JP4576148B2 - Scanning laser microscope equipment - Google Patents

Description

  The present invention combines laser beams of a plurality of wavelengths emitted from a plurality of laser light sources, scans the sample while irradiating the sample through an optical system that guides the combined laser light to the sample, and emits light emitted from the sample by irradiation. The present invention relates to a scanning laser microscope apparatus to be observed.

  In recent years, a scanning laser microscope apparatus that performs scanning while irradiating a specimen with a plurality of laser beams having different wavelengths and observing light emitted from the specimen has been widely used. Since the light intensity of the light emitted from the specimen is proportional to the light intensity of the laser light emitted to the specimen, in order to make the light intensity of the light emitted from the specimen appropriate, the light intensity of the laser light emitted to the specimen is appropriate. It is necessary to. Therefore, the laser light applied to the specimen is wavelength-separated using an acousto-optic device, wavelength selection filter, etc., and the light intensity is monitored for each wavelength, and the laser light is attached to a plurality of laser lights based on the monitored light intensity. A control method for controlling the acoustooptic device is proposed (Patent Document 1).

JP-A-11-174332

  However, when the light intensity of a plurality of laser beams is monitored by separating the wavelengths of the plurality of laser beams and the light intensity of the laser beams is controlled by an acousto-optic element, the control speed is higher than the speed at which the laser beams scan the specimen. There is a problem in that it is impossible to follow and an image obtained by irradiating a sample with laser light having an appropriate light intensity cannot be obtained.

  Further, when feedback control is performed and laser light having a desired light intensity is not emitted, the conventional apparatus has a problem that an abnormal part of the apparatus cannot be easily identified.

  The present invention has been made in view of the above, and provides a scanning laser microscope apparatus that can easily and accurately control the light intensities of laser beams having a plurality of wavelengths, or that can easily identify an abnormal portion of the apparatus. The purpose is to do.

In order to achieve the above object, a scanning laser microscope apparatus according to the present invention synthesizes a plurality of laser beams emitted from a plurality of laser light sources, and transmits the synthesized laser beams through an optical system that guides the sample to a specimen. In a scanning laser microscope apparatus that scans while irradiating a specimen and observes light emitted from the specimen by the irradiation, light intensity monitoring means for monitoring the light intensity of the synthesized laser light, and each laser light And a control means for controlling the light intensity of each laser beam by feeding back the light intensity monitored by the light intensity monitoring means and performing the time-sharing control of the emission.

The scanning laser microscope apparatus according to the present invention is the above-described invention, wherein the specimen is synthesized through an optical system that synthesizes a plurality of laser beams emitted from a plurality of laser light sources and guides the synthesized laser beams to the specimen. In a scanning laser microscope apparatus that scans while irradiating the laser beam and observes the light emitted from the specimen by the irradiation, each laser that outputs a light intensity corresponding to the light intensity of the laser light output from the emission port of each laser light source Detection light intensity monitoring means for monitoring the light intensity of the detection light from the detection port of the light source, and time division control of emission of each laser light, and feeding back the light intensity monitored by the detection light intensity monitoring means to each laser light And a control means for controlling the light intensity.

The scanning laser microscope apparatus according to the present invention is the above-described invention, wherein the specimen is synthesized through an optical system that synthesizes a plurality of laser beams emitted from a plurality of laser light sources and guides the synthesized laser beams to the specimen. In a scanning laser microscope apparatus that scans while irradiating and observing the light emitted from the specimen by the irradiation, light intensity monitoring means for monitoring the light intensity of the synthesized laser light, and emission of each laser light source Detection light intensity monitoring means for monitoring the light intensity of the detection light from the detection opening of each laser light source that outputs a light intensity corresponding to the light intensity of the laser light output from the opening, and time division control of emission of each laser light At the same time, the light intensity of the laser light or the detection light monitored by the light intensity monitoring means or the detection light intensity monitoring means is fed back to obtain the light intensity of each laser light. Control means for controlling, storage means for preliminarily storing a reference light intensity of the laser light, a reference light intensity of the reflected light, and a reference response ratio which is a response ratio of a control signal for the control means to control the light source; And the control means includes: the light intensity of the laser light, the light intensity of the detection light, and the response ratio of the control signal; the reference light intensity of the laser light stored in the storage means; and the reference light of the detection light. Based on the intensity and the reference response ratio, the presence / absence of an abnormality including the identification of an abnormal portion of the scanning laser microscope apparatus is determined.

