JP4792207B2 - Scanning laser observation apparatus, observation method, and observation program - Google Patents

Description

  The present invention relates to a scanning laser observation apparatus, an observation method, and an observation program for scanning a sample with light via an optical system and displaying an observation image of the sample on the screen based on reflected light or transmitted light from the sample, In particular, a scanning laser observation device typified by a microscope or an endoscope that excites a multiple-stained fluorescent sample using a plurality of excitation wavelengths and observes a plurality of fluorescence generated corresponding to each excitation wavelength, The present invention relates to an observation method and an observation program.

The operation of a conventional scanning laser observation apparatus will be described.
FIG. 16 is a diagram showing an overall configuration of a conventional scanning laser observation apparatus, and FIG. 17 is a view taken along arrow 105 of the turret 104 shown in FIG.

  As shown in FIG. 16, in the turret 104, an excitation filter 104a having a transmission characteristic at 488 nm is disposed on the optical path between the reflection mirror 103 and the excitation dichroic mirror 106, and the reflection mirror 104c passes through the focal pinhole 116. It is arranged on the light path.

  In FIG. 16, the laser light oscillated from the krypton argon laser 101 is enlarged to a desired diameter by the beam expander 102 and reflected downward by the reflection mirror 103. Then, the wavelength of 488 nm is selected by the excitation filter 104 a on the turret 104. Then, it is reflected by the excitation dichroic mirror 106, passes through the scanning optical system 107, then reflected by the excitation dichroic mirror 108, passes through the pupil projection lens 109, passes through the imaging lens 110, and is formed as a beam spot on the sample 112 by the objective lens 111. Image.

  Then, the fluorescence emitted by the fluorescein on the sample 112 passes through the excitation dichroic mirror 106 along the optical path. The fluorescence transmitted through the excitation dichroic mirror 106 passes through the confocal pinhole 116 via the imaging lens 114 and the mirror 115. At this time, since the reflection mirror 104c on the turret 104 is arranged on the traveling path of the fluorescence, the reflected light of 488 nm is reflected by the reflection mirror 104c and cut off by the barrier filter 119, and then detected by the photodetector 117. Is done.

  By shaking the galvanometer scanner mirror constituting the scanning optical system 107, the light beam is scanned in a two-dimensional direction on the sample 112, and the light amount detected by the photodetector 117 at each scanning position is converted into an electric signal. Convert.

  As described above, the turret 104 is rotated 90 degrees by the motor 202 in response to an instruction from the computer 200 immediately after the fluorescence emitted from the fluorescein is scanned once in the XY directions in the state of FIG. The excitation filter 104d for selecting 568 nm and the hole 104b are arranged on each optical path by rotating.

  FIG. 18 is a diagram showing a state in which TEXAS RED (trademark) is excited by 568 nm and the fluorescence is detected in the conventional scanning laser observation apparatus shown in FIG. 16, and FIG. 18 is a view of the turret 104 shown in FIG.

The state shown in FIG. 19 is a state in which the turret 104 is rotated 90 degrees from the state of FIG.
In this state, the wavelength of 568 nm among the wavelengths oscillated by the krypton argon laser 101 is selected by the excitation filter 104d and reflected by the excitation dichroic mirror 106 to form an image on the sample 112 as a beam spot. Then, the fluorescence emitted from the TEXAS RED (trademark) on the sample 112 passes through the excitation dichroic mirror 108 and passes through the confocal pinhole 116 via the imaging lens 114 and the mirror 115. At this time, since the air hole 104b on the turret 104 is disposed on the optical path of the fluorescence, the reflected light of 568 nm is cut by the barrier filter 120 and detected by the photodetector 118.

  Again, the light beam is scanned two-dimensionally on the sample 112 by shaking the galvanometer scanner mirror of the scanning optical system 107, and the light detected by the photodetector 118 at each scanning position is converted into an electrical signal. Enter 200.

Then, by rotating the turret 104 by 90 degrees for each frame scan in synchronization with the scanning of the scanning optical system 107, the two steps described with reference to FIGS. 16 to 18 are performed, and the photodetector 117 and the light detection are performed. The electric signals detected by the device 118 are sequentially taken into the computer 200. Then, for example, the color obtained by the photodetector 117 is processed in a pseudo color process in green, and the image obtained by the photodetector 118 is processed in a pseudo color process in red and displayed on the monitor 201 (see, for example, Patent Document 1).
JP-A-10-206745

  However, in the conventional configuration, in order to switch the selective excitation wavelength by the wavelength selection unit and the selection optical path by the optical path selection unit during light reception by one pixel in synchronization with scanning by the scanning mechanism, the rotating plate is moved to a desired position for each pixel reception. There is a problem that the light receiving sampling rate for each pixel cannot be increased.

  In addition, when the selective excitation wavelength is switched by the wavelength selection unit for each frame or line in synchronization with the scanning by the scanning mechanism, an observation time difference occurs for each wavelength as compared with the case of switching for each line or pixel. There was a problem.

Furthermore, when it is desired to observe multiple wavelengths at the same time, if scanning is performed while exciting all of the multiple wavelengths, there is a problem that it takes an enormous amount of time for image acquisition.
The present invention has been made in view of the above-described drawbacks of the prior art. It is possible to increase the light receiving sampling rate for each pixel, and one frame scan, one line scan or one scan in synchronization with the scan by the scanning mechanism. Scanning laser observation apparatus and observation method capable of minimizing the time difference for each wavelength when switching the selective excitation wavelength by the wavelength selection means during light reception of the pixel, and shortening the image acquisition time even when observing multiple wavelengths at the same time And to provide an observation program.

The present invention employs the following configuration in order to solve the above problems.
That is, according to one aspect of the present invention, the scanning laser observation apparatus of the present invention is a scanning laser observation apparatus that irradiates a sample with a light beam from a light source and scans the sample with the light beam by a scanning mechanism. A plurality of light source means for emitting a plurality of types of excitation light having different wavelengths, a wavelength selection means for selecting the excitation light having a predetermined wavelength from the plurality of types of wavelengths, and the plurality of types of wavelengths. A plurality of detection means for detecting a plurality of types of fluorescence, and a gate for switching excitation light selected by the wavelength selection means during one-frame scanning, one-line scanning, or one-pixel light reception in synchronization with scanning by the scanning mechanism Means.

In the scanning laser observation apparatus of the present invention, it is preferable that the wavelength selection unit selects the excitation light based on a predetermined rule regarding the type of wavelength and the order of detection.
In addition, the scanning laser observation apparatus of the present invention is configured so that the wavelength selection unit obtains a one-dimensional X, Y, Z, two-dimensional XY, XZ, YZ, or three-dimensional XYZ image every time T in advance. It is desirable to select excitation light by weighting one of the dimensions XYZT.

