JP2017068204A - Scanning type laser microscope system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve accuracy of time control to thereby guarantee repeatability of a control item, and to execute the control item at arbitrary timing by observers.SOLUTION: A scanning type laser microscope system 100 is provided that comprises: a microscope device 1 that causes a scanner to scan laser light from a laser light source on a sample; a hardware sequencer 4 that controls the microscope device 1 so as to cause an application program to execute a control item set in association with a time axis; a software sequencer 11 that manages control contents of a control items set to the application program; an external trigger input unit 20 that receives a trigger signal from an outside; and a start condition judgement unit 5 that judges a condition for causing the hardware sequencer 4 to start the control item to be managed by the software sequencer 11 on the basis of the trigger signal input into the external trigger input unit 20.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a scanning laser microscope system.

  2. Description of the Related Art Conventionally, a scanning laser microscope (hereinafter referred to as LSM) whose operation is controlled by an application program executed by a CPU provided in a computer is known. The application program manages a time flow in which control items such as image acquisition executed by the LSM are set along the time axis. Since the CPU executes other software at the same time as the application program, the variation in the execution time of each control item is as large as 100 milliseconds with respect to the set time flow, and the response of living cells in the order of 1 millisecond is observed. Time accuracy is insufficient.

  In order to cope with such problems, it is known that a software sequencer that operates separately from the CPU is installed in the computer, and that the software sequencer is configured to execute some of the functions that were conventionally performed by the CPU. (For example, refer to Patent Document 1). The software sequencer described in Patent Document 1 executes a preset time flow, and it is not assumed that the conditions of each control item are changed during execution of the time flow.

  Also, in the case of Patent Document 1, if the LSM function is increased by adding a scanner or the like, the control target of the software sequencer increases and the processing amount increases, so that the execution time of the control items varies as in the conventional case. There is a problem that it is difficult to ensure reproducibility of control items. In order to solve this problem, a scanning laser microscope system including a hardware sequencer that controls a microscope apparatus so that an application program executes control items set in association with a time axis is known (for example, (See Patent Document 2).

JP 2007-11300 A JP 2013-125069 A

  However, in the scanning laser microscope system of Patent Document 2, since each control item is executed at a timing determined by the hardware sequencer, there is an inconvenience that the control item cannot be executed at an arbitrary timing by the observer. is there.

  The present invention has been made in view of the above-described circumstances, and can improve the accuracy of time control to ensure the reproducibility of the control items, and can execute the control items at an arbitrary timing by the observer. An object of the present invention is to provide a scanning laser microscope system.

In order to achieve the above object, the present invention provides the following means.
According to one aspect of the present invention, a microscope apparatus that scans a sample with laser light from a laser light source and controls the microscope apparatus to cause an application program to execute control items set in association with a time axis A hardware sequencer, a software sequencer that manages the control contents of the control items set in the application program, an external trigger input unit that accepts an external trigger signal, and the control items managed by the software sequencer There is provided a scanning laser microscope system including a start condition determining unit that determines a start condition for starting a hardware sequencer based on the trigger signal input to the external trigger input unit.

  According to the present invention, when the observer inputs a trigger signal at the external trigger input unit, the start condition determination unit determines a condition for starting the control item. When the condition is satisfied, the hardware sequencer starts a control item managed by the software sequencer, and an image of the specimen is acquired by the microscope apparatus. By determining the start condition based on the trigger signal, the control item can be executed at an arbitrary timing by the observer.

  In addition, the hardware sequencer is responsible for executing the control items, and the software sequencer is responsible for managing the other control contents, so that the microscope apparatus can be operated as set in the application program. The reproducibility of the control items can be ensured by improving the control accuracy.

In the above aspect, when the trigger signal is input in a state where the condition setting executed by the software sequencer is completed prior to the execution of the control item, the start condition determination unit May be determined to be in place.
By doing in this way, even if a trigger signal is input during execution of a control item or setting a condition of a control item, the start condition determination unit can determine that the start condition is not ready.

Further, in the above aspect, when the start condition determining unit determines that the start condition is not satisfied at the time when the trigger signal is input, the control item cannot be started with the input trigger signal. This may be notified or recorded.
By doing in this way, even if a trigger signal is input during the execution of the control item or the condition setting of the control item, the start condition determining unit determines that the start condition is not ready and the control item is not started, By notifying or recording the fact that it could not be started, it is possible to collate later, and it is possible to know whether or not the observer was able to execute the control item at the intended timing.

