JP2010072230A - Microscope system, and method of observing observation object - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope system for achieving more excellent autofocus, and a method of observing an observation object. <P>SOLUTION: The microscope system includes: an objective lens 29; a stage 27; a holding part installed on the stage 27; a container-like observation sample held by the holding part and having a placing surface 22 on which the observation object 25 is placed in a bottom thereof; a focusing drive part 149 driving at least one of the objective lens 29 and the holding part in the optical axis direction of the objective lens 29; a control part 151 controlling the focusing drive part 149; and a reference member 15 which is provided in the vicinity of the placing surface 22 of the bottom of the observation sample and in which a reference mark 17 for autofocus is formed in a position facing the placing surface 22 in the optical axis direction of the objective lens 29. The control part 151 controls the focusing drive part 149, to perform the autofocus with respect to the reference mark 17, move at least one of the objective lens 29 and the holding part to a reference position and then, move at least one of the objective lens 29 and the holding part by a previously set offset amount. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a microscope system and an observation method of an observation body.

  When observing for a long time using a microscope, an autofocus device may be mounted for the purpose of preventing the specimen from being out of focus with time.

In order to increase the accuracy of autofocus, an infrared reflecting film or high-contrast marking may be provided on the microscope main body rather than an observation sample consisting of a specimen or a container. For example, a method is disclosed in which high-contrast marking is performed on a tray that holds an observation sample, and autofocus accuracy is increased by a known hill-climbing method (see Patent Document 1).
JP 2006-189470 A

  For example, when moving over a wide range such as a quadruple dish, the offset increases, and the error caused by the change in the ambient temperature also increases.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a microscope system and a method for observing an observation body that realize better autofocus.

  In order to solve the above problems, a microscope system of the present invention includes an objective lens, a stage, a holding unit installed on the stage, and a holding surface that is held by the holding unit and on which an observation object is placed. A container-like observation sample, a focusing drive unit that drives at least one of the objective lens and the holding unit in an optical axis direction of the objective lens, a control unit that controls the focusing drive unit, and the observation A reference member provided in the vicinity of the mounting surface at the bottom of the sample and provided with a reference mark for autofocus at a position facing the mounting surface in the optical axis direction of the objective lens, and the control unit Controlling the focusing drive unit, performing autofocus on the reference mark and moving at least one of the objective lens and the holding unit to a reference position, and then at least the objective lens and the holding unit on the other hand Characterized in that it moves by a preset offset amount.

  The observation object observation method of the present invention is the observation object observation method using the microscope system, wherein the reference position is updated every time auto-focusing is performed on the reference mark, and the objective lens is updated from the updated reference position. And observation at one or more observation points by moving at least one of the holding unit and a predetermined offset amount.

  ADVANTAGE OF THE INVENTION According to this invention, the microscope system and the observation method of an observation body which implement | achieve more favorable autofocus are provided.

  Hereinafter, a microscope system and an observation method of an observation body according to an embodiment of the present application will be described with reference to the drawings.

  FIG. 1 is a diagram illustrating a configuration of a microscope system according to an embodiment of the present application.

  FIG. 2 is an enlarged view showing a part of the microscope system of FIG.

  A microscope system according to an embodiment of the present application is substantially configured by combining a microscope and a culture apparatus for culturing specimens such as cultured cells. As shown in FIG. 1, the microscope system 100 includes a stage 27 on which the culture chamber 1 is placed and fixed, a moving block 9 inside the culture chamber 1 that can move in one horizontal direction, and a moving block 9. An objective lens 29 is provided so as to face the mounted quadruple dish 19 and the quadruple dish 19 that holds the specimen.

  The microscope system 100 includes a humidifier 101 that sends adjusted air into the culture chamber 1, a temperature controller 105 for controlling the temperature in the housing 103 that blocks the microscope system 100 and the outside air, and an imaging device 107.

The humidifier 101 includes a heater 109 and a temperature sensor 111, and CO ₂ 5% air is supplied from a tube 115 to a container 113 filled with distilled water maintained at a temperature suitable for culture (for example, 37 ° C.). introduced through sterile filter, to obtain a CO 2 _5% air high humidity about 95% by bubbling. High-humidity CO ₂ 5% air is supplied into the culture chamber 1 by an air supply tube 117 and discharged to the outside by an exhaust tube 119. In this way, an environment that matches the culture conditions of the specimen can be realized in the culture chamber 1.

  The temperature control device 105 includes a heater 121 for adjusting temperature in the housing 103, a fan 123 for blowing air, and a temperature sensor (not shown). Hot air is sent to the upper part of the microscope system 1 through the duct 125 by the fan 123, and the hot air is circulated in the housing 103. Further, the temperature in the housing 103 is detected by a temperature sensor (not shown), and the heater 121 is controlled so as to maintain a constant temperature, for example, 37 ° C. In this way, the temperature in the housing 103 can be adjusted.

  The imaging device 107 is connected to the housing 103 via a heat insulating member 127, and acquires sample image data via the objective lens 29, the mirror 129, the imaging lens 131, and the like.

