JP6153340B2 - Laser microscope equipment - Google Patents

Description

  The present invention relates to a laser microscope apparatus.

  Conventionally, a laser microscope apparatus that scans a specimen with pulsed laser light and generates an image of the specimen based on the luminance information of fluorescence from the specimen is known (for example, see Patent Documents 1 and 2). In such a laser microscope apparatus, if the pulse period of the pulse laser beam and the sampling period of the fluorescence are not synchronized, the number of fluorescent sampling differs for each pixel forming the image, and the image has unevenness (moire). There is a problem that occurs.

  The laser microscope apparatus described in Patent Document 1 measures the pulse period of pulsed laser light, generates a scanning clock by the scanning unit based on the pulse period, and synchronizes the pulse frequency and the sampling period, It is intended to prevent moiré from occurring in the image. In addition, the laser microscope apparatus of Patent Document 2 generates a designated pulse in which the number of fluorescent captures is equal in a pixel synchronization signal that determines the start and end timing of a cycle of one pixel, and matches the sampling effective period. This prevents the occurrence of moire in the image.

JP 2007-102235 A JP 2010-113062 A

  However, the laser microscope devices described in Patent Document 1 and Patent Document 2 have a disadvantage that the configuration is complicated. Further, in the laser microscope apparatus described in Patent Document 2, since the fluorescence signal is acquired with the sampling of the fewest pixels, the fluorescence signal is discarded with respect to the pixel that can capture a lot of fluorescence, and sufficient SN is obtained. There is an inconvenience that cannot be done. Furthermore, in the laser microscope apparatus described in Patent Document 2, the sampling pulse must be made sufficiently fast with respect to the laser pulse, and if the sampling pulse is made fast, the output becomes very small, so that a sufficient output can be obtained. There is no possibility.

  An object of the present invention is to provide a laser microscope apparatus capable of reducing the moire phenomenon occurring in an image with a simple configuration.

In order to achieve the above object, the present invention provides the following means.
The present invention relates to a light source that emits pulsed laser light whose intensity changes so as to have a peak at a predetermined period, a scanner that scans pulsed laser light emitted from the light source, and a pulsed laser light that is scanned by the scanner. An objective lens that irradiates the specimen, an observation light detector that detects the light intensity of the reflected light, fluorescence, or transmitted light from the specimen caused by the irradiation of the pulsed laser light, and photoelectrically converts it into a light intensity signal, and the observation An image information generation unit that generates image information by integrating light intensity signals of the reflected light, fluorescence, or transmitted light obtained by a light detection unit for each pixel period, and a pulse period of the pulsed laser light emitted from the light source noise and filter capable removed from the light intensity signal, said reflected light, fluorescence caused by the deviation between the sampling period of the light intensity signal by the observation light detector and Alternatively, a noise determination unit that determines whether or not the noise is included in the light intensity signal of transmitted light, and when the noise determination unit determines that the noise is included, the reflected light and fluorescence are filtered by the filter. Alternatively, a filter control unit that removes the noise from the light intensity signal of the transmitted light, a laser light detection unit that detects the light intensity of the pulsed laser light emitted from the light source and photoelectrically converts the light intensity signal, and the laser light A synchronization system control unit that synchronizes the detection unit and the sampling period of the observation light detection unit, and the noise determination unit is based on the light intensity signal of the pulsed laser light obtained by the laser light detection unit, Provided is a laser microscope apparatus that determines whether or not the noise is included in a light intensity signal of reflected light, fluorescence, or transmitted light .

  According to the present invention, when laser light emitted from a light source is irradiated onto a specimen via a scanner and an objective lens, reflected light from the specimen generated in synchronization with the peak of the laser light that appears periodically, fluorescence or The transmitted light is detected by the observation light detection unit and photoelectrically converted into a light intensity signal. Then, the image information generation unit integrates the light intensity signal for each pixel period for determining the light intensity of each pixel, and generates image information of the sample.

  In this case, if the pulse period of the pulsed laser light emitted from the light source and the sampling period of the light intensity signal by the observation light detection unit are not synchronized, the number of samplings for each pixel forming the image is different and the image is uneven ( Moire) occurs, but noise generated by the difference between the pulse period of the pulse laser beam and the sampling period of the light intensity signal that causes moire is removed from the light intensity signal by a filter, and is generated by the image information generation unit. Moire phenomenon that occurs in the image information of a specimen can be reduced.

