel data acquired in step S92 (step S93), and determines whether or not the calculated evaluation value is equal to or less than a predetermined threshold value. (Step S94).

  Here, when the gain of the light receiving element 30 is increased in step S91, the calculated evaluation value is increased. Therefore, in this example, the threshold value in step S94 is set to a value obtained by multiplying the gain ratio of the light receiving element 30 before and after the process in step S91 by the noise evaluation level Enl (FIGS. 7 and 10). The

  When the calculated evaluation value is equal to or less than the threshold value, the CPU 210 proceeds to the process of step S87 in FIG. On the other hand, when the calculated evaluation value is larger than a predetermined threshold value, the CPU 210 decreases the gain of the light receiving element 30 by a certain amount (step S95). The amount of gain decrease at this time is set equal to the amount of gain increase in step S91. Thereafter, the CPU 210 proceeds to the process of step S83 in FIG.

  As described above, in this example, when the evaluation value is equal to or less than the threshold value, the gain of the light receiving element 30 temporarily increases. Thereby, the value of the acquired plurality of pixel data is increased, and the evaluation value is increased. Thereby, even when the intensity of the laser beam irradiated on the surface of the unit region is low, the evaluation value and the noise evaluation level Enl can be compared. Thereby, the second pitch can be set sufficiently larger than the first pitch. As a result, the number of pixel data acquisition times can be sufficiently reduced, and the time for the boundary position search process is further shortened.

  In step S91, instead of increasing the gain of the light receiving element 30 by a certain amount, the CPU 210 may set the light amount of the laser light emitted from the laser light source 10 to be larger by a certain amount, or the attenuation of the ND filter 8. The rate may be set smaller by a certain amount. In this case, in step S95, the CPU 210 sets the light amount of the laser light emitted from the laser light source 10 to a small amount instead of decreasing the gain of the light receiving element 30 by a certain amount, or the attenuation factor of the ND filter 8 Is set larger by a certain amount. Even in these cases, the time for the boundary position search process can be further shortened.

(11) Other Embodiments (11-1) In the above embodiment, the upper and lower limit automatic setting process is executed in the confocal microscope system 500. However, the upper and lower limit automatic setting processing is not limited to this, and the state of the surface of the observation object is projected based on the digital signal that is projected to the observation object and acquired in accordance with the intensity of the light guided to the light receiving element 30. It can be applied to a microscope system for observing. As such a microscope system, for example, there is a microscope system using optical interferometry.

  (11-2) In the above embodiment, the relative position of the observation object S with respect to the objective lens 3 in the Z direction is changed by moving the objective lens 3 in the Z direction. However, the present invention is not limited to this. . The relative position of the observation object S with respect to the objective lens 3 in the Z direction may be changed by moving the stage 60 in the Z direction.

  (11-3) In the above-described embodiment, the laser beam is scanned on the observation object S in the X direction and the Y direction by controlling the XY scan optical system 20, but the present invention is not limited to this. The laser beam may be scanned on the observation object S in the X direction and the Y direction by moving the stage 60.

  Further, line light (for example, elongated light extending in the X direction) may be used as the laser light. In this case, instead of the XY scan optical system 20, a Y scan optical system that does not perform scanning in the X direction is used. Further, instead of the light receiving element 30, a line CCD camera or the like including a plurality of light receiving elements arranged in a direction corresponding to the X direction is used.

  The size of the light receiving surface in the direction corresponding to the Y direction of each light receiving element of the line CCD camera is generally several tens of μm. In this case, the light receiving surface of the line CCD camera is disposed at the focal position of the lens 2. When the surface of the observation object S is at the focal position of the objective lens 3, the line light reflected by the observation object S is collected on the light receiving surface of the line CCD camera. Thereby, most of the line light reflected by the observation object S enters the light receiving surface of the line CCD camera.

  On the other hand, when the observation object S is at a position deviating from the focal position of the objective lens 3, the line light reflected by the observation object S is condensed at a position before or after the light receiving surface of the line CCD camera. Thereby, only part of the line light reflected by the observation object S enters the light receiving surface of the line CCD camera. Therefore, it is not necessary to arrange the pinhole member 7 in front of the line CCD camera.

  (11-4) In the above embodiment, the CPU 210 of the PC 200 may have the function of the control unit 300. In this case, the control unit 300 may not be provided.

