JP4869562B2 - Scanning confocal microscope - Google Patents

Description

  The present invention relates to an optical scanning confocal microscope that irradiates a specimen while scanning light and detects detection light (fluorescence) from the specimen.

  As an optical scanning confocal microscope, a fluorescent dye is excited by irradiating a sample labeled with a fluorescent dye with a laser beam through an objective lens, and detection light (fluorescence) emitted from the sample by this excitation is sent to the optical system. Some of them are detected by a detector.

  Recently, multiple stained specimens in which a single specimen is labeled with a plurality of fluorescent dyes have been used, and in order to detect fluorescence corresponding to each fluorescent dye for such multiple stained specimens, It is also conceivable that the detection light from the light is dispersed into a plurality of channels for each fluorescent dye, and the detection light is captured for each channel.

Patent Document 1 is based on such an idea, and prepares a plurality of light sources that generate light having different wavelengths for each excitation wavelength of a sample part in which a fluorescent indicator or a fluorescent protein is expressed, and uses these light sources to prepare a sample. The fluorescent light that is excited and emits light is detected by a detector provided for each wavelength. In this case, an optical filter and a spectral dichroic mirror for selecting a wavelength for each fluorescence wavelength, and a dichroic mirror for separating excitation light and fluorescence are provided.
JP 2000-35400 A

  However, such a thing of patent document 1 requires a detection optical path and a detector according to the fluorescence wavelength to be detected. Therefore, in order to detect a plurality of fluorescence wavelengths, a detector is provided for each of these fluorescence wavelengths. Necessary. As a result, the number of wavelengths that can be separated is limited by the number of detectors, and when the number of detectors is increased, the entire detection optical unit becomes large and expensive.

  Further, it is necessary to select an optical element such as an optical filter according to the fluorescence wavelength of the specimen. For this reason, it is necessary to change the filter in accordance with the wavelength, and as a means for that purpose, it is possible to prepare a large number of filters in advance and switch these filters for each detection wavelength. However, in this method, the number of filters is limited, and there is a problem that the number of filters becomes insufficient as the number of types of samples increases.

  As a means for solving the increase in the number of filters, a spectroscopic detector is also considered. This method requires a mechanism for driving the spectroscope, a slit for selecting the fluorescence wavelength, and the like. However, these drives are mechanical, and these mechanical drives require a speed of 10 ms or more, so that high-speed switching is difficult.

  Further, in the spectroscopic method, a multi-anode PMT that enables multi-wavelength detection has been developed. However, the multi-anode PMT has a problem that it is difficult to finely tune the wavelength corresponding to various fluorescence wavelengths because the resolution of wavelength selection is not so high. In addition, this multi-anode PMT has a problem that a bright image cannot be obtained because the detection sensitivity cannot be expected so much.

  On the other hand, in order to separate excitation light and fluorescence, a plurality of dichroic mirrors are used, and these dichroic mirrors are switched according to the excitation wavelength, or the wavelength characteristic of the dichroic mirror reflects the laser light wavelength. It is necessary to use a dichroic mirror having a multi-wavelength band that transmits fluorescence. However, if a plurality of dichroic mirrors are used, the entire detection optical unit is increased in size, and those using multi-wavelength band dichroic mirrors are currently being developed. However, it requires a high level of technology in optical design, and it is necessary to make the film layer into a multilayer film, which makes the manufacturing difficult and expensive.

  Further, in a filter type that is not a spectroscopic method, a method of acquiring an image by switching laser wavelengths in a time division manner is also considered. However, in this method, it is necessary to switch the filter at a high speed, and if a mechanical driving method is employed, a speed problem occurs.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a scanning confocal microscope that can be configured at low cost and can efficiently use a wide wavelength region .

In order to achieve the above object, a scanning confocal microscope according to an aspect of the present invention includes an LED light source, a multimode optical fiber that transmits light from the LED light source, and light from the optical fiber as the sample. Optical scanning means for two-dimensional scanning above, an objective lens for converging the light scanned by the optical scanning means on the specimen and condensing detection light emitted from the specimen, and detection light condensed by the objective lens Is detected during two-dimensional scanning by the optical scanning means , a branching means for branching the light beam toward the detector before the optical fiber, a pinhole for confocal detection of the detection light branched by the branching means, The LED light source is controlled to increase the light amount of the LED light source at the timing of capturing light, and the light amount of the LED light source is decreased at the timing of not capturing detection light. Characterized by comprising control means for controlling the LED light source to the.