Moreover, in the said aspect, the said start condition determination part may be able to set the said start condition which differs separately for every said control item.
By doing so, it is possible to facilitate the setting when there are a plurality of external triggers input to the external trigger input unit and it is desired to start a control item under a condition in which these external triggers are combined. In addition, when it is desired to execute while sequentially switching different conditions as the start conditions of a plurality of control items, the software sequencer can set the start condition specific to the next control item in the start condition determination unit and execute it sequentially.

  Further, when the hardware sequencer completes the execution of the control items, the hardware sequencer generates an interrupt to the software sequencer, and the software sequencer then sends an interrupt to the microscope apparatus based on the interrupt from the hardware sequencer. The control contents of the control items to be executed may be set in the microscope apparatus and the hardware sequencer.

  In this way, when the control content differs for each control item, the control content can be set in advance by a quick interruption from the hardware sequencer before the next control item is started. Since the control content of the next control item is immediately set when the execution of the control item being executed is completed, it is possible to shift to a state where the next control item can be started as soon as possible.

Further, in the above aspect, a hardware timer that starts or stops counting based on a determination result by the start condition determination unit is provided, and the start condition determination unit has a predetermined time that is counted by the hardware timer. May be determined that the start condition is satisfied.
By doing in this way, the observation which mixed the control item started by an external trigger and the control item started after the predetermined time after the preset time or control item end can be performed.

  According to the present invention, the accuracy of time control is improved to ensure the reproducibility of control items, and the control items can be executed at an arbitrary timing by the observer.

1 is an overall configuration diagram of a scanning laser microscope system according to a first embodiment of the present invention. It is a time chart which shows an example of operation | movement of the scanning laser microscope system of FIG. 6 is a partial time chart showing a modification of the operation of the scanning laser microscope system of FIG. It is a whole block diagram of the scanning laser microscope system which concerns on the 2nd Embodiment of this invention. 6 is a time chart showing an example of the operation of the scanning laser microscope system of FIG.

The scanning laser microscope (LSM) system 100 according to the first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the LSM system 100 according to this embodiment includes a microscope apparatus 1, a computer 10 connected to the microscope apparatus 1 via an interface (not shown), and an observer inputs an external trigger. An external trigger input unit (external trigger input unit) 20 is provided.

The microscope apparatus 1 includes a main body 2 and a control unit 3 that controls the operation of each part of the main body 2.
The control unit 3 includes a plurality of hardware sequencers 4 a, 4 b, 4 c (hereinafter collectively referred to as a hardware sequencer 4) corresponding to each part included in the main body 2. And a stage controller 2j for controlling the position of a stage (not shown) on which a sample (not shown) is placed, as required.

  Specifically, one hardware sequencer 4a controls an observation unit group including an observation scan unit (scanner) 2a, detectors 2b and 2c, and an observation laser unit (laser light source) 2d to specialize the specimen. The main body 2 is made to acquire an optical image. That is, the observation laser light emitted from the observation laser unit 2d is scanned by the observation scan unit 2a, and special light such as fluorescence and Raman scattered light emitted from the specimen is detected by the detectors 2b and 2c. It has become.

  The other hardware sequencer 4b controls the stimulation unit group including the stimulation scan unit (scanner) 2e and the stimulation laser unit (laser light source) 2f to cause the main body 2 to perform optical stimulation of the specimen. . That is, the stimulation laser light emitted from the stimulation laser unit 2f is scanned by the stimulation scan unit 2e, thereby applying light stimulation to a predetermined region of the specimen.

The other hardware sequencer 4c controls the CCD observation unit group including the CCD camera 2g and the illumination unit 2h to execute acquisition of a bright field image of the specimen. That is, the illumination light from the illumination unit 2h is illuminated onto the specimen, and a bright field image of the illuminated specimen is photographed by the CCD camera 2g.
Note that a fluorescence image may be acquired by the CCD camera 2g by attaching a suitable fluorescence excitation filter to the microscope 2i using a fluorescence excitation illumination unit for the illumination unit 2h.