  Further, the scanning laser observation apparatus of the present invention provides an observation when the wavelength selection unit acquires a one-dimensional X, Y, Z, two-dimensional XY, XZ, YZ, or three-dimensional XYZ image every time T. It is desirable to select excitation light by weighting one of the dimensions XYZT according to the resolution of the resolution to be performed.

  Further, the scanning laser observation apparatus of the present invention provides a laser when the wavelength selection unit acquires a one-dimensional X, Y, Z, two-dimensional XY, XZ, YZ, or three-dimensional XYZ image every time T. It is desirable to select excitation light by performing weighting according to the wavelength at which the fluorescence reaction time due to light excitation differs.

  Further, the scanning laser observation apparatus of the present invention is arranged such that when the wavelength selection means acquires a one-dimensional X, Y, Z, two-dimensional XY, XZ, YZ or three-dimensional XYZ image every time T, It is desirable to select the excitation light by performing weighting according to the wavelength of the reaction intensity.

  The scanning laser observation apparatus of the present invention preferably further comprises display means for synthesizing and displaying each fluorescence wavelength obtained by switching between one frame scanning, one line scanning, or one pixel light reception by the gate means. .

  The scanning laser observation apparatus of the present invention preferably further includes setting means for setting a method for thinning out dimensions and wavelengths based on luminance data in a preview that is thinned out evenly in each dimension and in each wavelength.

  According to another aspect of the present invention, the observation method of the present invention includes an observation method performed by a scanning laser observation apparatus that irradiates a sample with a light beam from a light source and scans the sample with the light beam by a scanning mechanism. And selecting excitation light of a predetermined wavelength from a plurality of types of excitation light having different wavelengths emitted from a plurality of light source means, and detecting a plurality of types of fluorescence respectively excited by the plurality of types of wavelengths. The selected excitation light is switched during one-frame scanning, one-line scanning, or one-pixel light reception in synchronization with scanning by the scanning mechanism.

  Further, according to one aspect of the present invention, an observation program of the present invention causes a scanning laser observation apparatus that irradiates a sample with a light beam from a light source and scans the sample with the light beam by a scanning mechanism. An observation program, a procedure for selecting excitation light having a predetermined wavelength from a plurality of types of excitation light having different wavelengths emitted from a plurality of light source means, and a plurality of types of fluorescence that are respectively excited at the plurality of types of wavelengths. And a computer-executable observation program for executing a procedure for switching the excitation light selected during one-frame scanning, one-line scanning, or one-pixel light reception in synchronization with scanning by the scanning mechanism It is.

  According to the present invention, the light receiving sampling rate for each pixel can be increased, and the selective excitation wavelength by the wavelength selecting means during one frame scanning, one line scanning or one pixel light reception in synchronization with the scanning by the scanning mechanism. It is possible to provide a scanning laser observation apparatus, an observation method, and an observation program capable of suppressing the time difference of observation for each wavelength when switching between the two, and reducing the image acquisition time even when observing multiple wavelengths at the same time. .

Embodiments of the present invention will be described below with reference to the drawings.
First, a first embodiment of the present invention will be described with reference to FIGS.
FIG. 1 is a diagram showing a configuration of an optical system of a scanning laser observation apparatus to which the present invention is applied.

  In FIG. 1, a scanning laser observation apparatus 1 includes four laser light sources 1a, 1b, 1c, and 1d. The laser light sources 1a, 1b, 1c, and 1d emit different wavelengths λa, λb, λc, and λd, respectively. (Output. The laser beams emitted from the laser light sources 1a, 1b, 1c, and 1d pass through the shutters 2a, 2b, 2c, and 2d, or are blocked by the shutters 2a, 2b, 2c, and 2d. The laser light that has passed through the shutters 2a, 2b, 2c, and 2d is reflected by the mirror 3, or reflected and synthesized by the wavelength synthesis dichroic mirrors 4b, 4c, and 4d.

  The laser light reflected by the mirror 3 and reflected and synthesized by the wavelength synthesizing dichroic mirrors 4b, 4c, 4d is reflected by the excitation dichroic mirror 5 and has a function of transmitting fluorescence described later. Then, the laser beam reflected by the excitation dichroic mirror 5 is irradiated onto the sample 8 via the deflecting elements 6 x and 6 y and the objective lens 7.

  Then, the fluorescence, which is reflected or transmitted from the sample 8 irradiated with the laser light, again passes through the objective lens 7 and the deflecting elements 6x and 6y, and further passes through the excitation dichroic mirror 5 and the filter 9, thereby the spectral dichroic mirror 10a. 10b and 10c are spectrally reflected and reflected, and reflected by the mirror 11. Further, the reflected fluorescence is reflected by a photodetector (PMT) via the condenser lenses 12a, 12b, 12c, 12d, the confocal apertures 13a, 13b, 13c, 13d, and the barrier filters 14a, 14b, 14c, 14d. Detected at 15a, 15b, 15c, and 15d.

  Sample 8 fluoresces wavelength λa ′ (dash) when excited with wavelength λa, fluoresces wavelength λb ′ (dash) when excited with wavelength λb, and excites wavelength λc. The wavelength λc ′ (dash) is fluorescent, and when the wavelength λd is excited, the wavelength λd ′ (dash) is fluorescent.

FIG. 2 is a diagram showing a configuration of a control system and a display system of a scanning laser observation apparatus to which the present invention is applied.
In FIG. 2, the system control unit 16 controls the entire scanning laser observation apparatus 1 and generates a sampling clock indicating that one pixel has been moved by the preparation elements 6x and 6y. The laser control means 17 controls the power, ON / OFF, and the like of the laser light sources 1a, 1b, 1c, and 1d of the respective wavelengths. The power setting means 18a, 18b, 18c, and 18d set the power of the laser beam. The wavelength selecting unit 19 selects a wavelength in synchronization with the sampling clock. The laser ON / OFF means 20a, 20b, 20c, and 20d are configured so that laser light having a wavelength selected by the wavelength selecting means 19 is output or laser light having a wavelength not selected by the wavelength selecting means 19 is not output. The laser light emitted from the laser light sources 1a, 1b, 1c, and 1d is controlled on and off.

  The detection processing means 21a, 21b, 21c, and 21d process the fluorescence detection signals from the photodetectors 15a, 15b, 15c, and 15d in synchronization with the sampling clock, and store the output results in the memory 22. . The display means 23 performs display according to the amount stored in the memory 22.