  This eliminates the need for a complicated configuration to synchronize the pulse period of the pulsed laser light emitted from the light source and the sampling period of the light intensity signal by the observation light detection unit. The specimen can be observed with reduced

In the above invention, a noise determination unit that determines whether or not the noise is included in the light intensity signal of the reflected light, fluorescence, or transmitted light, and when the noise determination unit determines that the noise is included A filter control unit that removes the noise from the light intensity signal of the reflected light, fluorescence, or transmitted light by the filter .

  With this configuration, it is possible to automatically determine whether or not the moire phenomenon occurs in the sample image information by the noise determination unit. Further, even when the moire phenomenon occurs, the filter control unit can automatically suppress the occurrence of the moire phenomenon.

In the above invention, the laser light detection unit that detects the light intensity of the pulsed laser light emitted from the light source and photoelectrically converts it into a light intensity signal, and the sampling period of the laser light detection unit and the observation light detection unit A synchronization control unit that synchronizes, and the noise determination unit converts the light intensity signal of the reflected light, fluorescence, or transmitted light to the light intensity signal of the pulsed laser light obtained by the laser light detection unit. It is determined whether or not noise is included .

  Since sample information is included in reflected light, fluorescence, or transmitted light, if you try to determine whether the light intensity signals of these reflected light, fluorescence, or transmitted light actually contain noise that causes moire Will be affected. On the other hand, based on the light intensity signal of the pulse laser beam, it is affected by the specimen by determining whether the light intensity signal of reflected light, fluorescence, or transmitted light contains noise that causes moire. Therefore, the accuracy of determining the presence or absence of moire can be improved.

The present invention relates to a light source that emits pulsed laser light whose intensity changes so as to have a peak at a predetermined period, a scanner that scans pulsed laser light emitted from the light source, and a pulsed laser light that is scanned by the scanner. An objective lens that irradiates the specimen, an observation light detector that detects the light intensity of the reflected light, fluorescence, or transmitted light from the specimen caused by the irradiation of the pulsed laser light, and photoelectrically converts it into a light intensity signal, and the observation An image information generation unit that generates image information by integrating light intensity signals of the reflected light, fluorescence, or transmitted light obtained by a light detection unit for each pixel period, and a pulse period of the pulsed laser light emitted from the light source And a filter capable of removing from the light intensity signal noise generated by a deviation from the sampling period of the light intensity signal by the observation light detection unit, the reflected light, and fluorescence Alternatively, a noise determination unit that determines whether or not the noise is included in the light intensity signal of transmitted light, and when the noise determination unit determines that the noise is included, the reflected light and fluorescence are filtered by the filter. or comprising a filter controller for removing the noise from the light intensity signal of the transmitted light, and a laser beam detector which detects the light intensity of the pulse laser light emitted from the light source and photoelectrically converts the light intensity signal, the Based on the light intensity signal of the pulsed laser light obtained by the laser light detection unit and the sampling period of the observation light detection unit, a noise determination unit adds the noise to the light intensity signal of reflected light, fluorescence, or transmitted light. A laser microscope apparatus for determining whether or not is included .

  With this configuration, noise that causes moiré is generated in the light intensity signal of reflected light, fluorescence, or transmitted light based on the light intensity signal of the pulse laser light and the sampling period of the light intensity signal by the light detection unit. By determining whether or not it is included, it is possible to improve the accuracy of determining the presence or absence of moire without being affected by the sample.

In the invention as a reference example of the present invention, the noise determination unit is configured to reflect the reflected light, fluorescence, or transmitted light based on a light intensity signal of the reflected light, fluorescence, or transmitted light obtained by the observation light detection unit. It may be determined whether the noise is included in the light intensity signal.

In the above-mentioned invention, two or more observation light detection units are provided, and the filter is provided for each of the two or more observation light detection units, and the reflected light, fluorescence, or transmitted light obtained by each observation light detection unit. It may be possible to select whether to remove the noise for each light intensity signal.
With this configuration, moiré is reduced as necessary for each piece of image information based on the reflected light, fluorescence, and transmitted light intensity signals obtained for each of a plurality of observation detection units for the same specimen. You can do it or not.

  In the above invention, the observation light detection unit is a return light detection unit that detects the reflected light or the fluorescence, and a transmitted light detection unit that detects the transmitted light, and the filter is the return light detection unit. The noise is removed for each reflected light intensity signal or fluorescence intensity signal obtained by the return light detecting section and each transmitted light intensity signal obtained by the transmitted light detecting section. It may be possible to select whether or not to make it.