In the scanning laser microscope apparatus according to the present invention as set forth in the invention described above, the control means applies at least all of the laser beams for each irradiation position on the specimen irradiated with the synthesized laser beam. Switching control for completing time-division output is performed.

In the scanning laser microscope apparatus according to the present invention , in the above invention, the control unit compares the light intensity of the detection light with a reference light intensity of the detection light, and the light intensity of the detection light is If the intensity of the detection light is smaller than the reference light intensity, the laser light source is specified as abnormal.

Further, in the scanning laser microscope apparatus according to the present invention , in the above invention, when the light intensity of the detection light is not smaller than a reference light intensity of the detection light, the control means If the intensity of the laser light is compared with the reference light intensity of the laser light, and the light intensity of the laser light is smaller than the reference light intensity of the laser light, the optical system or the light intensity for guiding the laser light to the specimen It is characterized by specifying that a detection means is abnormal.

Further, in the scanning laser microscope apparatus according to the present invention , in the above invention, the control means is configured such that when the light intensity of the laser light is not smaller than the reference light intensity of the laser light, the response ratio and the A reference response ratio is compared, and when the response ratio is smaller than the reference response ratio, the laser light source is specified to be abnormal due to deterioration.

The scanning laser microscope apparatus according to the present invention is characterized in that, in the above invention, each of the plurality of laser light sources is a semiconductor laser capable of high-speed switching.

  The scanning laser microscope apparatus according to the present invention easily and accurately controls the light intensities of laser beams having a plurality of wavelengths by controlling a plurality of laser beams emitted from a plurality of laser light sources in a time-sharing manner, or There is an effect that an abnormal portion of the apparatus can be easily identified.

  Exemplary embodiments of a scanning laser microscope apparatus according to the present invention will be described below in detail with reference to the accompanying drawings.

(Embodiment 1)
FIG. 1 is a functional block diagram showing a schematic configuration of a scanning laser microscope apparatus 100 according to the first embodiment of the present invention. This scanning laser microscope apparatus 100 is a fluorescence microscope apparatus, and reflects LD units 1 and 2 that store semiconductor lasers (LD) 3 and 4 that are excitation light sources having different wavelengths, and laser light A emitted from the LD 3. A mirror 5 that combines the laser beam A emitted from the LD 3 and the laser beam B emitted from the LD 4 into a single beam laser beam AB, an optical fiber 7 that introduces the laser beam AB, A scanner unit 8 that receives the laser beam AB emitted from the optical fiber 7, a control unit 17 that controls the light intensity of the laser beams A and B using the laser beam AB 2 separated by the scanner unit 8, and the scanner unit 8 The microscope 14 for condensing the laser beam AB1 separated by the focus on the specimen 15 and the fluorescence emitted from the specimen 15 And a display unit 18 for displaying images through 8.

  Further, the scanner unit 8 splits the laser beam AB incident from the optical fiber 7 into laser beams AB1 and AB2, and converts the laser beam AB2 separated by the beam splitter 9 into an electric signal S2 corresponding to the light intensity. The photoelectric detector 16 for conversion, the excitation dichroic mirror 11 that reflects the laser light that has passed through the beam splitter 9, the galvano 10 that scans the sample 15 with the laser light reflected by the excitation dichroic mirror 11, and the sample 15 A confocal pinhole 12 that allows the fluorescence emitted only from the focal point of the excitation light to pass through, and a photodetector 13 that converts the fluorescence that has passed through the confocal pinhole 12 into an electrical signal corresponding to the light intensity.