  The microscope system 100 includes a transmission illumination device 133 and an epi-illumination device 135. The light source 137 of the transmission illumination device 133 provided above the culture chamber 1 is preferably an LED light source in which the temperature inside the housing 103 does not easily rise. Illumination light from an epi-illumination device 135, a part of which is shown, is introduced from the outside of the housing 103 through an optical fiber 139, and is irradiated onto the specimen via an optical system including the filter block 141, the objective lens 29, and the like. The microscope system 100 also includes a power supply device 143 and a humidity control device 145.

  The microscope system 100 includes a focusing drive unit 149 including an XY drive unit (not shown) that controls the position of the objective lens 29 in the XY direction and a Z drive unit 147 that controls the position of the objective lens 29 in the Z direction. . By controlling the focusing drive unit 149 by the drive system control device 151 which is a control unit, the position of the objective lens 29 in the Z direction can be electrically controlled for focusing.

  As shown in FIG. 2, the moving block 9 provided inside the culture chamber 1 having the chamber lid 3, the shield glasses 5, 7 and the like is cultured by a moving mechanism such as a guide mechanism, a ball screw 11 and a motor 13 (not shown). It can move in one direction along the stage 27 in the chamber 1. By controlling the moving means by the drive system controller 151, the position of the moving block 9 in one direction can be electrically controlled. The dish to be observed can be switched by moving the moving block 9 and moving an arbitrary dish in the quadruple dish 19 held by the moving block 9 to a position facing the objective lens 29. Further, a reference glass 15 provided with four reference marks 17 is fixed to the bottom of the moving block 9 in order to close the opening formed in the bottom and improve the accuracy of autofocus.

  The observation sample is a four-dish 19, a cover glass 21 fixed to the bottom of the four-dish 19, and an observation body arranged on a bottom surface 22 (hereinafter referred to as a placement surface) of a box-shaped part formed by them. 25 and a dish lid 23 that covers the upper part of the quadruple dish 19. The observation body 25 placed on the placement surface 22 is made of, for example, a specimen and a culture solution. Therefore, the observation body 25 is located above the cover glass 21 and the objective lens 29 is located below the cover glass 21.

  The observation body 25 can be placed on the four placement surfaces 22 respectively corresponding to the four dishes of the quadruple dish 19. Further, the four reference marks 17 on the reference glass 15 are provided at positions facing the four placement surfaces 22 in the optical axis direction of the objective lens 29.

  The drive system control device 151 of the microscope system 100 includes a known passive autofocus unit and controls the Z drive unit 147. That is, the autofocus unit optically detects the focus position of the observation optical system from the objective lens 29 and the imaging device 107 by a known technique, and sends the information to the Z drive unit 147. The Z drive unit 147 moves the objective lens 29 to the focus position of the observation sample according to the information from the autofocus unit.

  The procedure for setting the imaging conditions is as follows. First, the moving block 9 is moved, and the quadruple dish 19 is switched to a position where the first dish is observed. After switching, there is a reference mark 17 on the reference glass 15 within the XY movement range of the objective lens 29, the optical axis of the objective lens 29 is made to coincide with the reference mark 17, and auto-focusing with respect to the reference mark 17 is performed. I do. The position of the objective lens 29 in focus with respect to the reference mark 17 becomes the reference position. Each time autofocus is performed, the Z coordinate of the reference position is updated.

  Next, in order to focus the desired specimen, the observer first moves the objective lens 29 in the YX direction by the drive system controller 151 to determine the observation position, and then moves the objective lens 29 in the Z direction. And focus on the observation position. After focusing on the sample, the position of the objective lens 29 in the Z direction is registered. The distance between the reference position and the registered position is an offset. There is only one reference in the same dish, and it is a common reference for a plurality of observation points. In this way, an offset for one or more observation points in the first dish is set.

  Although it is important that the offset in the Z direction does not change in particular, as described above, in the present embodiment, the reference mark 17 is provided at the portion of the quadruple dish 19 that faces the mounting surface 22 of each dish. The offset value can be reduced. In addition, by fixing the four-dish 19 to the moving block 9 by an appropriate method, it is possible to easily prevent the offset from changing with respect to temperature change and time passage.

  Next, the moving block 9 is switched to the position of the second dish. After switching, there is a reference mark 17 on the reference glass 15 within the XY movement range of the objective lens 29, the optical axis of the objective lens 29 is made to coincide with the reference mark 17, and auto-focusing with respect to the reference mark 17 is performed. I do. Hereinafter, it is the same as the first dish. The same applies to the third and fourth dishes.

  For example, if you register the first observation point in the first dish, move to another dish, go back to the first dish again, and register the second observation point, Immediately after moving to the dish, the reference mark 17 is autofocused to update the reference position. That is, after moving the dish, it is necessary to perform autofocus on the reference mark 17 before registering the observation point.

  When performing time-lapse observation, first, the moving block 9 moves so that the first dish corresponds to the objective lens 29. The objective lens 29 moves to the reference position and performs autofocusing, and then moves to the observation point by a preset offset amount to take an image. If there are other observation points in the first dish, the image is moved by the same amount of offset.