  Since how moire is noticeable changes depending on the fluorescence and transmitted light, and how the moire is noticeable also changes depending on the type of fluorescence, this configuration allows the same specimen to be reflected by reflected light, fluorescence, and transmitted light. Moire can be reduced or not as necessary for each piece of image information.

In the above invention, the filter comprises a filter constant corresponding to the frequency of the noise included in the light intensity signal of the reflected light, fluorescence or transmitted light, and a filter strength for determining the strength for removing the noise, The filter strength may be changeable.
With this configuration, the amount of noise removal can be adjusted by changing the filter strength in accordance with the fluorescence lifetime for each specimen.

In the above invention, a table is provided in which a scanning speed per pixel by the scanner is associated with a frequency band to be removed by the filter, and the filter constant is determined by the table and the noise frequency. Also good.
With this configuration, the filter constant can be easily determined with reference to the table.

  According to the present invention, it is possible to reduce the moire phenomenon that occurs in an image with a simple configuration.

1 is a schematic configuration diagram illustrating a laser microscope apparatus according to a first embodiment of the present invention. It is a block diagram which shows the system control part periphery of the laser microscope apparatus of FIG. It is a block diagram which shows the system control part periphery of the laser microscope apparatus which concerns on the 3rd modification of 1st Embodiment of this invention. It is a block diagram which shows the system control part periphery of the laser microscope apparatus which concerns on the reference embodiment of invention as a reference example of this invention. It is a block diagram which shows the system control part periphery of the laser microscope apparatus which concerns on the modification of reference embodiment of invention as a reference example of this invention.

[First Embodiment]
A laser microscope apparatus according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, a laser microscope apparatus 100 according to the present embodiment observes a specimen S by irradiating a specimen S with a laser light source apparatus 1 that emits laser light and a laser light emitted from the laser light source apparatus 1. Microscope body 3, observation light detection unit (return light detection unit) 27, observation light detection unit (transmission light detection unit) 29, laser light source device 1, microscope body 3, and observation light detection units 27, 29 A system control unit 5 that controls the display, a display 7 that displays an image of the specimen S, and a PC (Personal computer) 9 that operates the system control unit 5 and the display 7.

  The laser light source device 1 includes a laser light source (light source) 11 that generates laser light, an AOM (acousto-optic element) 13 that adjusts the intensity of the laser light emitted from the laser light source 11, and laser light that has passed through the AOM 13. A dichroic mirror 15 that branches into one optical path, a laser intensity detector (laser light detector) 17 that detects the light intensity of the laser light that passes through one optical path branched by the dichroic mirror 15, and a laser beam that passes through the other optical path. And a scanner 19 for scanning on the specimen S.

The laser light source 11 generates laser light whose intensity changes so as to have a peak at a predetermined period, such as femtosecond pulse laser light, according to the wavelength and power set by the system control unit 5.
The AOM 13 can adjust the frequency of the laser light in accordance with ND and ON / OFF set by the system control unit 5.

  The dichroic mirror 15 reflects a specific wavelength of the laser light from the AOM 13 toward the laser intensity detector 17, while allowing other wavelengths to pass through and enter the scanner 19.

  The laser intensity detector 17 detects the light intensity of the laser light from the dichroic mirror 15 and photoelectrically converts it into a light intensity signal, and sends the obtained light intensity signal to the system control unit 5. The laser intensity detector 17 acquires a light intensity signal in accordance with the sampling period sent from the system control unit 5.

  As the scanner 19, for example, a galvanometer mirror, a resonant scanner, an acousto-optic deflection element, or the like is used. The scanner 19 includes a pair of galvanometer mirrors 19a and 19b that can swing around a predetermined swing axis (not shown) that intersects each other. The scanner 19 is driven by a raster scan method by changing the swing angle of the galvanometer mirrors 19a and 19b in accordance with the swing angle and swing speed set by the system control unit 5. As a result, the scanner 19 reflects the laser light transmitted through the dichroic mirror 15 by the galvanometer mirrors 19a and 19b, and can scan the sample S two-dimensionally.

  The microscope main body 3 irradiates the sample S with a stage 21 on which the sample S is placed, a dichroic mirror 23 that reflects the laser light scanned by the scanner 19, and the laser light reflected by the dichroic mirror 23. And an objective lens 25 that condenses the reflected light or fluorescence returned from the light source.

  The dichroic mirror 23 reflects the laser light from the scanner 19 toward the objective lens 25, and transmits reflected light or fluorescence from the sample S collected by the objective lens 25.