  In FIG. 1, LDs 3 and 4 emit laser beams A and B having different excitation wavelengths in a time-sharing manner in synchronization with time-divided control signals CA and CB, respectively. Since the laser beam A and the laser beam B are time-divided synchronously, even if they are combined by the combining dichroic mirror 6, they become a single beam laser beam AB without interfering with each other. The laser beam AB enters the scanner unit 8 through the optical fiber 7. The laser beam AB incident on the scanner unit 8 is separated by the beam splitter 9 into a laser beam AB1 directed to the excitation dichroic mirror 11 and a laser beam AB2 directed to the photoelectric detector 16.

  The laser beam AB1 irradiates the specimen 15, excites the specimen 15, and emits fluorescence. The fluorescence enters the excitation dichroic mirror 11 through the microscope 14 and the galvano 10 in order. Since the excitation dichroic mirror 11 has a characteristic of reflecting light having an excitation wavelength and transmitting light having a fluorescence wavelength, the fluorescence incident on the excitation dichroic mirror 11 is transmitted through the excitation dichroic mirror 11 and passes through the confocal pinhole 12. Through the photoelectric detector 13. The photoelectric detector 13 converts it into an electrical signal corresponding to the intensity of the incident fluorescence and outputs it to the display unit 18. The display unit 18 converts the input electrical signal into a display signal, converts it, and displays it on the display screen.

  On the other hand, the laser beam AB2 is converted by the photoelectric detector 16 into an electric signal S2 corresponding to the light intensity of the laser beam AB2. The electric signal S2 is output to the control unit 17. The control unit 17 converts the input electric signal S2 into electric signals SA and SB in a time-sharing manner in synchronization with the control signals CA and CB. Control signals CA and CB are output based on SB.

  FIG. 2 is a time chart showing the correspondence relationship between the time-divided laser beam A, laser beam B, laser beam AB2, and signal S2. As shown in FIG. 2, a laser beam A having a light intensity VA is emitted from the LD 3 in a pulse shape, and a laser beam B having a light intensity VB is emitted from the LD 4 in a pulse shape. Since the laser beams A and B are emitted so as not to interfere with each other, the laser beam AB synthesized by the synthesis dichroic mirror 6 is simply a combination of the laser beams A and B. Therefore, the electric signal S2 input to the control unit 17 corresponds to a simple combination of the electric signals SA and SB corresponding to the light intensities of the laser beams A and B.

  That is, since the control signals CA and CB and the laser beams A and B are time-divided synchronously, the laser beams A and B are combined into a single beam laser beam AB and converted into the electric signal S2. However, when the electric signal S2 is time-divided in synchronization with the control signals CA and CB and converted into the electric signals SA and SB, the electric signals SA and SB correspond to the laser beams A and B, respectively. Therefore, the control unit 17 can feedback control the light intensities of the laser beams A and B independently.

  In the first embodiment, the control unit 17 performs feedback control of the laser beams A and B independently by emitting the laser beams A and B in a time-sharing manner. Further, when a semiconductor laser is used as a laser light source, high-speed switching with respect to the laser light source is possible, and the above-described time division can be performed at high speed. As a result, the control unit 17 can control the emission of the laser beams A and B to the LDs 3 and 4 in nanosecond units, and the scanning time of the laser beam corresponding to one pixel of the display screen of the display unit 18 is about 2 Since it is microseconds, the light intensity of the laser light can be controlled in units of one pixel. That is, since it is possible to irradiate the sample 15 with laser light having a desired light intensity in units of one pixel, the display screen displayed on the display unit 17 is scanned with laser light having a desired light intensity. is there. Therefore, by measuring the brightness of the display screen, it is possible to quantitatively measure the fluorescence light intensity.

  Since the laser beam AB2 after passing through the optical system can be monitored by arranging the beam splitter 9 immediately before the excitation dichroic mirror 11, the control unit 17 takes into account the optical loss due to the optical system. Accurate feedback control can be performed.

  In the first embodiment, two laser light sources are used. However, three or more laser light sources may be used in accordance with the observation situation. Conversely, one laser light source may be used. Good.