Next, the operation of the scanning laser observation apparatus 1 configured as described above will be described.
FIG. 3 is a diagram showing the wavelength of the laser beam excited on the sample for each pixel in the scanning laser observation apparatus according to the first embodiment, and FIG. 4 is the excitation for the first two lines in FIG. It is the figure which showed the time change of the wavelength of the laser beam to perform.

FIGS. 3 and 4 show examples in which the fluorescence state of the wavelength λa ′ is observed in detail, and the other wavelengths λb ′, λc ′, and λd ′ are roughly observed.
As shown in FIG. 3, the excitation laser light is repeated for each pixel in the order of a combination of wavelength λa and wavelength λb, a combination of wavelength λa and wavelength λc, and a combination of wavelength λa and wavelength λd. In FIG. 3, for simplification of explanation, 9 pixels × 9 pixels are set on the XY plane, but in actual observation, a wide area such as 512 pixels × 512 pixels is observed in detail.

  Since the laser light having a wavelength to be excited in advance is determined as shown in FIG. 3, before the observation, two types of wavelengths to be excited by XY scanning, 9 pixels × 9 pixels, scanning speed, 1 pixel are sent to the system control means 16 (wavelength λa and wavelength λb)-> (wavelength λa and wavelength λc)-> (wavelength λa and wavelength λd) are set. (Wavelength λa and Wavelength λb)-> (Wavelength λa and Wavelength λc)-> (Wavelength λa and Wavelength λd) means that the first pixel excites the laser of wavelength λa and wavelength λb, and the next one pixel has wavelength This means that a series of operations such as exciting the lasers of λa and λc and exciting the lasers of wavelength λa and λd in the next one pixel is repeated.

  When these pieces of information are set to the system control means 16, the information is transmitted to the laser control means 17, and the period and laser power for outputting laser light of all wavelengths, (wavelength λa and wavelength λc)-> (wavelength λa and wavelength λc)-> (wavelength λa and wavelength λd) are set.

  When scanning is started by the system control means 16, X is the first pixel, Y is the first pixel, and is the first excitation within one pixel (hereinafter referred to as (X1 pixel / Y1 pixel / inside pixel). 1) is notified from the system control means 16 to the laser control means 17 and the detection processing means 21 using the output of the sampling clock or the like. The wavelength selection means 19 informed of the laser control means 17 (X1 pixel / Y1 pixel / first in the pixel) has the first wavelength λa out of the wavelength λa and the wavelength λb excited by the first pixel. Select a laser. By this selection, the laser ON / OFF means 20a turns on the laser light source 1a, and the laser ON / OFF means 20b, 20c, 20d turns off the laser light sources 1b, 1c, 1d. As a result, laser light having a wavelength λa is output from the laser light source 1a, but no laser light is output from the laser light sources 1b, 1c, and 1d.

  The laser light having the wavelength λa output from the laser light source 1a is reflected by the mirror 3, transmitted by the dichroic mirrors 4b and 4c, reflected by the dichroic mirror 4d, reflected by the excitation dichroic mirror 5, and X by the deflection element 6x. The sample 8 is scanned in the direction, scanned in the Y direction by the deflection element 6 y, passes through the objective lens 7, and is irradiated onto the sample 8. When the sample 8 is irradiated with the laser with the wavelength λa, the sample 8 generates fluorescence with the wavelength λa ′, and passes through the excitation dichroic mirror 5 via the objective lens 7 and the deflection element 6.

  The transmitted fluorescence having the wavelength λa ′ is transmitted through the filter 9 having a characteristic of cutting the wavelengths λa, λb, λc, and λd corresponding to the laser light and transmitting the fluorescence wavelengths λa ′, λb ′, λc ′, and λd ′. The spectral dichroic mirror 10a reflects the fluorescence having the wavelength λa ′. The fluorescence having the wavelength λa ′ reflected by the spectroscopic dichroic mirror 10a is detected by the photodetector 15a through the condenser lens 12a, the confocal aperture 13a, and the barrier filter 14a.

  The fluorescence value detected by the photodetector 15a is synchronized with the sampling clock that can be handled by the memory 22 by the detection processing means 21a, 21b, 21c, and 21d for each of the photodetectors 15a, 15b, 15c, and 15d. It is converted and stored in the memory 22 as (X1 pixel / Y1 pixel / first pixel) fluorescence data.

  Next, the fact that X is the first pixel and Y is the first pixel and the second excitation within one pixel is determined from the system control means 16 to the laser control means 17 and the detection processing means using the output of the sampling clock or the like. 21a, 21b, 21c, 21d. The wavelength selection means 19 informed of the laser control means 17 (X1 pixel / Y1 pixel / second pixel) has the second wavelength λb among the wavelengths λa and λb excited by the first pixel. Select a laser. By this selection, the laser ON / OFF means 20b turns on the laser light source 1b, and the laser ON / OFF means 20a, 20c, 20d turns off the laser light sources 1a, 1c, 1d. As a result, a laser having a wavelength λb is output from the laser light source 1b, but no laser light is output from the laser light sources 1a, 1c, and 1d. The laser light having the wavelength λb outputted from the laser light source 1b is reflected by the dichroic mirror 4b, reflected by the dichroic mirror 4c, reflected by the dichroic mirror 4d, reflected by the excitation dichroic mirror 5, and is deflected by the deflecting element 6x in the X direction. Is scanned in the Y direction by the deflection element 6y, passes through the objective lens 7, and is irradiated onto the sample 8.

  When the sample 8 is irradiated with laser light having a wavelength λb, fluorescence having a wavelength λb ′ is generated from the sample 8 and passes through the excitation dichroic mirror 5 via the objective lens 7 and the deflection element 6. The transmitted fluorescence having the wavelength λb ′ is transmitted through the filter 9 having a characteristic of cutting the wavelengths λa, λb, λc, and λd corresponding to the laser light and transmitting the fluorescence wavelengths λa ′, λb ′, λc ′, and λd ′. The light is transmitted by the spectroscopic dichroic mirror 10a, and the fluorescence having the wavelength λb 'is reflected by the spectroscopic dichroic mirror 10b.

  The fluorescence having the wavelength λb ′ reflected by the spectroscopic dichroic mirror 10b is detected by the photodetector 15b through the condenser lens 12b, the confocal aperture 13b, and the barrier filter 14b. The fluorescence value detected by the photodetector 15 is synchronized with a sampling clock that can be handled by the memory 22 by the detection processing means 21a, 21b, 21c, and 21d for each of the photodetectors 15a, 15b, 15c, and 15d. It is converted and stored in the memory 22 as fluorescence data of (X1 pixel, Y1 pixel, second pixel).