  According to the present invention, by using an acoustooptic device capable of determining the wavelength of light diffracted by the input frequency, the detection wavelength can be freely selected, and an optical scanning type that can easily detect multiple wavelengths with high accuracy. A focusing microscope can be provided.

Hereinafter, embodiments and reference examples of the present invention will be described with reference to the drawings.

(First reference example )
FIG. 1 shows a schematic configuration of an optical scanning confocal microscope according to a first embodiment of the present invention. In FIG. 1, reference numeral 1 denotes a laser light source unit as a light source. The laser light source unit 1 includes laser light sources 2, 3, and 4 that oscillate laser beams having different wavelengths.

  A reflection mirror 5 is disposed on the optical path of the laser light from the laser light source 4. Further, a dichroic mirror 6 is disposed on the intersection of the laser light reflected by the reflection mirror 5 on the optical path of the laser light from the laser light source 3. The dichroic mirror 6 combines these two laser light paths, and reflects the laser light from the laser light source 3 and transmits the laser light reflected by the reflection mirror 5. On the optical path of the laser beam from the laser light source 2, a dichroic mirror 7 is disposed at the intersection with the laser beam from the dichroic mirror 6. The dichroic mirror 7 combines these two laser light paths, transmits the laser light from the laser light source 3, and reflects the laser light from the dichroic mirror 6.

  On the optical path of the laser beam synthesized by the dichroic mirror 7, an acousto-optic variable filter (hereinafter referred to as AOTF) 8 is disposed as a wavelength switching unit, a laser beam ON / OFF unit, and a laser beam intensity control unit. The AOTF 8 performs wavelength selection, ON / OFF control, and intensity control on the laser light from the laser light sources 2, 3, 4.

  An incident end of a single mode fiber 9 is arranged as a fiber at the exit end of the AOTF 8, and the laser light from the laser light source unit 1 is introduced into the scanning unit 10 through the single mode fiber 9.

  In the scanning unit 10, a collimating lens 11 that converts laser light into parallel light is disposed on the emission side of the single mode fiber 9.

  An acoustooptic device 12 is disposed on the parallel light path of the collimator lens 11. Details of the acoustooptic device 12 will be described later.

  A condensing lens 13 is disposed on the optical path that has passed through the acoustooptic device 12. The condensing lens 13 condenses the parallel light transmitted through the acousto-optic element 12, and a pinhole 14 is disposed at this condensing position.

  A confocal lens 15 is disposed in the optical path transmitted through the pinhole 14. The confocal lens 15 converts the light transmitted through the pinhole 14 into parallel light again. A scanning optical unit 16 is disposed as an optical scanning unit in the optical path that is converted into parallel light by the confocal lens 15. The scanning optical unit 16 has two mirrors (not shown) for deflecting light in two orthogonal directions, and these mirrors scan laser light in a two-dimensional direction.

  An imaging lens 18 and an objective lens 19 are arranged via a relay lens 17 on the optical path of the laser light two-dimensionally scanned by the scanning optical unit 16. The relay lens 17 expands the light beam from the scanning optical unit 16 to about the pupil diameter of the objective lens 19, and has a role of filling the objective pupil position with light through the imaging lens 18.

  As a result, the laser light that is two-dimensionally scanned by the scanning optical unit 16 is imaged at the focal position of the specimen 20 via the relay lens 17, the imaging lens 18, and the objective lens 19. In addition, the fluorescence emitted from the specimen 20 returns to the objective lens 19, the imaging lens 18, the relay lens 17, the scanning optical unit 16, and the confocal lens 15.

  The fluorescence that has returned to the confocal lens 15 is collected in the pinhole 14. In this case, the pinhole 14 is arranged in a conjugate manner with the focal position on the surface of the specimen 20 so that a confocal effect can be obtained by the pinhole 14.

  Then, the fluorescence from the focal position on the surface of the specimen 20 that has passed through the pinhole 14 becomes parallel light by the condenser lens 13 and is guided to the acoustooptic device 12 again.

