JP4982974B2 - Confocal microscope system - Google Patents

Description

  The present invention irradiates a sample with laser light input to a confocal scanner through a microscope, captures a confocal image formed by imaging the return fluorescence from the sample with a camera, and captures imaging data at a predetermined time lapse period. The present invention relates to a confocal microscope system for storing captured images.

  FIG. 3 is a functional block diagram showing a configuration example of a conventional confocal microscope system. Reference numeral 1 denotes a high-speed video camera having a CCD sensor 1a. A confocal scanner 2 includes a dichroic mirror 2a. 3 is a microscope, 4 is an actuator, and shifts the objective lens 5 in the Z direction. Reference numeral 6 denotes a stage on which a sample 7 to be observed is mounted.

  An image processing unit 8 captures an image in a time-lapse manner in order to measure a long-term change of the sample. The video signal VS from the high-speed video camera 1 is sampled and captured at a time lapse period, and image processing is executed and stored in the image database 9. Reference numeral 10 denotes a time lapse cycle setting means, which outputs to the image processing means 8 an image capture command signal ST set with, for example, two different cycle setting values t1 and t2 in synchronization with the video signal VS.

  Reference numeral 11 denotes an arbitrary waveform generator, which receives the image capture command signal ST from the time lapse period setting means 10, increases by a fixed step every time ST is acquired, and returns to the original level when increasing to a predetermined step. SC is output to the actuator driver 12, and the actuator 4 of the objective lens 5 is scanned in the Z-axis direction. MP is an operation signal of the actuator driver 12.

  The high-speed video camera 1, the confocal scanner 2, the microscope 3, the actuator 4, and the objective lens 5 are disposed on the same optical axis. The confocal scanner 2 is an add-on type that has a nippo disk having 20,000 pinholes and a microlens array corresponding to the nippo disk and adopts a simple optical system, and is a camera port of the microscope 3. By attaching to the microscope, the handheld microscope 3 can be upgraded to a confocal microscope. The details of the Niipou disk type confocal scanner are disclosed in Japanese Patent Application Laid-Open No. 2004-228688.

  The laser beam R incident on the confocal scanner 2 is reflected by the dichroic mirror 2 a and irradiated on the sample 7 via the microscope 3, the actuator 4, and the objective lens 5. The return fluorescence from the sample 7 returns to the dichroic mirror 2a through the same path, passes through this, and forms a confocal image on the CCD sensor 1a of the high-speed video camera.

  The actuator 4 is mounted between the objective lens revolver of the microscope 3 and the objective lens 5, and the length of the focal direction (Z direction) of the image is changed in proportion to the level of the operation signal MP by piezo driving. 5 is controlled. The confocal microscope acquires a slice image of the sample 7 by scanning that shifts the focal position based on the operation signal MP.

  FIG. 4 is a time chart showing the concept of a conventional variable time lapse. In this example, image capture at the first time lapse period t1 is performed for a predetermined period T1, and then image capture at the second time lapse period t2 is performed for a predetermined period T2.

  Prior art documents related to the confocal microscope system include the following.

JP 2002-72102 A

  Prior art documents related to the Niipou disk type confocal scanner include the following.

Japanese Patent Laid-Open No. 5-60980

The time lapse period setting method in the conventional confocal microscope system has the following problems.
(1) The change with time of the sample (cell or the like) to be observed does not necessarily coincide with the period preset by the observer. For example, after a slow change for a long time, such as cell division, a high-speed division occurs, and then a slow change occurs again (1 to 24 hours for one division). If this phenomenon is executed in a time lapse period matched with high-speed splitting, the number of duplicate acquisition screens will be very large.

(2) Further, since the sample is irradiated with laser light for a long time, the fluorescence fades, and in the second half, the image becomes dark and cannot be seen.

(3) Conversely, if the time interval of image measurement for each time is increased, the number of captured images at the time of high-speed splitting decreases, and it becomes difficult to measure following the change.

    Therefore, the problem to be solved by the present invention is to realize a confocal microscope system that can automatically change to an optimal type lapse period in accordance with the intensity of temporal change of a sample to be observed. .

In order to achieve such a subject, the present invention has the following configuration.
(1) A sample is irradiated with laser light input to a confocal scanner through a microscope, a confocal image obtained by imaging the return fluorescence from the sample is captured by a camera, and the captured data is captured at a predetermined time lapse period. In a confocal microscope system for image processing and storage,
Difference value calculating means for calculating a difference value obtained from the difference between the captured image and the image captured a predetermined number of times ago;
Time-lapse control means for changing the time-lapse period based on the difference value;
Equipped with a,
The time lapse control means adjusts the time lapse period to be shorter when the difference value exceeds a predetermined threshold value and the difference value becomes larger than a previously calculated difference value, and is regarded as an acceleration period of change. In addition, the confocal microscope system is characterized in that when the difference value is smaller than the previously calculated difference value, the time lapse period is adjusted to be longer by considering the change deceleration period.

( 2 ) The confocal microscope system according to ( 1), wherein the confocal scanner is a Nipow disk system.

As is apparent from the above description, the present invention has the following effects.
(1) During long-time time lapse measurement, the time lapse cycle is automatically adjusted according to the degree of reaction and change of the sample. Detailed analysis is possible.

(2) When the reaction or change of the sample is settled, the time-lapse cycle can be lengthened to reduce the damage of the sample due to useless images or unnecessary excitation.

  Hereinafter, the present invention will be described in detail with reference to the drawings. FIG. 1 is a functional block diagram showing an embodiment of a confocal microscope system to which the present invention is applied. The same elements as those of the conventional system described with reference to FIG. Hereinafter, the characteristic part of the present invention will be described.