  As the observation light detection units 27 and 29, for example, a PMT (Photo Multiplier Tube) or an APD (Avalanche Photo Diode) can be used. The observation light detection unit 27 detects the light intensity of the fluorescence from the specimen S that has passed through the dichroic mirror 23, photoelectrically converts it into a light intensity signal, and sends the obtained light intensity signal to the system control unit 5. Yes. The observation light detection unit 29 detects the light intensity of the transmitted light that has passed through the sample S, photoelectrically converts it to a light intensity signal, and sends the obtained light intensity signal to the system control unit 5. These observation light detection units 27 and 29 are configured to acquire a light intensity signal in accordance with the sampling period sent from the system control unit 5.

  As shown in FIGS. 1 and 2, the system control unit 5 includes a filter 31 that removes specific noise from the light intensity signals obtained by the observation light detection units 27 and 29, and a light intensity signal that has passed through the filter 31. And an image information generating unit 33 that generates image information by integrating every pixel period.

  The filter 31 includes a filter constant corresponding to the frequency of noise included in the light intensity signal of reflected light, fluorescence, or transmitted light, and a filter strength that determines the strength for removing the noise. The filter strength can be changed. Therefore, the amount of noise removal can be adjusted by changing the filter strength according to the fluorescence lifetime for each specimen S.

  The image information generation unit 33 stores the coordinates of the condensing position of the laser light irradiated to the sample S and the light intensity signals from the observation light detection units 27 and 29 in association with each other, and stores the stored light intensity signal in the pixel cycle. The image information can be generated by integrating each time.

  As shown in FIGS. 1 and 2, the system control unit 5 includes a synchronous system control unit 41 that controls the sampling period of the laser intensity detector 17 and the observation light detection units 27 and 29, and an observation light detection unit 27, 29, a noise determination unit 43 that determines whether or not the specific light is included in the light intensity signal obtained by 29, a filter control unit 45 that controls the filter 31 according to the determination result of the noise determination unit 43, and a filter A filter setting UI (User Interface) 47 for controlling 31 is provided.

  The synchronous system control unit 41 inputs the same sampling cycle to the laser intensity detector 17 and the observation light detection units 27 and 29, and the sampling cycle of the light intensity signal of the laser beam by the laser intensity detector 17 and the observation light detection unit 27. The sampling period of the reflected light or fluorescent light intensity signal or the sampling period of the transmitted light intensity signal by the observation light detector 29 can be synchronized.

  Based on the light intensity signal sent from the laser intensity detector 17, the noise determination unit 43 determines the pulse period of the pulse laser light emitted from the laser light source 11 and the sampling period of the light intensity signal by the observation light detection units 27 and 29. It is determined whether or not the noise caused by the deviation is included in the light intensity signal obtained by the observation light detectors 27 and 29. Moreover, the noise determination part 43 can also determine the frequency of noise, when the noise is contained in a light intensity signal.

  Since the laser intensity detector 17 acquires the light intensity signal of the laser light in synchronization with the sampling period of the observation light detectors 27 and 29, the light intensity signal obtained by the laser intensity detector 17 is obtained by the observation light detectors 27 and 29. Information on the sampling period of reflected light, fluorescence or transmitted light is included. The determination result by the laser intensity detector 17 is sent to the filter control unit 45.

  When the noise determination unit 43 determines that the noise is included in the light intensity signals obtained by the observation light detection units 27 and 29, the filter control unit 45 determines the frequency of the noise, and one pixel by the scanner 19. Whether correction is performed to remove noise from the light intensity signal based on the per-scan speed (sec / pixel, hereinafter referred to as scan speed) and the size (number of pixels) of the image generated by the image information generation unit 33 It is to judge whether or not.

  The filter control unit 45 has a table 45a that associates the scan speed with the frequency band to be removed by the filter 31, and when correction of the light intensity signal is necessary, the table 45a and the noise frequency Thus, the filter constant of the filter 31 is selected and set in the filter 31.

  The filter setting UI 47 allows the user to set ON / OFF of the correction of the light intensity signal by the filter 31 and the intensity to remove noise from the light intensity signal, instead of controlling the filter 31 by the filter control unit 45. It is like that.

The operation of the laser microscope apparatus 100 configured as described above will be described.
In the present embodiment, in order to simplify the explanation, the case of observing the fluorescence from the specimen A will be described as an example. When the reflected light or the transmitted light from the specimen S is observed, the fluorescence from the specimen S will be described. Since the same processing as that in the case of observing the image may be performed, the description is omitted here. Therefore, it will be described that the dichroic mirror 23 transmits only the fluorescence from the sample S and does not transmit the reflected light from the sample S.