  The acoustooptic device 12 is a device that uses a phenomenon in which light is refracted by applying a ultrasonic wave to induce a change in refractive index, and determines the wavelength of the diffracted light according to the frequency of the ultrasonic wave, that is, the input frequency. It can be done. Specifically, the wavelength width of light that can be selected at a specific frequency is about 3 nm, but in order to obtain a general fluorescence wavelength, a width of about 10 nm to 40 nm is necessary. By simultaneously inputting a plurality of different frequencies to 12, it becomes possible to detect fluorescence wavelengths from 3 nm to several tens of nm at a time. That is, the detection wavelength width is determined by how many different frequencies are included. The intensity of the diffracted light can be controlled by controlling the magnitude of the input signal, for example, the voltage value, and the diffracted light intensity can be controlled in the range of 0 to 90% by the value of this voltage. .

  The fluorescence optical path diffracted by the acoustooptic device 12 is separated into two polarization components 201 and 202 of P component and S component.

  In the optical path of one polarization component 201, a detector 22 is arranged as a detection means. The detector 22 detects the fluorescence of the polarization component 201, and photoelectrically converts the detected light and outputs it. In addition, the detector 23 is disposed in the optical path of the other polarization component 202. The detector 23 detects the fluorescence of the polarization component 202, and photoelectrically converts the detected light and outputs it.

  A reflection mirror 24 is arranged in the optical path of the other polarization component 202. The reflection mirror 24 can be inserted into and removed from the optical path. When the reflection mirror 24 is inserted in the optical path (shown in the figure), the reflection mirror 24 reflects the fluorescence of the polarization component 202 and introduces it into the detector 22. Yes. That is, in a state where the reflection mirror 24 is inserted in the optical path (shown state), the fluorescence of the two polarization components 201 and 202 of the P component and the S component is detected by the detector 22, and the reflection mirror 24 is retracted from the optical path. In this state, the fluorescence of the two polarization components 201 and 202 of the P component and the S component is detected by the detectors 22 and 23, respectively.

  A control unit 25 is connected to the AOTF 8, the scanning optical unit 16, the acoustooptic device 12, the detectors 22 and 23, and the reflection mirror 24. A personal computer (hereinafter, PC) 26 is connected to the control unit 25.

  The control unit 25 executes control of each device in accordance with an instruction from the PC 26, and controls the wavelength switching of the laser light and the light intensity with respect to the AOTF 8. In synchronization with the scanning of the scanning optical unit 16, the laser light ON / OFF by the AOTF 8 is also controlled. This is effective when detecting fluorescence only in a desired region on the specimen 20. Further, in the scanning confocal laser microscope, when scanning the scanning optical unit 16, there are a case of reciprocal scanning and a case of single-sided scanning. Control to turn off the laser beam.

  Control of the AOTF 8 by the control unit 25 is performed by an electric signal from the control unit 25. The AOTF 8 is turned on / off by turning on / off a frequency signal input to the AOTF 8. Further, the wavelength selection of the laser light is determined by the frequency to be put in the AOTF 8. For example, if two or more frequencies are input to the AOTF 8, laser light having two or more wavelengths can be turned on / off.

  The control unit 25 also performs various controls with the acoustooptic device 12. In this case, if it is desired to increase the detection wavelength width of fluorescence, a plurality of different frequencies are input at a time. Further, in order to switch the detection wavelength range, control is performed so as to selectively switch different input frequencies. Further, the diffraction intensity (transmission amount) of light can be changed by changing the amplitude (intensity) of the input signal. For example, when detecting a large amount of reflected light as light from the sample 20, the light from the laser light source unit 1 is introduced into the sample 20 and reflected from the sample 20 by setting the transmission amount to about 50%. It is possible to direct light to the detector 22 (23). In this case, the reflected light from the specimen 20 returns to the detector 22 (23), but the light from the laser light source unit 1 is opposite to the direction in which the reflected light from the specimen 20 is introduced, and to the detector 22 (23). Since it does not return, it can function as an optical isolator.

  Furthermore, the control unit 25 also controls insertion / removal of the reflection mirror 24 into / from the optical path. In this case, when the reflection mirror 24 is inserted into the optical path, the two polarization components 201 and 202 of the fluorescence and the S component can be detected by the detector 22, and when the reflection mirror 24 is retracted from the optical path, the fluorescence The two polarization components 201 and 202 of the P component and the S component can be detected by the detectors 22 and 23, respectively.

Next, the operation of the reference example configured as described above will be described.

  In this case, the control unit 25 switches the wavelength of the laser beam by the AOTF 8 and switches the input frequency of the acoustooptic device 12 at a high speed, and also detects the timing of fluorescence detection by the detector 22 (23) and the scanning optical unit 16. Control to synchronize the two-dimensional scanning.