  The control unit 3 also performs tasks (control items) by the hardware sequencer 4 based on the external trigger input to the external trigger input unit 20 and a condition setting completion notification output from the software sequencer 11 of the computer 10 described later. Is provided with a start condition determination unit 5 for controlling the start of That is, the start condition determination unit 5 outputs a notification from the software sequencer 11 indicating that the conditions for executing the next task have been set in the hardware sequencer 4, the microscope 2i, or the stage controller 2j. In addition, it is determined that the condition for starting the task has been met, both due to the input of the external trigger.

The hardware sequencer 4 holds the control data (control content) output from the software sequencer 11 and triggers the start control signal output from the start condition determination unit 5 to each corresponding part according to the control data. The main body 2 is caused to execute a task by starting output of a synchronization signal for instructing driving.
The hardware sequencer 4 outputs an interrupt signal for starting the setting of conditions for the next task to the software sequencer 11 every time one task is completed.

  The computer 10 includes a CPU (not shown) and a software sequencer 11 that operates separately from the CPU. The software sequencer 11 includes an MCU (Microcontroller) and its peripheral circuits on a PCI board connected to a PCI bus (not shown) inside the computer 10. Further, a logic circuit, a memory, or the like may be configured inside using an FPGA (Field Programmable Gate Array) instead of the MCU.

  The CPU causes an application program stored in a storage device (not shown) included in the computer 10 to be executed. The application program displays a control table in which a time axis and a task are set in association with each other on a monitor (not shown) connected to the computer 10 and controls the control table by an input means (not shown) such as a mouse or a keyboard. A graphical user interface (GUI) that allows an operator to register tasks and input conditions for each task is provided.

  The task conditions include, for example, the scanning method of the scan units 2a and 2e, the sensitivity of the detectors 2b and 2c, the ND filter inserted into the optical path of the laser light, and the switching of the optical path. The operator can register the task in the control table using the input means, or can rewrite the control table by changing the condition of the task already registered.

  The software sequencer 11 reads the control table from the storage device via the PCI (Peripheral Component Interconnect) board 12, and among the tasks registered in the control table, the main body 2 is executed based on the contents of the task to be executed next. The control data which is the control content of each part of 2 is calculated.

  When the software sequencer 11 receives the interrupt signal from the hardware sequencer 4, the software sequencer 11 reads the control table from the storage device, calculates the control data of the task to be executed next by the main body 2, and the control data as the final calculation result Is set in the corresponding hardware sequencer 4 and main body 2. When the condition setting is completed, the software sequencer 11 notifies the start condition determination unit 5 to that effect. That is, the setting of control data in the hardware sequencer 4 is executed during an interval period from the completion of one task to the start of the next task. In this way, the software sequencer 11 manages the task conditions and execution order set in the control table by the operator.

Next, the operation of the LSM system 100 configured as described above will be described with reference to the drawings.
Here, a sequence in which the three tasks are executed in order will be described as an example. The first task is an observation task using the observation unit group, the second task is a stimulation task using the stimulation unit group, and the third task is a CCD observation task using the CCD observation unit group.

  In these series of sequences, settings made by the observer using the GUI on the computer 10 are set in the software sequencer 11 via the PCI board 12. The software sequencer 11 disassembles the control into each task, and manages the settings of the hardware sequencer 4 and the microscope 2i or the stage controller 2j and the control state.

The condition setting for the first task is executed by the software sequencer 11 at the start of the sequence, and a trigger waiting state is waited for the input of an external trigger for starting the first task.
When an external trigger is input via the external trigger input unit 20, the start condition determination unit 5 determines whether or not the start condition is satisfied. If the start condition is satisfied, the hardware sequencer 4 starts a task.

  As shown in FIG. 2, the start condition determination unit 5 sets a flag when it receives a notification of completion of condition setting, and enters an external trigger input waiting state. Then, when the observer inputs an external trigger from the external trigger input unit 20, the start condition determination unit 5 determines that the start condition is satisfied, sends a start control signal to the hardware sequencer 4, and the first task starts. Is done. When the first task is started, the flag is reset.

Even if an external trigger is input, if the start condition determination unit 5 determines that the start condition is not satisfied, the start control signal from the start condition determination unit 5 is not output to the hardware sequencer 4.
For example, since the flag is reset during the execution of the task or during the task condition setting, the start condition determination unit 5 does not start the condition even if an external trigger is input. Judge that it is not in place.