  In order to observe the sample S with the laser microscope apparatus 100 according to the present embodiment, first, the sample S is placed on the stage 21, and the system control unit 5 performs laser source 11, AOM 13, laser intensity detector 17, scanner 19, The observation light detection unit 27 and the display 7 are driven under predetermined setting conditions. In this embodiment, the synchronous system control unit 41 sets the laser intensity detector 17 and the observation light detection unit 27 to the same sampling period.

  The frequency of the laser light emitted from the laser light source 11 is adjusted by the AOM 13 and then branched into two optical paths by the dichroic mirror 15. The laser beam reflected by the dichroic mirror 15 enters the laser intensity detector 17. In the laser intensity detector 17, the light intensity of the incident laser light is detected and photoelectrically converted into a light intensity signal in accordance with the sampling period set by the synchronous system control unit 41, and sent to the noise determination unit 43.

  On the other hand, the laser light transmitted through the dichroic mirror 15 is reflected by the galvano mirrors 19 a and 19 b of the scanner 19 that swings around the swing axis, and then is reflected by the dichroic mirror 23 and is irradiated onto the sample by the objective lens 25. . Thereby, the laser beam is scanned two-dimensionally on the specimen S in accordance with the swing angle of the galvanometer mirrors 19a and 19b.

  On the focal plane of the objective lens 25 in the specimen S, the photon density of the laser light is increased, and the fluorescent substance in the specimen S is excited by the multiphoton excitation effect to generate fluorescence. The generated fluorescence is collected by the objective lens 25, passes through the dichroic mirror 23, and enters the observation light detection unit 27. In the observation light detection unit 27, the light intensity of the fluorescence from the specimen S is detected and photoelectrically converted into a light intensity signal according to the sampling period set by the synchronous system control unit 41.

  The fluorescence light intensity signal acquired by the observation light detection unit 27 is sent to the image information generation unit 33 via the filter 31. In the image information generating unit 33, the coordinates of the condensing position of the laser light irradiated on the specimen S and the light intensity signal sent from the observation light detecting unit 27 are stored in association with each other, and the stored light intensity signal is stored. The image information of the sample S is generated by integrating every pixel period. The image information of the specimen S generated by the image information generation unit 33 is displayed on the display 7 via the PC 9. Thereby, the user can observe the sample S on the image displayed on the display 7.

  In this case, if the pulse period of the pulsed laser light emitted from the laser light source 11 and the sampling period of the light intensity signal by the observation light detection unit 27 are not synchronized, the number of sampling differs for each pixel forming the image. As a result, unevenness (moire) occurs in the image.

  In contrast, in the present embodiment, the noise determination unit 43 converts the fluorescence light intensity signal obtained by the observation light detection unit 27 based on the light intensity signal of the laser light transmitted from the laser intensity detector 17. It is determined whether or not the noise that causes moiré is included.

  When the noise determination unit 43 determines that the noise is not included in the fluorescence light intensity signal, the light intensity signal passes through the filter 31 without being corrected and is input to the image information generation unit 33. On the other hand, if it is determined that the noise is included in the fluorescence light intensity signal, the noise determination unit 43 also determines the frequency of the noise, and the noise control and the determination result of the noise frequency are filter-controlled. Sent to the unit 45. For example, if the intensity change of the light intensity signal is periodic and the amplitude of the change is equal to or greater than a certain threshold, the noise determination unit 43 determines that there is noise and can calculate the frequency. The frequency range to be determined and the intensity change range are determined in advance.

  The filter controller 45 performs correction to remove the noise from the light intensity signal based on the frequency of noise included in the fluorescence light intensity signal, the scan speed, and the size of the image generated by the image information generator 33. It is determined whether or not to do so. For example, the filter control unit 45 can calculate the number of moire fringes per line from the scan speed and the image size, and can determine whether or not the moire fringes appear in the image. In other words, from the “noise frequency”, “scan speed”, and “image size”, the number of shades generated on one screen and the difference between the shades and shades can be calculated, and by setting a threshold for the number of shades and the amplitude, Whether or not correction is necessary can be determined.

  When the filter control unit 45 determines that it is not necessary to correct the fluorescence light intensity signal, the light intensity signal passes through the filter 31 without being corrected and is input to the image information generation unit 33. In this case, an image that is not substantially affected by moire is generated by the image information generation unit 33 and displayed on the display 7.