  From this state, when the laser light from the laser light sources 2, 3, 4 of the laser light source unit 1 is sequentially switched via the AOTF 8, it enters the collimating lens 11 of the scanning unit 10 via the single mode fiber 9 and is parallel. It becomes light and is guided to the acoustooptic device 12.

  The laser light emitted from the acoustooptic device 12 is condensed by the condenser lens 13, passes through the pinhole 14, becomes parallel light by the confocal lens 15, enters the scanning optical unit 16, and is two-dimensionally scanned. Laser light that is two-dimensionally scanned by the scanning optical unit 16 is imaged at the focal position of the specimen 20 via the relay lens 17, the imaging lens 18, and the objective lens 19.

  In this case, when a multiple staining sample labeled with a plurality of fluorescent dyes is used as the sample 20, fluorescence with different wavelengths is generated by excitation of laser light of different wavelengths from the laser light sources 2, 3, and 4. These fluorescences return to the confocal lens 15 via the objective lens 19, the imaging lens 18, the relay lens 17, and the scanning optical unit 16, and are collected in the pinhole 14.

  The fluorescence confocally detected by the pinhole 14 becomes parallel light by the condenser lens 13 and is guided to the acoustooptic device 12 again.

  In this case, the acoustooptic device 12 switches the input frequency in accordance with the wavelength switching of the laser light in the AOTF 8 by the control unit 25, and switches the wavelength range of fluorescence detection at high speed. As a result, the fluorescence having different wavelengths from the specimen 20 is sequentially diffracted by the acoustooptic device 12 and guided to the detector 22 (23). At this time, if the reflection mirror 24 is inserted in the optical path, the two polarization components 201 and 202 of the fluorescence of each wavelength and the S component can be detected by the detector 22, and the reflection mirror 24 is retracted from the optical path. In this case, the two polarization components 201 and 202 of the fluorescence P component and the S component can be detected by the detectors 22 and 23, respectively.

  Here, as a specific example, the case where FITC and rhodamine are used as a plurality of fluorescent dyes labeled on the specimen 20 will be described as an example. FITC generates fluorescence of about 515 nm by excitation at 490 nm. Fluorescence of about 580 nm is generated by excitation of. For explanation, as the laser light source of the laser light source unit 1, 488 nm of argon laser is used for FITC and 543 nm of HeNe laser is used for rhodamine.

  First, when laser light from an argon laser is condensed on the specimen 20 via the acoustooptic element 12 and the objective lens 19, a portion labeled with FITC in the specimen 20 is excited. Fluorescence having a wavelength of 520 nm is generated from the excited portion, and this fluorescence is introduced into the acoustooptic device 12. At this time, when a frequency corresponding to 520 nm is input to the acoustooptic device 12, only the light of 520 nm is diffracted, and the diffracted light is detected and imaged by the detector 22 (23).

  Next, when the laser light from the HeNe laser is condensed on the specimen 20 via the acousto-optic element 12 and the objective lens 19, a portion labeled with rhodamine in the specimen 20 is excited. Fluorescence having a wavelength of 580 nm is generated from the excited portion, and this fluorescence is introduced into the acoustooptic device 12. At this time, when a frequency corresponding to 580 nm is input to the acoustooptic device 12, only the light of 580 nm is diffracted, and the diffracted light is detected and imaged by the detector 22 (23).

  In this example, the images of the site labeled with FITC and the site labeled with rhodamine are separately acquired. However, in the scanning optical unit 16, line scanning is performed twice at the same position in the X direction. By switching the wavelength of the laser light at the AOTF 8 and switching the input frequency to the acousto-optic device 12 so as to detect each fluorescence of rhodamine in the first scan and FITC in the first scan, almost simultaneously Two types of fluorescence can be detected. In this case, a frame image can be obtained by generating an image while shifting the Y direction by one line for the data detected for each line in the X direction.

  Moreover, if each fluorescence detection of FITC and rhodamine is performed not for each line but for each pixel, two images can be acquired almost simultaneously. In such fluorescence detection for each pixel, it is necessary to switch the frequency of the input signal of the acoustooptic device 12 on the order of μsec.

In the above description, an example in which two wavelengths of fluorescence can be detected almost simultaneously even with a single detector is described. For example, when the specimen 20 is labeled with two or more fluorescent dyes, Two or more detectors are required. However, by switching the input frequency to the acoustooptic device 12 at high speed, a single detector can detect multiple wavelengths. In other words, an image having almost simultaneous multi-wavelength fluorescence can be detected by switching the input frequency of the acoustooptic element 12 to the acoustooptic element 12 at high speed in accordance with the switching of the wavelength of the laser light source. Is possible.