(Embodiment 2)
Next, a second embodiment of the present invention will be described. In the first embodiment, the laser beam AB is separated into the laser beams AB1 and AB2 by the beam splitter 9, and the laser beam AB2 obtained by combining the laser beams A and B is monitored. , LDs 3 and 4 are provided with photodiodes (PD) 21 and 22, and the light intensity corresponding to the laser beams A and B is monitored by the PDs 21 and 22.

  FIG. 3 is a functional block diagram showing a schematic configuration of a scanning laser microscope apparatus 200 according to the second embodiment of the present invention. This scanning laser microscope apparatus 200 is a fluorescence microscope apparatus, and the beam splitter 9 and the photoelectric detector 16 in the scanner unit 8 shown in FIG. 1 are deleted, and the detection ports of the LDs 3 and 4 are respectively connected to the PDs 21 and 22. The control unit 17 is replaced with the control unit 23. The PDs 21 and 22 output electric signals SA1 and SB1 corresponding to the light intensity of the monitored laser light to the control unit 23. The controller 23 controls the LDs 3 and 4 by outputting control signals CA and CB based on the input signals SA1 and SB1. In addition, the same code | symbol is attached | subjected to the component same as FIG.

  When the laser beams are emitted from the emission port, the laser beams are detected from the detection port at a predetermined ratio. Since this predetermined ratio is constant in each of the LDs 3 and 4, when the PDs 21 and 22 are attached to the detection ports of the LDs 3 and 4, respectively, and the light intensity of the detected laser light is monitored, The light intensity of the emitted laser beams A and B is indirectly monitored.

  Therefore, when the control unit 23 inputs the electrical signals SA1 and SB1 monitored by the PDs 21 and 22 and outputs the control signals CA and CB based on the electrical signals SA1 and SB1, the control unit 23 performs feedback control of the LDs 3 and 4. can do.

  In the second embodiment, the control unit 23 monitors the laser light detected from the detection ports of the LDs 3 and 4, thereby performing feedback control without attenuating the light intensity of the laser light AB irradiated on the specimen 15. Like to do.

  In the second embodiment, the PDs 21 and 22 are individually arranged with respect to the LDs 3 and 4. However, for example, a module in which an LD chip and a monitor PD are integrated may be used. .

(Embodiment 3)
Next, a third embodiment of the present invention will be described. In the first and second embodiments, a semiconductor laser is used as a laser light source and two laser beams are emitted in a time-sharing manner, and feedback control is performed by one photoelectric detector 16, but this third embodiment Then, the control unit controls the LD based on the signal input from the photoelectric detector, compares a plurality of predetermined threshold values with the value of the signal input / output by the control unit, and determines an abnormal portion of the apparatus. I try to identify.

  FIG. 4 is a block diagram showing a configuration of a scanning laser microscope apparatus 300 according to the third embodiment of the present invention. This scanning laser microscope apparatus 300 is a fluorescence microscope apparatus, and PDs 21 and 22 are arranged in the LDs 3 and 4 shown in FIG. 1, respectively, the control unit 17 is replaced with the control unit 19, and an abnormal portion of the laser microscope apparatus 300 is further provided. And a storage unit 20 for storing a plurality of threshold values determined in advance for identification. In addition, the same code | symbol is attached | subjected to the component same as FIG.

  The control unit 19 inputs the electric signal S2 from the photoelectric detector 16, converts the electric signal S2 to time signals and converts them into electric signals SA and SB. The control signals CA and CB are respectively converted based on the electric signals SA and SB. It outputs and controls LD3 and 4. Further, the control unit 19 compares the input electrical signals SA, SB, SA1, and SB1, the output control signals CA and CB, and a threshold value stored in advance in the storage unit 20, and identifies an abnormal portion of the apparatus. .