  (11-5) In the above embodiment, the evaluation value is the sum of the values of all the pixel data for the unit area. However, the evaluation value is not limited to this, and may be an average value of all pixel data values for the unit area. Further, the evaluation value may be a sum or average value of some pixel data values in the unit area.

(12) Correspondence between each component of claim and each part of embodiment The following describes an example of a correspondence between each component of the claim and each part of the embodiment. It is not limited.

  In the above embodiment, the observation object S is an example of the observation object, the confocal microscope system 500 is an example of the microscope system, and the objective lens 3 is an example of the optical system.

  The unit regions s1 to s4 are examples of unit regions, the lens driving unit 63 is an example of a moving unit, the control unit 300 and the CPU 210 of the PC 200 are examples of control units, and the PC 200 is an example of an observation range setting unit. The control unit 300 is an example of a pixel data output unit.

  Furthermore, the range defined by the upper limit position UP and the lower limit position BP is an example of the movement range set in the height image data generation process.

  The lower end position zt1 is an example of a predetermined first distance, the upper end position zt2 is an example of a predetermined second distance, and the first pitch is an example of a first movement amount. The second pitch is an example of the second movement amount.

  Further, a new lower end position that is lower than the lower end position zt1 (FIGS. 7, 9, and 10) set when the upper / lower limit automatic setting process fails in step S5 of FIG. 11 is the third distance. It is. Furthermore, a new upper end position that is a fixed distance higher than the upper end position zt2 (FIGS. 7, 9, and 10) set when the upper / lower limit automatic setting process fails in step S5 in FIG. 11 is an example of the fourth distance. It is.

  As each constituent element in the claims, various other elements having configurations or functions described in the claims can be used.

  The present invention can be effectively used in various microscope systems.

DESCRIPTION OF SYMBOLS 1, 2 Lens 3 Objective lens 4-6 Half mirror 7 Pinhole member 8 ND filter 10 Laser light source 20 XY scan optical system 30 Light receiving element 40 White light source for illumination 50 Color CCD camera 60 Stage 61 Stage operation part 62 Stage drive Part 63 Lens drive part 200 PC
210 CPU
220 ROM
230 Working Memory 240 Storage Device 300 Control Unit 400 Display Unit 410 Image Display Area 420 Condition Setting Area 421 Range Setting Button 422 Acquisition Start Button 500 Confocal Microscope System AZ1, AZ2, AZ3 Range BP Lower Limit Position Emax, max Output Upper Limit Value Enl Noise evaluation level Ez0 Evaluation peak position Ez1, Ez2, ma1, ma2, mb1, mb2, zn1, zn2, zn3, zn4, zs1, zs2 Position fp Focal position nl Noise level s1 to s4 Unit area ov Overlapping part pi1, pi2 Value UP Upper limit position S Observation target Sa1 First surface Sa2 Second surface Sa3 Third surface z0 Peak position zp1 First Z position zp2 Second Z position zp3 Third Z position zt1 Lower end position zt2 Upper end position

Claims (7)