During these processes, the deflecting element 6X shifts the irradiation position on the sample 8 by shifting the optical axis of the laser beam applied to the sample 8 toward the X2 pixel and the Y1 pixel.
When this irradiation position reaches the X2 pixel and the Y1 pixel, the system control means 16 moves to the laser control means 17, the detection processing means 21a, 21b, 21c, 21d, the display means 22, etc. (X2 pixel / Y1 pixel). (1) in the pixel is transmitted using an output such as a sampling clock. The wavelength selection means 19 informed of (the X2 pixel, the Y1 pixel, and the first in the pixel) via the laser control means 17 is the wavelength λa and the wavelength λc that are excited by the second pixel set in advance. The laser with the first wavelength λa is selected. By this selection, the laser ON / OFF means 20a turns on the laser light source 1a, and the laser ON / OFF means 20b, 20c, and 20d turn off the laser light sources 1b, 1c, and 1d. As a result, laser light having a wavelength λa is output from the laser light source 1a, but no laser light is output from the laser light sources 1b, 1c, and 1d.

  The laser light of wavelength λa output from the laser light source 1a is reflected by the mirror 3, transmitted by the dichroic mirrors 4b and 4c, reflected by the dichroic mirror 4d, reflected by the excitation dichroic mirror 5, and X direction by the deflecting element 6x. Is scanned in the Y direction by the deflection element 6y, passes through the objective lens 7, and is irradiated onto the sample 8.

  When the sample 8 is irradiated with the laser with the wavelength λa, the sample 8 generates fluorescence with the wavelength λa ′, and passes through the excitation dichroic mirror 5 via the objective lens 7 and the deflection element 6. The transmitted fluorescence having the wavelength λa ′ is transmitted through the filter 9 having a characteristic of cutting the wavelengths λa, λb, λc, and λd corresponding to the laser light and transmitting the fluorescence wavelengths λa ′, λb ′, λc ′, and λd ′. The spectral dichroic mirror 10a reflects the fluorescence having the wavelength λa ′.

  The fluorescence having the wavelength λa ′ reflected by the spectroscopic dichroic mirror 10a is detected by the photodetector 15a through the condenser lens 12a, the confocal aperture 13a, and the barrier filter 14a. The fluorescence value detected by the photodetector 15a is converted into a format that can be handled by the memory 22 by the detection processing means 21a, 21b, 21c, and 21d for each of the photodetectors 15a, 15b, 15c, and 15d. Accumulated as fluorescence data (X2 pixel, Y1 pixel, first pixel).

  Next, X is the second pixel, Y is the first pixel, and the second excitation in one pixel is processed in the same manner, and the memory 22 stores (X2 pixel, Y1 pixel, second pixel). Accumulated as fluorescence data.

Also during these processes, the deflection element 6x shifts the irradiation position on the sample 8 by shifting the optical axis of the laser beam irradiated to the sample 8 toward the X3 pixel and the Y1 pixel.
When this irradiation position reaches the X3 pixel and the Y1 pixel, the same processing is performed, and the fluorescence data (X3 pixel, Y1 pixel, first pixel) and the (X3 pixel, Y1 pixel) are stored in the memory 22. -Accumulated as second fluorescence data in the pixel.

  The X4 pixel and the Y1 pixel are returned to the state of exciting the laser light of (wavelength λa and wavelength λb), and a series of (wavelength λa and wavelength λb)-> (wavelength λa and wavelength λc)-> (wavelength The operation of λa and wavelength λd) is repeated, and scanning in the X direction is performed up to the set X9 pixels.

  When the pixel reaches the X9 pixel, it moves to the X1 pixel and the Y2 pixel, and the same operation as the operation for one line corresponding to the Y1 pixel, that is, the first line is repeated again at the Y2 pixel, that is, the second line. .

Similarly, scanning in the Y direction is performed up to the set Y9th pixel.
When the fluorescence data for one screen is accumulated in the memory 22 in this way, the system control means 16 reads the fluorescence data from the memory 22 and displays it on the display means 23. Here, the fluorescence data in the memory 22 is displayed after being accumulated for one screen, but it may be for an arbitrary number of lines or an arbitrary number of pixels depending on the processing load of the system control means 16.

By repeating the series of fluorescence data display for one screen as described above, temporal changes on the XY plane can be observed.
Here, the laser light source and the shutter are not limited to the laser light sources 1a, 1b, 1c, and 1d that are semiconductor lasers and the shutters 2a, 2b, 2c, and 2d, but a plurality of lasers that oscillate single-line laser light. A combination of a gas laser and an AOTF (Acousto-Optic Tunable Filter), or a combination of a gas laser that oscillates a multi-line laser beam and an AOTF can be used, and laser light ON / OFF control and light amount control are possible. What is necessary is just to take the structure.

  Here, the fluorescence of wavelengths λb ′, λc ′, and λd ′ to be roughly observed is excited once every three pixels in the X direction, and compared with the case where it is excited once every pixel, The detection time is reduced to 1/3, and the capacity of the memory 22 is also reduced to 1/3. In addition, the fluorescence of the wavelength λa ′ to be observed in detail is excited once for each pixel, has a resolution of one pixel, and can be observed in detail with good XY resolution.

  Furthermore, since the excitation wavelength existing in the optical path is a single wavelength in any time zone among the laser beams having a plurality of wavelengths, the problem of fluorescence crosstalk can be eliminated. In addition, since a plurality of laser wavelengths are switched for each pixel, there is an effect that temporal deviation between fluorescence wavelengths can be suppressed as much as possible.

Next, a second embodiment of the present invention will be described with reference to FIGS.
In the second embodiment, the change in Z is not so large at any wavelength, but an example is shown in which observation is performed with high XY resolution.

  FIG. 5 is a diagram showing the wavelength of the laser beam excited on the sample for each pixel in the scanning laser observation apparatus according to the second embodiment, and showing the Z plane of the three-dimensional XYZ. FIG. 6 is a diagram showing, for each pixel, the wavelength of the laser beam excited on the sample in the scanning laser observation apparatus according to the second embodiment, and is a diagram showing the wavelength of the laser beam on the odd Z plane for each pixel. It is. FIG. 7 is a diagram showing the wavelength of the laser light excited on the sample for each pixel in the scanning laser observation apparatus according to the second embodiment, and shows the wavelength of the laser light on the even Z plane for each pixel. There is a figure.