  In FIG. 1, reference numeral 100 denotes a time lapse management means introduced in the present invention. In the image database 9, G1 to Gn are image data captured and stored every time lapse period, G1 is the latest captured image data, and Gn is the captured image data n times before.

  In the time lapse management means 100, 101 is a difference value calculation means, which inputs the image signal S1 of the latest captured image data G1 and the image signal Sn of the captured image data Gn n times before, and determines a predetermined value based on the difference between the two images. The difference value ΔS quantified based on the image comparison algorithm is calculated and output.

  Reference numeral 102 denotes time lapse control means for inputting the difference value ΔS and outputting the time lapse period change signal t to the time lapse period setting means 10. SH is a preset threshold value, and when the difference value ΔS exceeds the threshold value, the time lapse period is automatically adjusted.

If the difference between the images becomes larger than the previously calculated difference value , it is regarded as the acceleration period of change, and the time lapse period is shortened. If the difference between the images becomes smaller than the previously calculated difference value , the time lapse cycle is lengthened by considering it as a change deceleration period. The adjustment factor for the time lapse period is set in advance as a parameter.

  The time lapse control means 102 calculates and outputs an optimum time lapse period t based on the difference value ΔS by a predetermined algorithm, or accesses a table holding a plurality of time lapse periods based on the difference value ΔS to obtain the optimum time lapse period t. Selects and outputs the time lapse period.

  By introducing such time-lapse management means, it is possible to automatically adjust the time-lapse period according to the degree of sample reaction and change during long-time time-lapse measurement, and sample reaction and change are severe. Sometimes, detailed images can be obtained by acquiring images at fine time intervals. Conversely, when the change is slow, it is possible to reduce unnecessary image acquisition by taking a long time lapse period.

  FIG. 2 is a flowchart showing a signal processing procedure of the time lapse management means 100. When the condition setting routine starts in step S1, the image signals (S1, S2) are read in step S2.

  If the end time is not reached in the end time check in step S3, a difference value ΔS from the image n times before is calculated in step S4. In step S5, it is checked whether or not the difference value ΔS is larger than the threshold value. If larger, the process proceeds to step S6.

In step S6, it is checked whether or not the change of the sample is an acceleration period. If it is determined that the difference is an acceleration period in which the difference is larger than the previously calculated difference value, an adjustment to increase the time lapse period is executed in step S7. . If it is determined that the difference is a deceleration period in which the difference is smaller than the previously calculated difference value, adjustment is performed to shorten the time lapse period in step S8, and the process proceeds to step S9.

  In step S9, a predetermined waiting time is provided after the adjustment is executed in step S7 or step S8 until the system starts capturing an image with a new time lapse period. When this time has elapsed, the process returns to step S2.

  If the end time has been reached in step S2, the process proceeds to step S10, and the condition setting routine ends. If it is determined in step S5 that the difference value ΔS is smaller than the threshold value, the process jumps to step S9 without adjusting the time lapse period.

  In the embodiment of FIG. 1, an example of a system that performs long-time observation that captures a three-dimensional slice image of the sample 7 by Z-direction shift control of the actuator 4 is illustrated, but the present invention is not limited to this, and the focal position The present invention can also be applied to a system for observing a real-time image of a sample in which is fixed.

  In the present invention, the high-speed video camera 1 is not limited to this, and normal camera means may be used. Further, the confocal scanner 2 is not limited to the Nipow disk system.

  As in the embodiment, according to the combination of the high-speed video camera having an imaging period sufficiently shorter than the time lapse period and the Niipou disc confocal scanner, the Nipo disc rotation synchronization control, the video signal acquisition start timing by the image processing means, and the optical The scanning start timing of the focus position of the objective lens by the control system is all synchronized with the video signal, the position accuracy of the confocal image is improved, and there is variation in individual acquisition time when acquiring multiple slice images Therefore, a highly reliable slice image can be obtained.

It is a functional block diagram which shows one Embodiment of the confocal microscope system to which this invention is applied. It is a flowchart which shows the procedure of the signal processing of a time lapse management means. It is a functional block diagram which shows the structural example of the conventional confocal microscope system. It is a time chart which shows the concept of the conventional variable time lapse.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 High-speed video camera 1a CCD sensor 2 Confocal scanner 2a Dichroic mirror 3 Microscope 4 Actuator 5 Objective lens 6 Stage 7 Sample 8 Image processing means 9 Image database 10 Time lapse period setting means 11 Arbitrary waveform generator 12 Actuator driver 100 Time lapse management means 101 Difference value calculation means 102 Time lapse control means

Claims (2)

A sample is irradiated with laser light input to the confocal scanner through a microscope, a confocal image is formed by imaging the return fluorescence from the sample, the captured data is captured at a predetermined time lapse period, and image processing is performed. In a confocal microscope system
Difference value calculating means for calculating a difference value obtained from the difference between the captured image and the image captured a predetermined number of times ago;
Time-lapse control means for changing the time-lapse period based on the difference value;
Equipped with a,
The time lapse control means adjusts the time lapse period to be shorter when the difference value exceeds a predetermined threshold value and the difference value becomes larger than a previously calculated difference value, and is regarded as an acceleration period of change. In addition, the confocal microscope system is characterized in that when the difference value is smaller than the previously calculated difference value, the time lapse period is adjusted to be longer by considering the change deceleration period.   The confocal microscope system according to claim 1, wherein the confocal scanner is a Nipow disk system.

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