Here, it will be described how the control unit 19 specifies an abnormal portion such as an optical system through which the laser beam emitted from the LD 3 or LD 3 passes. For example, when the LD 3 is defective, the control unit 19 does not receive the predetermined electric signal SA1 from the PD 21 with respect to the predetermined control signal CA. The storage unit 20 stores a predetermined threshold value SA1 _TH corresponding to the electrical signal SA1 when the LD 3 is normal. When the electrical signal SA1 is input from the PD 21, the control unit 19 extracts the threshold value SA1 _TH from the storage unit 20, comparing the electrical signal SA1 and the threshold SA1 _TH, it is determined whether LD3 is defective.

Also, the LD 3 is normal, and either the optical system (mirror 5, composite dichroic mirror 6, optical fiber 7, beam splitter 9) through which the laser light emitted from the LD 3 passes or the photoelectric detector 16 is defective. In this case, the control unit 19 does not receive the predetermined electrical signal SA from the photoelectric detector 16 with respect to the predetermined control signal CA. Storage unit 20 stores a predetermined threshold SA _TH of an optical system and a photoelectric detector 16 corresponds to an electrical signal SA in the normal, control unit 19 inputs the electrical signal SA from the photoelectric detector 16, stores The threshold value SA _TH is extracted from the unit 20, and the electric signal SA is compared with the threshold value SA _TH to determine whether the optical system or the photoelectric detector 16 is defective.

Further, when the optical system and the photoelectric detector 16 are normal and the LD 3 is deteriorated, the control unit 19 does not receive the predetermined electric signal SA with respect to the predetermined control signal CA. The storage unit 20 stores a predetermined threshold (SA / CA) _TH that is a ratio of the control signal CA and the electrical signal SA when the LD 3 is normal, and the control unit 19 outputs the control signal CA and the electrical signal SA. , The ratio (SA / CA) is calculated, the threshold value (SA / CA) _TH is extracted from the storage unit 20, the ratio (SA / CA) is compared with the threshold value (SA / CA) _TH, and the LD 3 Judge whether it has deteriorated or not.

FIG. 5 is a flowchart showing a procedure for the control unit 19 to identify an abnormal part of the apparatus. As shown in FIG. 5, when the electric signal SA1 is input, the control unit 19 extracts the threshold value SA1 _TH from the storage unit 20 and compares it with the electric signal SA (step S101). If the electrical signal SA1 is smaller than the threshold SA1 _TH (step S101, Yes), it is determined that the LD3 is defective (step S102). If the electrical signal SA1 is the threshold value SA1 _TH or more (step S101, No), it extracts the threshold SA _TH from the storage unit 20, compares the electric signal SA (step S103). When the electrical signal SA is smaller than the threshold value SA _TH (step S103, Yes), it is determined that the optical system or the photoelectric detector 16 is defective (step S104). When the electrical signal SA is equal to or greater than the threshold value SA1 _TH (step S103, No), the control unit 19 extracts the threshold value (SA / CA) _TH from the storage unit 20 and compares it with the ratio (SA / CA) (step S105). ). When the ratio (SA / CA) is smaller than the threshold (SA / CA) _TH (step S105, Yes), it is determined that the LD3 has deteriorated (step S106), and the ratio (SA / CA) is the threshold ( If SA / CA) _TH or more (step S105, No ), it is determined that the apparatus is normal (step S107).

  In the third embodiment, the scanning laser microscope apparatus 300 includes a storage unit 20 that stores a plurality of threshold values, and the control unit 19 performs feedback control when fluctuations in the light intensity of the laser light occur. The laser light intensity is controlled to a desired light intensity, and an abnormal portion of the apparatus is specified based on the value of the fluctuation.

  In the third embodiment, the determination of abnormality determination of the optical system and the like through which the laser beam A emitted from the LD 3 and the LD 3 passes has been described. However, the optical through which the laser beam B emitted from the LD 4 and the LD 4 passes. The same identification can be made for systems and the like. In the third embodiment, the control unit 19 outputs the control signal CA based on the electrical signal SA and controls the LD 3. However, the control unit 19 controls the LD 3 based on the electrical signal SA 1. Alternatively, the LD 3 may be controlled based on the two electric signals SA and SA1.