  On the other hand, when the filter control unit 45 determines that correction of the fluorescence light intensity signal is necessary, the filter control unit 45 refers to the noise frequency information and a table in which the preset noise frequency and the filter constant are associated with each other. The filter 31 is set. As a result, noise that causes moire is removed from the fluorescent light intensity signal by the filter 31 and sent to the image information generation unit 33. The image information generation unit 33 generates an image with reduced moire phenomenon, and the display 7 Is displayed.

  As described above, according to the laser microscope apparatus 100 according to the present embodiment, the filter 31 removes the noise causing the moire from the fluorescent light intensity signal, so that the moire phenomenon that occurs in the image of the sample S is eliminated. Can be reduced. This eliminates the need for a complicated configuration for synchronizing the pulse cycle of the pulsed laser light emitted from the laser light source 11 and the sampling cycle of the light intensity signal by the observation light detection unit 27, and generates the image with a simple configuration. The specimen S can be observed with reduced moire phenomenon.

  Further, the noise determination unit 43 can automatically determine whether or not a moire phenomenon occurs in the image of the sample S. Further, even when the moire phenomenon occurs, the filter control unit 45 can automatically suppress the occurrence of the moire phenomenon.

  Further, since the reflected light, fluorescence, or transmitted light includes information on the specimen S, it is determined whether or not noise that causes moire is actually included in the light intensity signal of the reflected light, fluorescence, or transmitted light. Then, the sample S is affected. On the other hand, based on the light intensity signal of the pulsed laser light and the sampling period of the light intensity signal by the observation light detection unit 27, it is determined whether or not the light intensity signal of the fluorescence includes noise that causes moire. Thus, it is possible to improve the determination accuracy of the presence or absence of moire without being affected by the sample S. The same applies when the specimen S is observed with reflected light or transmitted light.

This embodiment can be modified as follows.
In the present embodiment, the noise determination unit 43 determines whether noise is included in the fluorescence light intensity signal, and the filter constant of the filter 31 is set by the filter control unit 45 (automatic mode). As a first modification, the user may determine the presence or absence of moire while viewing the image of the sample S displayed on the display 7 and set the filter constant of the filter 31 using the filter setting UI 47 (manual mode). Good.
According to the manual mode, it is possible to correct even a minute moire that cannot be determined in the automatic mode.

  In the present embodiment, the filter control unit 45 has the table 45a that associates the scan speed with the frequency band to be removed by the filter 31, and selects the filter constant of the filter 31 according to the table 45a. As a second modified example, the filter constant of the filter 31 may be calculated by calculation processing based on the scan speed and the frequency band to be removed by the filter without using the table 45a.

  In the present embodiment, a single filter 31 is used for the plurality of observation light detection units 27 and 29. However, as a third modification, as shown in FIG. A filter 31A for removing specific noise from the reflected light or fluorescent light intensity signal obtained by the unit 27, and a filter 31B for removing specific noise from the transmitted light intensity signal obtained by the observation light detecting unit 29. It is good also as adopting.

  In this case, whether or not noise is removed for each of the light intensity signal obtained by the observation light detection unit 27 and the light intensity signal obtained by the observation light detection unit 29 by controlling the filters 31A and 31B by the filter control unit 45. It is good also as selecting. In addition, as shown in FIG. 3, the image information generation unit 33 </ b> A that generates an image based on the light intensity signal from the observation light detection unit 27 and the image based on the light intensity signal from the observation light detection unit 29 are generated. The image information generation unit 33B may be employed.

  Since the moire conspicuousness changes depending on the fluorescence and the transmitted light, and the moire conspicuousness varies depending on the type of the fluorescent light. By doing so, the same specimen S is reflected by the reflected light, the fluorescent light, and the transmitted light. Moire can be reduced or not as necessary for each image.

[ Reference embodiment]
Next, a microscope apparatus according to a reference embodiment of the invention as a reference example of the present invention will be described.
As shown in FIG. 4, the laser microscope apparatus 200 according to the present embodiment is different from the first embodiment in that the dichroic mirror 15 and the laser intensity detector 17 are not provided.
In the following, portions having the same configuration as those of the laser microscope apparatus 100 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

In the laser microscope apparatus 200 according to the present embodiment, all of the laser light that has passed through the AOM 13 enters the scanner 19.
The synchronous system control unit 41 inputs the same sampling period to the observation light detection units 27 and 29, the sampling period of the reflected light or fluorescent light intensity signal by the observation light detection unit 27, and the transmitted light by the observation light detection unit 29. The sampling period of the light intensity signal can be synchronized.