  Therefore, in this way, the detection wavelength can be freely selected by controlling the input frequency of the acoustooptic device 12 in accordance with the wavelength switching of the laser light from the laser light source unit 1 by the AOTF 8, so that it can be emitted from the multiple stained specimen. Fluorescence having different wavelengths can be easily and accurately detected by the single detector 22. Thereby, conventionally, when detecting fluorescence of different fluorescence wavelengths, the configuration can be simplified compared to those having a detection optical path and a detector for each fluorescence wavelength to be detected or having means for switching a plurality of optical filters, The price can be reduced.

  Control of the AOTF 8 and the acousto-optic element 12 can be easily changed by creating a menu for switching the AOTF 8 and switching the input frequency of the acousto-optic element 12 in the PC 26. . Even if a new fluorescent reagent having a fluorescence wavelength characteristic that does not exist at present is developed, this can easily cope with detection of these fluorescence wavelengths.

In addition to not requiring maintenance work such as adding an optical filter , the user can freely change the wavelength, so if you want to do a new experiment, do not purchase a filter and immediately perform an experiment. You can start.

  Furthermore, since the number of optical elements such as dichroic mirrors (DM) and optical filters can be greatly reduced, the optical system is simplified and the apparatus can be miniaturized. In addition, when there are many optical elements, there is a disadvantage that the fluorescence intensity decreases each time the optical elements pass, but since the optical system is simple, it is possible to always detect a bright image.

  In FIG. 1, a common pinhole 14 is included in the optical path for introducing laser light from the laser light source unit 1 and the detection optical path for fluorescence from the specimen 20, and light is imaged on the specimen 20 in the introduced optical path. In addition, the detection optical path has a role of detecting only the focused light, but the introduction optical path and the detection optical path may be separated. Is possible. When the pinhole 14 is shared, there is an advantage that it can be configured at low cost, and when it is separated, there is an advantage that the configuration of the apparatus can be freely set.

(Modification of the first reference example)
In the first reference example , the laser light from the laser light source unit 1 is introduced to the 0th order side which is not diffracted even when the input frequency of the acoustooptic device 12 is applied, and the detector 22 is provided on the side where the 1st order diffracted light is obtained. Although (23) is arranged, laser light may be introduced from the primary light of the acousto-optic element 12, and the detector 22 may be arranged on the zero-order light side.

In this way, it is possible to detect the fluorescence with the detector 22 while selecting the wavelength of the laser beam with the acoustooptic device 12. In this case, it is impossible to cope with the case where the sample 20 generates two fluorescences at one wavelength, but an image can be acquired without selecting the input frequency by the acoustooptic device 12. Further, in such a configuration, 100% of the diffraction efficiency of the acoustooptic device 12 cannot be ensured, so it is desirable to put a barrier filter in accordance with the laser wavelength before the detector 22. The rest is the same as the first reference example described above.

(Second reference example )
Next, a second reference example of the present invention will be described.

  In this case, since the schematic configuration of the optical scanning confocal microscope shown in FIG. 1 is the same, the same figure is used.

In the second reference example , the single mode fiber 9 is provided in the laser light introduction path to the scanning unit 10, but a multimode fiber 31 is provided instead of the single mode fiber 9 as shown in FIG. ing.

  In general, in a laser microscope, an optical fiber is often used to guide laser light output from a laser light source to the microscope body. In order to maximize the resolution in a laser microscope, it is necessary to illuminate the specimen with an ideal point light source. When the laser light includes a plurality of modes of light, ideal imaging is hindered. Therefore, a single mode fiber that propagates only light has been used as an optical fiber used for laser beam transmission. By using the single mode fiber, the laser light is guided to the specimen while remaining in a single mode. In particular, by using a single mode fiber, it is possible to obtain about 1.3 times higher resolution than a general conventional microscope.