  When the first task is completed, the hardware sequencer 4 notifies the software sequencer 11 by an interrupt signal. The software sequencer 11 immediately reads the control table from the storage device, calculates the control data for the second task, and sets conditions for setting the control data in the hardware sequencer 4 and the main body 2. Thus, by setting the condition of the next task promptly after the task execution and completing it, the dead time that cannot be started even if the next external trigger is input can be shortened as much as possible.

  Thereafter, when the external trigger is input in the same manner, the second task is executed. After the task is finished, the condition of the third task is set immediately, and the third task is executed by the external trigger to complete the series of sequences. To do.

  Here, when an external trigger is input in a state where the flag is not set, the external condition may be ignored by the start condition determination unit 5 not outputting the start control signal, or the software sequencer 11 may be notified of an interrupt to notify that an external trigger that could not be started is input. Alternatively, in association with the fact that the start condition has not been established, it is possible to leave a record in a recorder (not shown), collate the record of the recorder with application software, and detect afterwards that there was an external trigger input that could not be started. You may do it.

  Furthermore, as shown in FIG. 3, another flag (second flag) that is set by the input of the external trigger when the flag (first flag) is not set is prepared. When the first flag is set while the flag 2 is set, the task may be started immediately. Thereby, the next task can be started without missing the external trigger.

  As described above, according to the present embodiment, among the functions necessary for executing the task, the function related to time management is assigned to the hardware sequencer 4 and the other functions are assigned to the software sequencer 11. As a result, the hardware sequencer 4 can cause the main body 2 to execute the task set in the control table according to the schedule, and the time control accuracy of each part operation of the microscope 2i that starts the operation with the external trigger input as a starting point. To improve task reproducibility.

  In addition, the software sequencer 11 does not need to manage time and only needs to perform processing according to an interrupt signal or notification from the hardware sequencer 4, so that the load on the software sequencer 11 can be reduced. Further, by reducing the load on the software sequencer 11 as described above, one software sequencer 11 is responsible for managing control data of the plurality of hardware sequencers 4a and 4b provided independently for each of the scan units 2a and 2e. Even so, sufficient time accuracy can be ensured.

  Further, according to the present embodiment, since the task is started by the external trigger input by the observer via the external trigger input unit 20, there is an advantage that the task can be started at a timing intended by the observer.

Next, a scanning laser microscope system 200 according to a second embodiment of the present invention will be described below with reference to the drawings.
In the description of the present embodiment, portions having the same configuration as those of the scanning laser microscope system 100 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

As shown in FIG. 4, the scanning laser microscope system 200 according to the present embodiment includes a hardware timer 21 that is turned on / off according to the determination result by the start condition determination unit 5, and is counted by the hardware timer 21. Tasks and condition settings may be started according to time.
For example, in the example shown in FIG. 5, when the software sequencer 11 sets the condition of the first task, the time counted by the hardware timer 21 as a condition for the hardware sequencer 4 to start the first task is the time T0. In some cases, the flag is set to enter the external trigger waiting state, and the time counted by the hardware timer 21 as the interrupt generation condition for starting the condition setting of the second task is time T1. Set it. As the time T1, a time exceeding the time required for the first task is calculated and set in advance.

  The hardware timer 21 initializes the count value when the condition setting of the first task is completed, and starts counting. Here, if the initial value of the counting time of the hardware timer 21 is T0, the start condition determination unit 5 immediately matches the time T0 when the first task condition setting is completed. Therefore, the flag is set immediately after the first task condition setting is completed. At the same time, the start condition determination unit 5 turns off the hardware timer 21 when the flag is set, and keeps the stop state at the time T0.

In this state, when an external trigger is input from the external trigger input unit 20, the start condition determination unit 5 starts the first task and turns on and stops the hardware timer 21 on the assumption that the condition is satisfied. The counted time is restarted from T0, and the flag is reset.
In this way, in the actual time, the task execution process is suspended until the external trigger is input after the sequence starts, the first task is started after the external trigger is input, and the interrupt notification is made after the first task ends. Has occurred. On the other hand, when the time of the hardware timer 21 is mainly viewed, the time T0 from the counted time T0 to the input of the external trigger remains the time T0, and after the external trigger is input, the first task is executed, An interrupt is notified at the counting time T1 after the end of the task. Therefore, it can be assumed that there is no waiting time until the external trigger is input on the hardware timer 21, and the first task execution and the second task condition setting start interrupt notification are scheduled. Equivalent to being performed sequentially according to the time.