The observation light detection unit 27 sends the acquired fluorescence or reflected light intensity signal together with the filter 31 to the noise determination unit 43, and the observation light detection unit 29 also acquires the acquired transmitted light intensity signal together with the filter 31 and the noise determination unit. 43 to send.
The noise determination unit 43 determines whether or not the light intensity signal transmitted from the observation light detection units 27 and 29 actually includes noise that causes moire.

The operation of the thus configured laser microscope apparatus 200 will be described.
Also in the present embodiment, in order to simplify the description, a case where fluorescence from the specimen S is observed will be described as an example.
The fluorescence light intensity signal acquired by the observation light detection unit 27 is sent to the filter 31 and the noise determination unit 43. If the noise determination unit 43 determines that the noise causing the moire is not included in the fluorescence light intensity signal, the fluorescence light intensity signal sent to the filter 31 passes through without being corrected and is image information. Input to the generation unit 33.

  On the other hand, when the noise determination unit 43 determines that noise that causes moire is included in the fluorescence light intensity signal, the noise determination unit 43 also determines the frequency of the noise, and includes noise. The determination result of the noise frequency is sent to the filter control unit 45.

  Next, the filter control unit 45 determines whether or not the fluorescence light intensity signal needs to be corrected in the same manner as in the first embodiment. When correction is necessary, the filter control unit 45 selects the filter constant of the filter 31 based on the table 45 a and the noise frequency, and sets the filter constant in the filter 31. As a result, noise that causes moire is removed from the fluorescent light intensity signal by the filter 31 and input to the image information generation unit 33.

  As described above, according to the laser microscope apparatus 200 according to the present embodiment, the moiré phenomenon generated in the image is reduced with a simpler and less expensive configuration because the dichroic mirror 15 and the laser intensity detector 17 are not employed. The specimen S can be observed.

This embodiment can be modified as follows.
That is, also in this embodiment, it is good also as controlling the filter 31 by manual mode instead of automatic mode similarly to the 1st modification of 1st Embodiment.
In the present embodiment, similarly to the second modification of the first embodiment, the filter control unit 45 does not employ the table 45a, but based on the scan speed and the frequency band to be removed by the filter. These filter constants may be calculated by arithmetic processing.

  Similarly to the third modification of the first embodiment, as shown in FIG. 5, a filter 31A for removing specific noise from the reflected light or fluorescent light intensity signal obtained by the observation light detection unit 27; A filter 31B that removes specific noise from the light intensity signal of the transmitted light obtained by the observation light detection unit 29 is employed, and the light intensity signal obtained by the observation light detection unit 27 and the light obtained by the observation light detection unit 29 are used. It is good also as selecting whether noise is removed for every intensity | strength signal.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included. For example, the present invention is not limited to those applied to each of the above embodiments, and may be applied to embodiments in which these embodiments are appropriately combined, and is not particularly limited.

  Moreover, in the said embodiment, although the aspect provided with the observation light detection part 27 as a return light detection part and the observation light detection part 29 as a transmitted light detection part was illustrated and demonstrated, it replaced with this, for example, is reflected, for example The aspect provided with either one of the observation light detection part which detects light or fluorescence, and the observation light detection part which detects transmitted light may be sufficient. Moreover, the aspect in which both the observation light detection part which detects reflected light or fluorescence, and the observation light detection part which detects transmitted light may exist in multiple numbers, respectively, either one of these observation light detection parts is one and the other There may be a mode in which a plurality of exist. In this case, a filter 31 is provided for each observation light detection unit, and the filter control unit 45 selects whether to remove noise for each intensity signal of reflected light, fluorescence, or transmitted light obtained by each observation light detection unit. You can do that.

  Further, instead of the observation light detection units 27 and 29, for example, a multi-channel having a plurality of channels (observation light detection units) that detect the intensity of reflected light, fluorescence, or transmitted light from the specimen S and convert it into a light intensity signal. An observation light detection device (not shown) such as a PMT (Photomultiplier Tube) may be employed. Also in this case, a filter 31 is provided for each channel, and the filter control unit 45 can select whether noise is removed for each intensity signal of reflected light, fluorescence, or transmitted light obtained by each channel. That's fine.

  Further, in the above-described embodiment of the present invention, a mode in which the sampling period of the laser intensity detector 17 and the observation light detection units 27 and 29 is controlled to be synchronized has been shown, but such control is not performed, for example, The noise determination unit 43 may determine whether noise is included in the light intensity signal based on the light intensity signal sent from the laser intensity detector 17 and the sampling periods of the observation light detection units 27 and 29. .