  However, the optical scanning confocal microscope is not only required for high resolution but also used for internal observation of multiple stained specimens as described above, and can transmit laser beams having different wavelengths over a wide wavelength region. is there. In the field of biological research, special observation methods such as two-photon excitation method and release of caged reagent are used together with various fluorescent reagents for labeling specimens. For observation using these, laser light of various wavelengths is used as irradiation light. However, the single mode fiber is limited in the wavelength band that can be propagated due to its characteristics. In the conventional laser microscope, when using such a wide wavelength range (for example, visible, ultraviolet, infrared), in order to transmit the laser light of each band in a single mode from the above-mentioned viewpoint, Single mode fiber was used. Along with this, a precise optical system for synthesizing the laser beams was necessary in order to finally integrate the laser beams transmitted through the plurality of optical fibers accurately into one microscope optical axis. For this reason, there are problems in that the apparatus is expensive due to the use of a plurality of expensive single-mode fibers, the precision adjustment of the combining optical system, and the light loss.

  On the other hand, since the multimode fiber 31 can be a single mode fiber having a core diameter of about 2 to 5 μm and a large diameter of 2 to 2000 μm, and therefore has a wide wavelength of about 300 to 2000 nm. A state where the light transmittance is good over the region can be ensured, and optical transmission with less loss becomes possible. For this reason, the number of fibers to be used can be greatly reduced compared to the case of using a single mode fiber, and the cost can be reduced.

  Multimode fiber is less expensive than single mode fiber itself. Since the multimode fiber transmits laser light in a plurality of modes, an ideal point light source cannot be obtained when the multimode fiber is used. However, the provision of the pinhole 14 ensures a confocal effect that is important for observation of a three-dimensional specimen. Although the resolution is lower than when a single mode fiber is used, it is possible to provide a scanning confocal microscope that is inexpensive and has a simple structure for observation applications that do not require the resolution to the limit level.

(Third reference example )
Next, a third reference example of the present invention will be described.

  In this case, since the schematic configuration of the optical scanning confocal microscope shown in FIG. 1 is the same, the same figure is used.

Also in this case, as in the second reference example , a multimode fiber 31 is used instead of the single mode fiber 9 as shown in FIG. When such a multimode fiber 31 is used, the advantages described in the second reference example can be obtained.

  When the multimode fiber 31 is used, the core diameter can be increased as described above. Therefore, although the laser light sources 2, 3, and 4 are used as the light source, various light sources other than the laser light source can be used. For example, in a single mode fiber having a core diameter of about 2 to 5 μm, it is difficult to introduce a light source such as an LED, a xenon lamp or a mercury lamp, and even if it can be introduced, 0% or 1% cannot be transmitted. In addition, the single mode fiber can only be manufactured with a wavelength in the visible region and a wavelength width of about 200 nm.

  On the other hand, when the multimode fiber 31 is used, the core diameter can be increased to 2 to 2000 μm as described above, and the light transmittance can be secured well over a wide wavelength region of 300 to 2000 nm. Even when a light source having a wide wavelength range such as an LED, a xenon lamp, or a mercury lamp is used as the light source of the unit 1, an efficient system can be realized.

  Conventionally, the fibers are introduced separately for the ultraviolet wavelength and the visible wavelength, but the multimode fiber 31 eliminates the necessity, and the optical components can be reduced correspondingly, resulting in an inexpensive and small system. Can be built. In addition, light sources such as LEDs, mercury lamps, and xenon lamps can be obtained at a lower cost than laser light sources, so an inexpensive system can be constructed.

  When a mercury lamp or a xenon lamp is used as the light source, a wavelength selection element for selecting an excitation wavelength (a liquid crystal shutter can be used for high-speed switching on the optical path from the light source to the objective lens. ) Is desirable. When an LED is used as the light source, the excitation wavelength can be selected by electrically turning on / off the LED. In this case, since the wavelength characteristic of the LED is broader than that of the laser light source, it is desirable to provide a filter in accordance with the excitation wavelength on the optical path of the light from the LED.

(Fourth reference example )
Next, a fourth reference example of the present invention will be described.

  In this case, since the schematic configuration of the optical scanning confocal microscope shown in FIG. 1 is the same, the same figure is used.

  In this case, if the multimode fiber 31 is used instead of the single mode fiber 9, an LED can be used as the light source. In an optical scanning confocal microscope using such an LED, efficient image acquisition can be performed by switching the amount of light emitted from the LED in accordance with the scanning of the scanning optical unit 16. The LED has a smaller amount of light than other light sources such as mercury lamps and xenon lamps, but can switch the amount of light at a high speed. For example, when capturing an image, increase the amount of light and not capture an image. If the control is performed so that the amount of light is reduced, the average power can be lowered. In other words, the rated power is determined for the LED, but this rated power is determined in units of time, so if the ratio of the amount of light when capturing an image and the amount of light when not capturing an image is 2: 1, It is possible to increase the current value when increasing the amount of light to twice the current value at the time of average power. By doing so, it is possible to capture an image in a state where a current twice as large as the rated current of the LED is supplied and the amount of light is sufficiently increased, so that efficient image acquisition can be performed.