  Although the change in Z is not so large at any wavelength, when it is desired to observe with a high resolution of XY, as shown in FIG. 6, after the first line is excited with the wavelength λa on the odd Z plane, the same 1 The line is excited with the wavelength λc, and then the Y1 line is moved, and the second line is similarly excited with the wavelength λa, and then the same second line is excited with the wavelength λc so as to be repeated for each line. . Also, as shown in FIG. 7, in the even Z plane, after the first line is excited with the wavelength λb, the same first line is excited with the wavelength λd and then moved by Y1 line, and the second line is the same. Then, the same second line is excited with the wavelength λd after being excited with the wavelength λb.

  The configuration of the optical system, control system, and display system is the same as that of the first embodiment shown in FIGS. 1 and 2, but in the second embodiment, the fluorescence data for one screen is stored in the memory 22. Is stored, the position of the sample 8 in the Z direction to be observed is changed, and the three-dimensional XYZ fluorescence state is observed.

  Since the laser having the wavelength to be excited in advance is determined as shown in FIG. 6 and FIG. 7, the system control unit 16 is excited by XYZ scanning, 8 pixels × 8 pixels × 8 pixels, scanning speed, 1 line before observation. Two types of wavelengths to be set, (wavelength λa and wavelength λc)-> (wavelength λb and wavelength λd), etc. are set. (Wavelength λa and Wavelength λc)-> (Wavelength λb and Wavelength λd) means that all the lines in the first Z1 plane are excited by switching the laser light of wavelength λa and wavelength λc for each line. This means that a series of operations such as switching and exciting the laser light of wavelength λb and wavelength λd for each line is repeated in all the lines.

  When these pieces of information are set to the system control means 16, the information is transmitted to the laser control means 17, and the period and laser power for outputting laser light of all wavelengths, (wavelength λa and wavelength λc)-> (wavelength λb and wavelength λd) are set.

  When scanning is started by the system control means 16, the wavelength λa is first excited while X scanning is performed on the Y1 line of the Z1 plane, and the data of the wavelength λa ′ is stored in the memory 22. Next, the wavelength λc is excited while X scanning of the same Y1 line is performed, and the fluorescence data of the wavelength λc ′ is stored in the memory 22. Here, since the two types of wavelengths to be excited by one set line have been scanned, the system control means 16 moves by one line, performs X scanning while exciting the Y2 line separately for wavelengths λa and λc, and wavelength λa Fluorescence data is stored in the memory 22 for each of ′ and λc ′. Similarly, X scanning for each of the wavelengths λa and λc is repeated up to the Y8th line.

  When the X-scan of the Y8th line is completed, the data in the memory 22 is displayed, and the position of the specimen 8 in the Z direction to be observed is changed based on the set XYZ scan information, so that the Y1th line of the Z2 plane is changed. The wavelength λb is excited while X scanning is performed at, and the fluorescence data of the wavelength λb ′ is stored in the memory 22.

  Next, the wavelength λd is excited while X scanning is performed on the same Y1 line, and the fluorescence data of the wavelength λd ′ is stored in the memory 22. Here, since the two types of wavelengths to be excited with one set line have been scanned, the system control means 16 moves by one line, performs X scanning while exciting the Y2 line separately for wavelengths λb and λd, and wavelength λb Fluorescent data is stored in the memory 22 for each of ′ and λd. Similarly, X scanning for each of the wavelengths λb and λd is repeated until the Y8th line.

  When the X scan of the Y8th line is completed, the data in the memory 22 is displayed, and the Z3 plane is scanned in the same manner as the Z1 plane, and the fluorescence data of the wavelengths λa ′ and λc ′ is accumulated. After the scanning of the Z3 plane, the data in the memory 22 is displayed, and the Z4 plane is scanned in the same manner as the Z2 plane, and the fluorescence data of the wavelengths λb ′ and λd ′ is accumulated.

  The same operation is repeated every time the Z plane is changed, and after scanning up to the Z8 plane, the process returns to the Z1 plane and the operation from the Z1 plane to the Z8 plane is repeated again to observe the temporal change in the three-dimensional XYZ fluorescence state. .

  Here, the fluorescence in the Z direction to be roughly observed is excited by two pixels evenly for each wavelength, and the detection time is halved compared to the case of being excited once for each pixel. And the capacity of the memory 22 can be halved. In addition, the fluorescence on the XY plane to be observed in detail is excited once for each pixel equally for each wavelength, has a resolution of one pixel, and can be observed in detail with good XY resolution.

Further, among the lasers having a plurality of wavelengths, the excitation wavelength existing in the optical path is one wavelength in any time zone, so that the problem of fluorescence crosstalk can be eliminated.
When the fluorescence in the Z direction is excited once by two pixels, the image acquisition sequence is decimated by half. However, the Z1 surface has a wavelength λa, the Z2 surface has a wavelength λb, and the Z3 surface has a wavelength λc. If the Z direction is thinned down to ¼ like the wavelength λd on the Z4 surface, the image acquisition time is shortened to ¼, which is suitable for applications such as preview.

Next, a third embodiment of the present invention will be described with reference to FIG.
The third embodiment shows an example in which the change in XY is not so large at any wavelength, but the observation is made with a high resolution of Z.

FIG. 8 is a diagram showing, for each pixel, the wavelength of the laser excited on the sample by the scanning laser observation apparatus according to the third embodiment.
The change in XY is not so great at any wavelength, but when it is desired to observe with a high resolution of Z, as shown in FIG. 4, the odd-numbered line is excited at wavelength λa and then at wavelength λb on all Z planes. Switch excitation for each pixel, then move for Y1 line, switch even-numbered line for excitation with wavelength λc and then for excitation with wavelength λd, then move for Y1 line Then, the process shifts to the odd line again, and the same operation is repeated.

  The configurations of the optical system, the control system, and the display system are the same as those of the first embodiment shown in FIGS. 1 and 2, but in the third embodiment, the fluorescence data for one screen is stored in the memory 22. Is stored, the position of the sample 8 in the Z direction to be observed is changed, and the three-dimensional XYZ fluorescence state is observed.

  Further, since the laser having a wavelength to be excited in advance is determined as shown in FIG. 8, the wavelength 1 to be excited by XYZ scanning, 8 pixels × 8 pixels × 8 pixels, scanning speed, 1 pixel to the system control means 16 before observation. The type, odd wavelength line (wavelength λa)-> (wavelength λb), even line number (wavelength λc)-> (wavelength λd) are set. (Wavelength λa)-> (wavelength λb) means that a series of operations such as exciting the laser light of wavelength λa in the first pixel and exciting the laser light of wavelength λb in the next pixel are repeated for one line. (Wavelength λc)-> (wavelength λd) means that a series of operations such as exciting the laser of wavelength λc in the first pixel and exciting the laser of wavelength λd in the next pixel. It means repeating for the line.