It is a functional block diagram of the scanning laser microscope apparatus concerning Embodiment 1 of this invention. It is a wave form diagram which shows a response | compatibility with the control signal concerning Embodiment 1 of this invention, and an electrical signal. It is a functional block diagram of the scanning laser microscope apparatus concerning Embodiment 2 of this invention. It is a functional block diagram of the scanning laser microscope apparatus concerning Embodiment 3 of this invention. It is a flowchart which shows the judgment procedure of the control part concerning Embodiment 3 of this invention.

Explanation of symbols

1, 2 LD unit 3, 4 LD
DESCRIPTION OF SYMBOLS 5 Mirror 6 Synthesis | combination dichroic mirror 7 Optical fiber 8 Scanner unit 9 Beam splitter 10 Galvano 11 Excitation dichroic mirror 12 Confocal pinhole 13, 16 Photoelectric detector 14 Microscope 15 Sample 17, 19, 23 Control part 18 Display part 20 Storage part 21 , 22 PD
100, 200, 300 Laser microscope apparatus

Claims (5)

Different wavelengths emit laser beams of a plurality of combining a plurality of laser beams having different emitted wavelengths from the laser light source is scanned while irradiating the sample through an optical system that guides the synthesized laser beam on the sample from each other, In a scanning laser microscope apparatus for observing light emitted from the specimen by the irradiation,
An optical fiber included in the optical system for guiding the combined laser beam;
Light intensity monitoring means for monitoring the light intensity of the combined laser light emitted from the optical fiber ;
Detection light intensity monitoring means for monitoring the light intensity of the detection light from the detection opening of each laser light source that outputs the light intensity corresponding to the light intensity of the laser light output from the emission opening of each laser light source;
The laser light emission from the plurality of laser light sources is controlled in a time-sharing manner, and the light intensity of the laser light or the detection light monitored by the light intensity monitoring means or the detection light intensity monitoring means is applied to the plurality of laser light sources. Control means for controlling the light intensity of each laser beam by feedback;
A confocal pinhole that allows light emitted from the focal point of the excitation light on the specimen to pass;
A photoelectric detector that detects the light passing through the confocal pinhole by converting it into an electrical signal;
With
The control means, the control signal for controlling the light intensity of the light intensity monitoring means and the light intensity of the detection light detected by the light intensity of the laser light detected the detection light intensity monitoring means by the laser beam and the judges whether there is an abnormality of the laser scanning microscope apparatus on the basis, and identifies among the plurality of positions, including a laser light source and the optical system anomaly of the plurality of cases where there is abnormal Scanning laser microscope device. Storage means for storing in advance the reference light intensity of the laser light detected by the light intensity monitor means and the reference light intensity of the detection light detected by the detection light intensity monitor means;
The control means compares the light intensity of the detection light with the reference light intensity of the detection light. If the light intensity of the detection light is smaller than the reference light intensity of the detection light, the laser light source is abnormal. The scanning laser microscope apparatus according to claim 1 , wherein the scanning laser microscope apparatus is specified. When the light intensity of the detection light is not smaller than the reference light intensity of the detection light, the control means compares the light intensity of the laser light with the reference light intensity of the laser light, and 3. The optical system or the light intensity detecting means for guiding the laser light to the specimen is specified as abnormal when the light intensity is smaller than a reference light intensity of the laser light. The scanning laser microscope apparatus described. The storage means stores in advance a reference response ratio that is a reference value of a response ratio of the light intensity of the laser light or the light intensity of the detection light with respect to a control signal for the control means to control the light source;
The control means, when the light intensity of the detection light is not small compared to the reference light intensity of the detection light, and the light intensity of the laser light is not small compared to the reference light intensity of the laser light, compared to the response ratio and the reference ratio response, if the response ratio is smaller than the reference response ratio degradation by the claim 3, wherein the laser light source is specified as being abnormal Scanning laser microscope device. Wherein the plurality of laser light sources, laser scanning microscope apparatus according to any one of claims 1-4, characterized in that each is a semiconductor laser capable of high-speed switching.