Of course, also in this case, by using the multimode fiber 31, the advantages described in the second reference example can be obtained.

(Fifth reference example )
Next, a fifth reference example of the present invention will be described.

  In this case, since the schematic configuration of the optical scanning confocal microscope shown in FIG. 1 is the same, the same figure is used.

  In biological experiments, there is a technique called FRAP. This FRAP is a technique for analyzing a series of flows by fading the fluorescence of a specific part of a cell. In other words, there is a phenomenon in which a fading site is restored with time because protein movement is constantly occurring in the cell, and this is a technique of analyzing with this series of flows.

  However, when detecting the process from fluorescence fading to restoration of a specific part of a cell, it is necessary to increase the intensity of light from the light source in order to discolor the fluorescence. The light from the light source returns to the detector, causing a problem that the detection accuracy is lowered. For this reason, it is necessary to remove the return of light from the light source as much as possible.

  Therefore, conventionally, a dichroic mirror and a filter for selecting a wavelength are arranged in the optical path so that the light from the light source does not return, or the dynamic range of the detector is controlled by the voltage applied to the detector. Yes. However, if dichroic mirrors and filters are arranged in the optical path, the number of optical components used is increased and the cost becomes expensive. Also, the voltage to be controlled is not limited to the voltage applied to the detector. Because of the high pressure, switching can be performed only in the mS order, and accurate control is difficult.

On the other hand, in the fourth reference example , the laser light from the laser light source unit 1 does not return to the detector 22 (23) side, but emits strong fluorescent light almost in proportion to the power intensity of the laser light. It is necessary to detect with the detector 22 (23). In this case, if the intense fluorescence is incident on the detector 22 (23) as it is, the dynamic range of the detector 22 (23) may be insufficient.

  In this case, if the light diffracted by the acousto-optic element 12 is weakened, it can be detected within the dynamic range of the detector 22 (23). For this purpose, the magnitude of the input signal to the acoustooptic device 12, for example, the voltage value is controlled. By controlling the voltage value, the intensity of the diffracted light can be controlled in the range of 0 to 90%, and strong fluorescence can be detected within the dynamic range of the detector 22 (23).

  Further, if the image is acquired at the level at which the color fading is determined by observing the degree of color fading while observing the level of the electrical signal after photoelectrically converting the fluorescence detected by the detector 22 (23), the color fading is obtained. Since the immediately following image can be acquired, the time lapse after fading can be measured without waste.

  In the above description, the intensity of the diffracted light is changed by controlling the voltage value to the acoustooptic device 12. However, since the fluorescence wavelength returns at a broad wavelength, the base of the fluorescence wavelength is reduced. If detected, the same effect can be realized. That is, in the configuration of FIG. 1, the wavelength range can be varied at high speed in the acoustooptic device 12, so that it is sufficient to detect at the base of the fluorescence wavelength during fading and to detect the fluorescence image during image acquisition. If switching between image acquisition and image acquisition during fading is determined from the luminance information during color fading and a fluorescent image is acquired, an FRAP experiment can be performed.

( One embodiment )
Next, an embodiment of the present invention.

FIG. 2 shows a schematic configuration of a laser scanning microscope according to an embodiment of the present invention. In FIG. 2, 41 is a laser light source unit, and this laser light source unit 41 also has laser light sources 42, 43, and 44 that oscillate laser beams of different wavelengths. Further, a laser beam path synthesizing unit having a reflection mirror 45 and dichroic mirrors 46 and 47 is provided in the optical path of the laser beam from these laser light sources 42, 43 and 44.

  An incident end of the multimode fiber 48 is disposed in the optical path from the laser beam path combining means of the laser light source unit 41. In this case, the multimode fiber 48 has a core diameter of 2 to 2000 μm and can obtain a good light transmittance over a wide wavelength region of about 300 to 2000 nm.

  Dichroic mirrors 49 and 50 are disposed in the optical path of light from the emission end of the multimode fiber 48. The dichroic mirror 49 combines the laser light from the laser light source 51 with the laser light from the multimode fiber 48. In this case, the laser light source 51 generates laser light having a wavelength outside the wavelength region of the multimode fiber 48. The dichroic mirror 50 has such characteristics that it transmits laser light from each of the laser light sources 42 to 44 and 51 and reflects detection light (fluorescence) from a specimen 57 described later.