  When these pieces of information are set to the system control means 16, the information is transmitted to the laser control means 17, and the period and laser power for outputting lasers of all wavelengths, and the odd-numbered lines are (wavelength λa)-> (wavelength (λb), the even-numbered line is set such as (wavelength λc)-> (wavelength λd). Here, the odd-numbered line is set in advance as (wavelength λa)-> (wavelength λb), and the even-numbered line is set as (wavelength λc)-> (wavelength λd). (Wavelength λa)-> (wavelength λb) is reset when changing to the eye, and (wavelength λc)-> (wavelength λd) is reset when changing from the odd-numbered line to the even-numbered line. The setting may be changed in accordance with the operation status to operate.

When scanning in such a state, it is possible to observe a temporal change in the three-dimensional XYZ fluorescence state.
Here, the fluorescence of the XY plane to be roughly observed is excited once for each of the four pixels evenly for each wavelength, and the detection time is reduced to ¼ compared to the case of being excited once for each pixel. And the capacity of the memory 22 can be reduced to ¼. Further, the fluorescence in the Z direction that is desired to be observed in detail is excited once for each pixel evenly for each wavelength, has a resolution of one pixel, and can be observed in detail with good Z resolution. This speeds up the acquisition of multi-wavelength images and makes it easier to observe changes in Z.

Next, a fourth embodiment of the present invention will be described with reference to FIG.
In the fourth embodiment, an example in which the fluorescence state of the wavelength λa ′ is observed in detail, the wavelengths λc ′ and λd ′ are roughly observed, and the wavelength λb ′ is moderately observed is shown.

FIG. 9 is a diagram showing, for each pixel, the wavelength of the laser excited on the sample in the scanning laser observation apparatus according to the fourth embodiment.
If the fluorescence state of the wavelength λa ′ is observed in detail, the wavelengths λc ′ and λd ′ are roughly observed, and the wavelength λb ′ is moderately observed, as shown in FIG. In the odd-numbered line, the combination of the wavelength λa and the wavelength λb is repeated in the order of the combination of the wavelength λa and the wavelength λc. In the even-numbered line, the combination of the wavelength λa and the wavelength λd is repeated Like.

  The configurations of the optical system, the control system, and the display system are the same as those of the first embodiment shown in FIGS. 1 and 2, but in the fourth embodiment, wavelengths λc ′ and λd that are desired to be roughly observed are used. The fluorescence of ′ is excited once every four pixels. In addition, the fluorescence of the wavelength λa ′ that is desired to be observed in detail is excited once per pixel, has a resolution of one pixel, and can be observed in detail with good Z resolution. Further, the fluorescence having the wavelength λb ′ to be observed at a medium level is excited once per two pixels.

  Thereby, it is possible to perform thinning weighting for excitation between wavelengths having different fluorescence reaction times by excitation of laser light or between wavelengths having different intensity of fluorescence reaction, and image acquisition can be appropriately thinned out.

Next, a fifth embodiment of the present invention will be described with reference to FIG.
FIG. 10 is a diagram showing, for each pixel, the wavelength of the laser excited on the sample by the scanning laser observation apparatus according to the fifth embodiment.

  In the fifth embodiment, as shown in FIG. 10, with respect to the wavelengths λb, λc, and λd, one display pixel is composed of X2 pixels × Y2 pixels = 4 pixels for one excitation or detection pixel. I am letting.

  The configurations of the optical system, the control system, and the display system are the same as those in the first embodiment described with reference to FIGS. 1 and 2, and the operation from the output of the laser light to the storage in the memory 22 is the fourth. This is the same as the embodiment. However, wavelength λb is assigned blue, wavelength λc is assigned green, and wavelength λd is assigned red.

  The fluorescence data stored in the memory 22 is extracted by the system control means 16 and displayed with the wavelength λa. In addition, for the wavelengths λb, λc, and λd, fluorescence data is first extracted for each color by the system control means 16 so that X2 pixels × Y2 pixels = 4 pixels and one display pixel, and these light amounts are combined. Then, color data is generated, and color image data is displayed separately from the fluorescence data by the wavelength λa.

This makes it easier to grasp the general tendency of the observation contents in a sensuous manner rather than analyzing the observation contents for each specific wavelength.
Next, a sixth embodiment of the present invention will be described with reference to FIGS.

FIG. 11 is a configuration diagram of a control system and a display system of a scanning laser observation apparatus according to the sixth embodiment.
The components having the same functions as those shown in FIG. 2 used for the description of the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.

That is, in FIG. 11, the scanning laser observation apparatus 30 includes a setting unit 31 and an automatic thinning unit 35 in addition to the components included in the scanning laser observation apparatus 1 illustrated in FIG. 2.
The setting unit 31 includes a preview mode setting unit 32, a scan-with-preview operation unit 33, and a non-preview scan operation unit 34. An automatic thinning-out unit 35 includes a preview data storage memory 36, a mode-specific data processing unit 37, and a thinning-out unit. Automatic setting means 38 is provided.

  The mode setting means 32 for preview is automatically selected at the time of preview, such as mode A for thinning out an axis with a large difference, mode B for thinning out an axis with a small difference, and mode C for confirming a wavelength of a brightness suddenly generated from a background level with high resolution. Set the thinning mode. The scanning operation means 33 with a preview operates a scan start / stop with a preview. The non-preview scan operating means 34 operates a scan start / stop without a preview.

  The preview data storage memory 36 is a memory for storing preview data, and stores brightness data at the time of preview in which X / Y / λ in the scan with preview is evenly thinned. The mode-specific data processing unit 37 calculates a difference between adjacent pixels on each of the X / Y axes in the mode A and extracts an axis having a large difference. In the mode B, the data processing unit 37 extracts the adjacent pixels on the X / Y axes. In mode C, when the observation is started, a wavelength that is lower than a certain background level but has a high luminance at a specific time is extracted. Then, the thinning automatic setting unit 38 performs setting for appropriately thinning out X / Y / λ according to the axis and wavelength extracted by the mode-specific data processing unit 37.