11 Laser light source 17 Laser intensity detector (laser light detector)
19 Scanner 25 Objective Lens 27 Observation Light Detection Unit (Return Light Detection Unit)
29 Observation light detector (transmitted light detector)
31 Filter 33 Image Information Generation Unit 43 Noise Determination Unit 45 Filter Control Unit 100, 200 Laser Microscope Device

Claims (6)

A light source that emits pulsed laser light whose intensity changes so as to have a peak at a predetermined period;
A scanner that scans the pulsed laser light emitted from the light source;
An objective lens for irradiating the sample with pulsed laser light scanned by the scanner;
An observation light detection unit that detects the light intensity of reflected light, fluorescence, or transmitted light from the specimen generated by the irradiation of the pulsed laser light and photoelectrically converts it into a light intensity signal;
An image information generation unit that generates image information by integrating the light intensity signals of the reflected light, fluorescence, or transmitted light obtained by the observation light detection unit for each pixel period;
A filter capable of removing, from the light intensity signal, noise caused by a deviation between a pulse period of the pulsed laser light emitted from the light source and a sampling period of the light intensity signal by the observation light detection unit ;
A noise determination unit that determines whether or not the noise is included in the light intensity signal of the reflected light, fluorescence, or transmitted light;
A filter control unit that removes the noise from the light intensity signal of the reflected light, fluorescence, or transmitted light by the filter when the noise determination unit determines that the noise is included;
A laser light detector that detects the light intensity of the pulsed laser light emitted from the light source and photoelectrically converts it into a light intensity signal;
A synchronization system control unit that synchronizes the sampling period of the laser light detection unit and the observation light detection unit,
The noise determination unit determines whether or not the noise is included in the light intensity signal of reflected light, fluorescence, or transmitted light based on the light intensity signal of the pulsed laser light obtained by the laser light detection unit. Laser microscope device. A light source that emits pulsed laser light whose intensity changes so as to have a peak at a predetermined period;
A scanner that scans the pulsed laser light emitted from the light source;
An objective lens for irradiating the sample with pulsed laser light scanned by the scanner;
An observation light detection unit that detects the light intensity of reflected light, fluorescence, or transmitted light from the specimen generated by the irradiation of the pulsed laser light and photoelectrically converts it into a light intensity signal;
An image information generation unit that generates image information by integrating the light intensity signals of the reflected light, fluorescence, or transmitted light obtained by the observation light detection unit for each pixel period;
A filter capable of removing, from the light intensity signal, noise caused by a deviation between a pulse period of the pulsed laser light emitted from the light source and a sampling period of the light intensity signal by the observation light detection unit ;
A noise determination unit that determines whether or not the noise is included in the light intensity signal of the reflected light, fluorescence, or transmitted light;
A filter control unit that removes the noise from the light intensity signal of the reflected light, fluorescence, or transmitted light by the filter when the noise determination unit determines that the noise is included;
A laser light detection unit that detects the light intensity of the pulsed laser light emitted from the light source and photoelectrically converts it into a light intensity signal;
Based on the light intensity signal of the pulsed laser light obtained by the laser light detection unit and the sampling period of the observation light detection unit, the noise determination unit converts the light intensity signal of reflected light, fluorescence, or transmitted light to the light intensity signal. A laser microscope apparatus that determines whether or not noise is included . Comprising two or more observation light detectors;
The filter is provided for each of the two or more observation light detection units, and selects whether to remove the noise for each light intensity signal of the reflected light, fluorescence, or transmitted light obtained by each observation light detection unit The laser microscope apparatus according to claim 1 or 2 , which can be used. The observation light detection unit includes a return light detection unit that detects the reflected light or the fluorescence, and a transmitted light detection unit that detects the transmitted light,
The filter is provided for each of the return light detection unit and the transmitted light detection unit, and the reflected light intensity signal or the fluorescence intensity signal obtained by the return light detection unit and the transmitted light obtained by the transmitted light detection unit. The laser microscope apparatus according to claim 3 , wherein it is possible to select whether to remove the noise for each light intensity signal. The filter includes a filter constant corresponding to the frequency of the noise included in the light intensity signal of the reflected light, fluorescence, or transmitted light, and a filter intensity that determines the intensity for removing the noise, and the filter intensity can be changed. The laser microscope apparatus according to any one of claims 1 to 4 , wherein: 6. The laser according to claim 5 , further comprising a table in which a scanning speed per pixel by the scanner is associated with a frequency band to be removed by the filter, and the filter constant is determined by the table and the noise frequency. Microscope device.

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