Next, the software sequencer 11 sets a flag and waits for an external trigger when the second task start condition is the counting time T2 of the hardware timer 21 in the second task condition setting. Set. As the time T2, a time exceeding the time required for setting the condition of the second task currently being executed is calculated and set in advance.
Also, the interrupt generation condition for starting the setting of the condition of the third task is set to be the counting time T3 of the hardware timer 21. As the time T3, a time exceeding the time required for the second task is calculated and set in advance.

  After the second task condition is set, the flag is set when the counting time T2 of the hardware timer 21 is reached. At the same time, the start condition determination unit 5 turns off the hardware timer 21 when the flag is set, and keeps the stop state at the time T2.

  In this state, when an external trigger is input from the external trigger input unit 20, the start condition determination unit 5 starts the second task and turns on and stops the hardware timer 21 assuming that the conditions are satisfied. The counted time is restarted from T2, and the flag is reset. Then, when the count time T3 of the hardware timer 21 after the end of the second task is reached, an interrupt for starting condition setting for the third task is output.

  Furthermore, the software sequencer 11 sets only the count time T4 of the hardware timer 21 as the start condition of the third task in the condition setting of the third task. As the time T4, a time exceeding the time required for setting the condition of the third task currently being executed is calculated and set in advance.

  Thereby, when the counting time T4 of the hardware timer 21 is reached, the start condition determination unit 5 assumes that the condition is satisfied, and immediately starts the third task without waiting for the input of the external trigger. Since the third task is the final task, after the end, the software sequencer 11 is notified of an interrupt signal, and the software sequencer 11 ends a series of sequences.

Thus, by dynamically switching between time management by the hardware timer 21 and condition setting by the start condition determination unit 5, the first task and the second task are started by an external trigger input, and the third task These tasks can be automatically executed after a predetermined time has elapsed after the end of the second task.
Further, according to such a configuration, depending on the condition setting by the software sequencer 11, it is possible to execute all tasks only by the counting time by the hardware timer 21 without using an external trigger.

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 2 Main body 2a Scan unit for observation (scanner)
2b, 2c Detector 2d Observation laser unit (laser light source)
2e Stimulus scan unit (scanner)
2f Stimulation laser unit (laser light source)
2g CCD camera 2h Illumination unit 2i Microscope 2j Stage controller 3 Control unit 4, 4a, 4b, 4c Hardware sequencer 5 Start condition determination unit 10 Computer 11 Software sequencer 12 PCI board 20 External trigger input unit (external trigger input unit)
21 Hardware timer 100, 200 Scanning laser microscope system

Claims (6)

A microscope apparatus that scans a laser beam from a laser light source on a specimen with a scanner;
A hardware sequencer that controls the microscope apparatus so that an application program executes control items set in association with a time axis;
A software sequencer for managing the control contents of the control items set in the application program;
An external trigger input that accepts external trigger signals;
A scanning laser comprising: a start condition determining unit that determines a start condition for causing the hardware sequencer to start the control item managed by the software sequencer based on the trigger signal input to the external trigger input unit Microscope system.   When the trigger signal is input in a state where the condition setting executed by the software sequencer is completed prior to the execution of the control item, the start condition determining unit is satisfied with the start condition The scanning laser microscope system according to claim 1 for determination.   The start condition determination unit notifies or records that the control item cannot be started with the input trigger signal when it is determined that the start condition is not satisfied at the time when the trigger signal is input. The scanning laser microscope system according to claim 1 or 2.   The scanning laser microscope system according to any one of claims 1 to 3, wherein the start condition determination unit can set the start condition that is different for each control item. When the hardware sequencer completes execution of the control item, it generates an interrupt to the software sequencer,
5. The software sequencer according to claim 1, wherein the control sequence of the control item to be executed next by the microscope apparatus is set in the microscope apparatus and the hardware sequencer based on an interrupt from the hardware sequencer. A scanning laser microscope system according to claim 1. A hardware timer that starts or stops counting based on the determination result by the start condition determination unit,
The scanning according to any one of claims 1 to 5, wherein the start condition determination unit determines that the start condition is satisfied when a time counted by the hardware timer reaches a predetermined time. Type laser microscope system.

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