Next, the operation of the scanning laser observation apparatus 30 configured as described above will be described.
FIG. 12 is a diagram showing the wavelength to be excited for each pixel in XY scanning, 8 pixels × 8 pixels (in preview), and FIG. 13 is for XY scanning, excitation for each pixel in 8 pixels × 8 pixels. FIG. 14 is a diagram showing wavelengths (when the X-axis is evenly thinned), and FIG. 14 is a diagram showing wavelengths to be excited for each pixel of XY scanning, 8 pixels × 8 pixels (Y-axis is evenly thinned). FIG. 15 is a diagram (wavelength λa is extracted with high resolution in mode C) showing the wavelength excited for each pixel of XY scanning and 8 pixels × 8 pixels.

  In the sixth embodiment configured as described above, when mode A is selected by the preview mode setting means 32 and the scan-with-preview scanning means 33 is started to scan, all wavelengths are first evenly X-axis. / X2 pixel × Y2 pixel area where pixels are adjacent to each other by switching wavelengths λa, λb, λc, and λd for each pixel as a preview in which the Y-axis pixels are thinned out evenly In this case, the wavelengths λa, λb, λc, and λd are irradiated once, fluorescence data is collected, and scanning is stopped when a data amount appropriate for the preview is collected.

  The fluorescence data is displayed on the display means 23 and stored in the preview data storage memory 36. The mode-specific data processing means 37 calculates the difference between adjacent pixels on each of the X / Y axes, and the axis having the large difference. , And the thinning automatic setting means 38 sets an appropriate thinning method so as to thin out X-axis or Y-axis pixels having a large difference.

  Specifically, when the axis having a large difference is the X axis, thinning is performed as shown in FIG. 13, and when the axis having the large difference is the Y axis, thinning is performed as shown in FIG. In such a state, the scanning is started again, the luminance data is displayed on the display means 23, and the series of operations is repeated until the scanning is stopped by the scanning operation means 33 with preview.

  When mode B is selected by the preview mode setting means 32, the mode-specific data processing means 37 calculates the difference between adjacent pixels on each of the X / Y axes to extract an axis with a small difference. The thinning automatic setting means 38 sets an appropriate thinning method so as to thin out small X-axis or Y-axis pixels, and scans again.

  When the mode C is selected by the preview mode setting means 32, the mode-specific data processing means 37 calculates the change in the passage of time for each wavelength, and the high luminance that appears suddenly from the background level acquired in advance. The wavelength is extracted, the wavelength extracted by the thinning automatic setting means 38 is set so as to have a high resolution, and scanning is performed again. More specifically, the case where the wavelength λa is extracted is thinned as shown in FIG.

  As described above, in each of the embodiments of the present invention, the case of the configuration of the four-wavelength fluorescence by the four-wavelength excitation has been described. However, the present invention is not limited to this. Needless to say, etc. are configured by an appropriate number of appropriate types and applied to multi-wavelength observation.

  Further, in the present invention, the concept of changing the scanning wavelength for each line to a different wavelength on different planes of XY, the concept of changing the scanning wavelength for each pixel to a different wavelength for different lines of Y, 1 It also includes the concept that the wavelength to be switched and scanned for each pixel is different on different XY planes.

  Further, the scanning laser observation apparatus to which the present invention is applied is not limited to the above-described embodiments and the like as long as the function is executed, and a plurality of apparatuses may be used even if they are a single apparatus. Needless to say, the system or the integrated device may be a system in which processing is performed via a network such as a LAN or a WAN.

  It can also be realized by a system comprising a CPU, ROM or RAM memory connected to the bus, input device, output device, external recording device, medium driving device, portable recording medium, and network connection device. That is, a ROM or RAM memory, an external recording device, or a portable recording medium that records the program code of the software that realizes the system of each of the embodiments described above is supplied to the scanning laser observation device, and the scanning laser Needless to say, this can also be achieved by the computer of the observation apparatus reading and executing the program code.

  In this case, the program code itself read from the portable recording medium or the like realizes the novel function of the present invention, and the portable recording medium or the like on which the program code is recorded constitutes the present invention. .

  Examples of portable recording media for supplying program codes include flexible disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, DVD-ROMs, DVD-RAMs, magnetic tapes, and non-volatile memories. Various recording media recorded through a network connection device (in other words, a communication line) such as a card, a ROM card, electronic mail or personal computer communication can be used.

  In addition, the functions of the above-described embodiments are realized by executing the program code read out on the memory by the computer, and the OS running on the computer is actually executed based on the instruction of the program code. The functions of the above-described embodiments are also realized by performing part or all of the process.

  Furthermore, a program code read from a portable recording medium or a program (data) provided by a program (data) provider is provided in a function expansion board inserted into a computer or a function expansion unit connected to a computer. The CPU of the function expansion board or function expansion unit performs part or all of the actual processing based on the instruction of the program code, and the functions of the above-described embodiments are also performed by the processing. Can be realized.

  That is, the present invention is not limited to the embodiments described above, and can take various configurations or shapes without departing from the gist of the present invention.