  Next, the moving block 9 moves so that the second dish corresponds to the objective lens 29. Similarly, autofocus is performed, and the image is taken while moving to each observation point by a preset offset amount. The same applies to the third and fourth dishes. In this way, it is possible to cancel the positioning reproducibility error that occurs when the moving block 9 moves.

  Since the movable block 9 is movable and the reference mark 17 is provided at a position facing the mounting surface 22 of each dish of the quadruple dish 19, the distance between the reference mark 17 and the observation body 25 can be reduced. Therefore, the range of movement of the objective lens 29 is minimized when performing offset driving, and for example, the influence on the change in offset when the ambient temperature changes can be reduced.

  Furthermore, if the movement range of the objective lens 29 is small, the movement time is short, and the throughput during time lapse can be improved.

  Next, a modification of the microscope system according to this embodiment will be described.

  FIG. 3 is an enlarged view showing a part of a modification of the microscope system according to the present embodiment.

  This modification is an example in which the above-described characteristic configuration relating to offset driving is applied to a general-purpose microscope that does not include a culture chamber or a moving block. The same members as those in the above-described embodiment are denoted by the same reference numerals, and redundant description is omitted. In the above-described embodiment, the observation sample is held in the holding unit including the stage 27, the culture chamber 1, the moving block 9, the moving unit, and the like. A series of well plates 33 are held directly on the stage 27.

  The observation sample includes a well plate 33, a cover glass 21 fixed to the bottom of the well plate 33, an observation body 25 disposed on the mounting surface 22 formed by them, and a well plate that covers the top of the well plate 33. And a lid 35. The reference glass 15 provided with the four reference marks 17 is fixed to the stage 27 so as to close the opening formed in the stage 27. Other configurations are the same as those in the above-described embodiment, and the observation method of the observation body is also the same.

  Also by this modification, the same effect as the above-mentioned embodiment can be acquired.

  As described above, according to the present embodiment, the accuracy of autofocus is improved, and, for long-time observation, while maintaining a high throughput, for example, a focus state due to a change in ambient temperature is suppressed, and a sample state is obtained with a clear image. It is possible to provide a microscope system 100 and a method for observing an observation body using the microscope system 100.

  The microscope system 100 of the present embodiment is configured to move the objective lens 29 in the XYZ directions, but may be configured to move the stage 27 or may be configured to move both. . In either case, the same effect can be obtained.

  Furthermore, this embodiment is only an example and is not limited to this configuration or shape. Modifications and changes can be made in a timely manner within the scope of the present invention. For example, the shape and the number of mounting surfaces are not limited to the above-described form as long as the observation sample has a container shape having a mounting surface on which the observation body is placed at the bottom. For example, an eight-dish is also possible, and in this case, the moving block can be configured to move in two directions of XY. A plurality of reference marks may be provided for one dish.

It is a figure which shows the structure of the microscope system which concerns on one Embodiment of this application. It is a figure which expands and shows a part of microscope system of FIG. It is a figure which expands and shows a part of modification of the microscope system which concerns on this embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Culture chamber 3 Chamber lid 5 Shield glass 7 Shield glass 9 Moving block 11 Ball screw 13 Motor 15 Reference glass 17 Reference mark 19 Quadruple dish 21 Cover glass 22 Placement surface 23 Dish lid 25 Observation body 27 Stage 29 Objective lens 100 Microscope System 151 Drive system controller (including AF)
107 Imaging Device 147 Z Drive Unit 149 Focusing Drive Unit

Claims (3)

An objective lens, a stage, and a holding unit installed on the stage;
A container-like observation sample held on the holding part and having a mounting surface on the bottom for placing an observation body;
A focusing drive unit that drives at least one of the objective lens and the holding unit in an optical axis direction of the objective lens;
A control unit for controlling the focusing drive unit;
A reference member provided near the placement surface at the bottom of the observation sample, and provided with a reference mark for autofocus at a position facing the placement surface in the optical axis direction of the objective lens;
The control unit controls the focusing drive unit, performs auto focus on the reference mark, moves at least one of the objective lens and the holding unit to a reference position, and then holds the objective lens and the holding unit. A microscope system, wherein at least one of the first and second units is moved by a preset offset amount. The holding unit includes a culture chamber fixed on the stage, a moving block provided in the culture chamber, to which the reference member is fixed, and a moving unit that horizontally moves the moving block in the culture chamber. Including
The observation sample has a plurality of dishes and the placement surface described above for each dish, and is held by the moving block,
The reference mark is provided at a position facing the mounting surface described above for each dish in the optical axis direction of the objective lens,
2. The microscope system according to claim 1, wherein the control unit controls the moving unit to move the moving block in a horizontal direction to switch a dish to be observed. In the observation method of the observation body by the microscope system according to claim 1 or 2,
The reference position is updated each time auto-focusing is performed on the reference mark, and at least one of the objective lens and the holding unit is moved from the updated reference position by a preset offset amount. An observation method of an observation body characterized by performing observation at an observation point.

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