A microscope system for measuring the surface state of a measurement object,
An observation range setting unit for setting a plurality of unit areas as an observation range on the surface of the measurement object;
A light receiving element;
For each unit region set by the observation range setting unit, it irradiates the light while converging to the unit region through the objective lens, an optical system for guiding light irradiated to the unit region in the light receiving element ,
For each unit region set by the observation range setting unit, so that the irradiation of light by the optical system at a plurality of positions along the direction perpendicular to the unit region is performed, the initial position determined for each unit area A moving unit that moves the focal point of the optical system to a plurality of positions in the optical axis direction of the optical system by moving the objective lens relative to the measurement object in the optical axis direction of the optical system; ,
A pixel data output unit that outputs a plurality of pixel data respectively corresponding to a plurality of pixels based on an output signal of the light receiving element;
A control unit for controlling the light receiving element and the moving unit ,
The controller is
For each unit region set by the observation range setting unit, the objective lens is in the initial position determined for the unit region, and based on the pixel data output from the pixel data output unit, the light receiving element A first process for adjusting the gain;
The objective lens is moved by the moving unit in the optical axis direction from the initial position determined for the unit region, and a plurality of pieces of pixel data output from the pixel data output unit at each position in the optical axis direction. A second process of calculating an evaluation value using pixel data values corresponding to a predetermined number of pixels and automatically setting an upper limit position and a lower limit position in the optical axis direction based on the calculated evaluation value;
The objective lens is moved by the moving unit within a moving range determined by the upper limit position and the lower limit position set by the second processing, and a plurality of pixels output from the pixel data output unit at each position in the optical axis direction. A third process for generating, as height image data, data representing peak positions in the optical axis direction of a plurality of pixel data for a plurality of pixels corresponding to the unit region, based on the pixel data;
Concatenation that connects a plurality of height image data generated by repeatedly executing the first process, the second process, and the third process for a plurality of unit areas set by the observation range setting unit A microscope system characterized by executing processing . The control unit automatically executes a process of generating ultra-depth image data based on a plurality of pixel data for each of the plurality of unit regions set by the observation range setting unit, and is generated for each of the plurality of unit regions. Concatenating multiple ultra-deep image data,
The microscope system according to claim 1, wherein the ultra-deep image data is data representing peak intensity in the optical axis direction of a plurality of pixel data for a plurality of pixels corresponding to each unit region. When the control unit moves the objective lens to a plurality of positions in the optical axis direction in the second processing, the pixel data value output from the pixel data output unit is a predetermined output upper limit value. The microscope system according to claim 1, wherein the gain of the light receiving element is decreased so as to be smaller than the minimum. The second processing, the pixel data output at each position of the objective lens while moving the objective lens in a direction and away from the direction of approaching the objective lens is the observation object from the determined initial position for each unit area Including a process of acquiring a plurality of pixel data output from the unit,
The control unit is acquired when the objective lens is moving in the direction in which the objective lens and the observation object approach from the determined initial position in the second processing for each unit region. The objective lens at a time point when the level of the output signal of the light receiving element changes below the predetermined level from a state in which the level of the output signal of the light receiving element is higher than a predetermined level based on the pixel data. Based on the acquired pixel data when the objective lens is moved in a direction in which the objective lens and the observation object move away from the determined initial position. The level of the output signal of the light receiving element is determined from the state in which the level of the output signal of the light receiving element is higher than the predetermined level. Characterized in that detecting the position of the objective lens at the time that changes below the bell as a second relative position, sets the movement range of the unit region on the basis of the first and second relative position detected The microscope system according to any one of claims 1 to 3. The second processing, the pixel data output at each position of the objective lens while moving the objective lens in a direction and away from the direction of approaching the objective lens is the observation object from the determined initial position for each unit area Including a process of acquiring a plurality of pixel data output from the unit,
In the second process for each unit region, the control unit determines that the relative distance between the objective lens and the observation object from the determined initial position is greater than the first distance from the first distance. The objective lens is moved relative to the object to be observed over a predetermined range until the second distance becomes greater, and the level of the output signal of the light receiving element is determined based on the acquired pixel data. Detecting a plurality of positions of the objective lens at a plurality of points in time when the level of the output signal of the light receiving element changes below the predetermined level from a state where is higher than a predetermined level. The microscope system according to claim 1, wherein the movement range for the unit region is set so as to include the position of the unit region. In the second process for each unit region, the control unit determines whether the level of the output signal of the light receiving element is higher than a predetermined level based on the acquired pixel data, When the level of the output signal of the light receiving element is larger than the predetermined level, the objective lens is moved relative to the observation object by a first movement amount, and the output signal of the light receiving element is When the level is equal to or lower than the predetermined level, the objective lens is moved relative to the observation object by a second movement amount;
The microscope system according to claim 5, wherein the second movement amount is larger than the first movement amount. In the second processing for each unit region, the control unit moves the objective lens relative to the observation object over a predetermined range from the first distance to the second distance. When the movement range is not set even if the movement is performed, the relative distance between the objective lens and the observation object is smaller than the first distance from the determined initial position. The objective lens is moved relative to the observation object over a predetermined range until the fourth distance is larger than the second distance, and the light reception is performed based on the acquired pixel data. wherein the plurality of time the level of the output signal changes below said predetermined level of said light receiving element from a state higher than the level at which the level reaches a predetermined output signal of the element objective Lens of the plurality of positions respectively detected, the microscope system according to claim 5 or 6, wherein the setting the movement range of the unit region so as to include the detected plurality of positions.

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