It is a figure which shows the structure of the optical system of the scanning laser observation apparatus to which this invention is applied. It is a figure which shows the structure of the control system and display system of the scanning laser observation apparatus to which this invention is applied. It is the figure which showed the wavelength of the laser beam excited to a sample for every pixel with the scanning laser observation apparatus which concerns on 1st Embodiment. It is the figure which showed the time change of the wavelength of the laser beam excited for the head 2 lines in FIG. It is the figure which showed the wavelength of the laser beam excited to a sample with the scanning laser observation apparatus which concerns on 2nd Embodiment for every pixel, and is a figure which shows Z surface of three-dimensional XYZ. It is the figure which showed the wavelength of the laser beam excited to a sample for every pixel with the scanning laser observation apparatus which concerns on 2nd Embodiment, and is the figure which showed the wavelength of the laser beam in an odd-numbered Z surface for every pixel. It is the figure which showed the wavelength of the laser beam excited to a sample for every pixel with the scanning laser observation apparatus which concerns on 2nd Embodiment, and is the figure which showed the wavelength of the laser beam in an even-numbered Z surface for every pixel. It is the figure which showed the wavelength of the laser excited to a sample with the scanning laser observation apparatus which concerns on 3rd Embodiment for every pixel. It is the figure which showed the wavelength of the laser excited to a sample with the scanning laser observation apparatus which concerns on 4th Embodiment for every pixel. It is the figure which showed the wavelength of the laser excited to a sample with the scanning laser observation apparatus which concerns on 5th Embodiment for every pixel. It is a block diagram of the control system and display system of the scanning laser observation apparatus which concerns on 6th Embodiment. It is the figure (at the time of a preview) which showed the wavelength excited for every pixel in XY scanning and 8 pixels x 8 pixels. It is the figure which showed the wavelength excited for every pixel in XY scanning and 8 pixels x 8 pixels (when the X-axis was thinned out uniformly). It is the figure which showed the wavelength excited for every pixel by XY scanning and 8 pixels x 8 pixels (when the Y-axis was thinned out uniformly). It is the figure which showed the wavelength excited for every pixel in XY scanning and 8 pixels x 8 pixels (when wavelength lambda a was extracted with high resolution in mode C). It is a figure which shows the whole structure of the conventional scanning laser observation apparatus. FIG. 17 is a 105 arrow view of the turret 104 shown in FIG. 16. FIG. 17 is a diagram showing a state in which TEXAS RED (trademark) is excited by 568 nm and the fluorescence is detected in the conventional scanning laser observation apparatus shown in FIG. 16. It is the 105 arrow directional view of the turret 104 shown in FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Scanning laser observation apparatus 1a Laser light source 1b Laser light source 1c Laser light source 1d Laser light source 2a Shutter 2b Shutter 2c Shutter 2d Shutter 3 Mirror 4b Wavelength synthesis dichroic mirror 4c Wavelength synthesis dichroic mirror 4d Wavelength synthesis dichroic mirror 5 Excitation dichroic mirror 6x 6y deflection element 7 objective lens 8 sample 9 filter 10a spectral dichroic mirror 10b spectral dichroic mirror 10c spectral dichroic mirror 11 mirror 12a condenser lens 12b condenser lens 12c condenser lens 12d condenser lens 13a confocal aperture 13b confocal aperture 13c Focal aperture 13d Confocal aperture 14a Barrier filter 14b Barrier filter 14c Barrier filter 14d Barrier filter 15 a Photodetector (PMT)
15b Photodetector (PMT)
15c Photodetector (PMT)
15d Photodetector (PMT)
16 system control means 17 laser control means 18a power setting means 18b power setting means 18c power setting means 18d power setting means 19 wavelength selection means 20a laser ON / OFF means 20b laser ON / OFF means 20c laser ON / OFF means 20d laser ON / OFF means 20d laser ON / OFF OFF means 21a Detection processing means 21b Detection processing means 21c Detection processing means 21d Detection processing means 22 Memory 23 Display means 30 Scanning laser observation device 31 Setting means 32 Preview mode setting means 33 Preview scanning operation means 34 Preview non-scanning operation means 35 Automatic thinning means 36 Preview data storage memory 37 Mode-specific data processing means 38 Thinning automatic setting means 101 Krypton argon laser 102 Beam expander 103 Mirror 104 Turret 104a Excitation filter 104b Air hole 104c Reflection mirror 104d Excitation filter 106 Excitation dichroic mirror 107 Scanning optical system 108 Excitation dichroic mirror 109 Pupil projection lens 110 Imaging lens 111 Objective lens 112 Sample 114 Imaging lens 115 Mirror 116 Confocal pin Hall 117 Photo detector 118 Photo detector 119 Barrier filter 120 Barrier filter 200 Computer 201 Monitor 202 Motor (M)

Claims (10)

In a scanning laser observation apparatus that irradiates a sample with a light beam from a light source and scans the sample with the light beam by a scanning mechanism,
A plurality of light source means for emitting a plurality of types of excitation light having different wavelengths;
Wavelength selection means for selecting excitation light of a predetermined wavelength from the plurality of types of wavelengths;
A plurality of detection means for detecting a plurality of types of fluorescence respectively excited at the plurality of types of wavelengths;
A blocking means for switching excitation light selected by the wavelength selection means during one frame scanning, one line scanning, or one pixel light reception in synchronization with scanning by the scanning mechanism ;
The wavelength selection unit selects excitation light by performing predetermined weighting when acquiring a one-dimensional X, Y, Z, two-dimensional XY, XZ, YZ, or three-dimensional XYZ image. Scanning laser observation device.   The scanning laser observation apparatus according to claim 1, wherein the wavelength selection unit selects the excitation light based on a predetermined rule regarding the type of wavelength and the order of detection. It said wavelength selection means, laser scanning observation apparatus according to claim 1 or 2, characterized in that selecting the excitation light by weighting to one of the pre-Me dimension xyzt. It said wavelength selection means, laser scanning observation apparatus according to claim 1 or 2, characterized in that selecting the excitation light by weighting any dimension XYZT depending on the crude fine resolution of Judging watch. Said wavelength selection means, laser scanning observation apparatus according to claim 1 or 2, characterized in that the time of the fluorescent reaction by excitation of Les laser light selecting excitation light by weighting according to different wavelengths . Said wavelength selection means, laser scanning observation apparatus according to claim 1 or 2, characterized in that the intensity of the fluorescence reaction selects excitation light by weighting according to different wavelengths.   7. The display device according to claim 1, further comprising display means for combining and displaying each fluorescence wavelength obtained by switching between one frame scanning, one line scanning, or one pixel light reception by the gate means. The scanning laser observation apparatus described in 1.   8. The method according to claim 1, further comprising setting means for setting a thinning method of dimensions and wavelengths based on luminance data in a preview that is thinned equally for each dimension and for each wavelength. Scanning laser observation device. An observation method performed by a scanning laser observation apparatus that irradiates a sample with a light beam from a light source and scans the sample with the light beam by a scanning mechanism,
Select excitation light of a predetermined wavelength from a plurality of types of excitation light with different wavelengths emitted from a plurality of light source means,
Detecting a plurality of types of fluorescence respectively excited at the plurality of types of wavelengths,
The one-dimensional X, Y, Z, two-dimensional XY, XZ, YZ, or three-dimensional XYZ image represents the excitation light selected during one-frame scanning, one-line scanning, or one-pixel light reception in synchronization with scanning by the scanning mechanism. An observation method characterized in that the selection is made on the basis of a predetermined weighting when acquiring . An observation program for causing a scanning laser observation apparatus that irradiates a sample with a light beam from a light source and scans the sample with the light beam by a scanning mechanism,
A procedure for selecting excitation light of a predetermined wavelength from a plurality of types of excitation light having different wavelengths emitted from a plurality of light source means;
Detecting a plurality of types of fluorescence respectively excited at the plurality of types of wavelengths;
The one-dimensional X, Y, Z, two-dimensional XY, XZ, YZ, or three-dimensional XYZ image represents the excitation light selected during one-frame scanning, one-line scanning, or one-pixel light reception in synchronization with scanning by the scanning mechanism. A procedure for selecting based on a predetermined weight when acquiring
A computer-executable observation program for executing the program.