  A scanning optical unit 52 is disposed on the transmitted light path of the dichroic mirror 50. This scanning optical unit 52 has two mirrors 52a and 52b for deflecting light in two orthogonal directions, and these mirrors 52a and 52b scan laser light in a two-dimensional direction. .

  A pupil projection lens 53, a reflection mirror 54, an imaging lens 55, an objective lens 56, and a specimen 57 are arranged on the optical path of the laser light that is two-dimensionally scanned by the scanning optical unit 52. In this case, the laser light that is two-dimensionally scanned by the scanning optical unit 52 passes through the pupil projection lens 53, the reflection mirror 54, the imaging lens 55, and the objective lens 56, and on the sample 57 held on a stage (not shown). The detection light (fluorescence) collected and emitted from the specimen 57 passes through the objective lens 56, the imaging lens 55, the reflection mirror 54, the pupil projection lens 53, and the scanning optical unit 52, and returns to the dichroic mirror 50. It has become.

  In the reflected light path of the dichroic mirror 50, a barrier filter 58, a confocal lens 59, and a pinhole 60 are disposed.

  The barrier filter 58 has a characteristic that cuts each wavelength of the laser light from the laser light sources 42 to 44 and 51 and transmits the wavelength range of the detection light (fluorescence) from the sample 57. The confocal lens 59 collects the detection light reflected from the dichroic mirror 50 in the pinhole 60. The pinhole 60 is arranged in a conjugate manner with the focal position on the specimen 57 so that a confocal effect can be obtained with respect to the light condensed on the pinhole 60.

  A dichroic mirror 61 as an optical path dividing unit is disposed in the optical path that has passed through the pinhole 60. In one optical path divided by the dichroic mirror 61, a detector 63 is disposed as a first detection means via a bandpass filter 62, and in the other optical path, a bandpass filter 64 is provided as a second detection means. A detector 65 is arranged via the. The detector 63 detects detection light (fluorescence) having a specific wavelength transmitted through the bandpass filter 62, and the detector 65 detects detection light (fluorescence) having a specific wavelength transmitted through the bandpass filter 64. It is.

  According to such a configuration, when the laser light from the laser light sources 42, 43, 44 of the laser light source unit 41 is output, it enters the dichroic mirror 50 via the multimode fiber 48. The laser light transmitted through the dichroic mirror 50 enters the scanning optical unit 52 and is two-dimensionally scanned. The laser light that is two-dimensionally scanned by the scanning optical unit 52 is imaged at the focal position of the sample 57 via the pupil projection lens 53, the reflection mirror 54, the imaging lens 55, and the objective lens 56.

  In this case, when a multiple staining sample labeled with a plurality of fluorescent dyes is used as the sample 57, fluorescence having different wavelengths is generated by excitation of laser light having different wavelengths from the laser light sources 42, 43, and 44.

  These fluorescences are returned to the dichroic mirror 50 through the objective lens 56, the imaging lens 55, the reflection mirror 54, the pupil projection lens 53, and the scanning optical unit 52. Then, the light is reflected by the dichroic mirror 50, the detection light (fluorescence) from the specimen 57 is transmitted by the barrier filter 58, and is condensed on the pinhole 60 by the confocal lens 59.

  Then, the detection light obtained by the confocal effect in the pinhole 60 is divided into two optical paths by the dichroic mirror 61 and detected by the detector 63 via the bandpass filter 62, and at the same time, the bandpass filter 64 And detected by the detector 65.

Therefore, in this way, the advantage described in the second reference example can be obtained by using the multimode fiber 48 as a fiber for introducing the laser light from the laser light source unit 41. In addition, the multimode fiber 48 having a large diameter of 2 to 2000 μm can be used, although the core diameter of the single mode fiber is about 2 to 5 μm. As a result, a good light transmittance can be ensured over a wide wavelength region of about 300 to 2000 nm, so that transmission with less loss is possible for laser light having different wavelengths from the laser light sources 42, 43, 44. . For this reason, the number of fibers to be used can be greatly reduced compared to the case of using a single mode fiber, and the cost can be reduced.

  In the above description, the laser light sources 42, 43, and 44 are used as the light sources, but various light sources other than these laser light sources can be used. For example, in a single mode fiber having a core diameter of about 2 to 5 μm, it is difficult to introduce a light source such as an LED, a xenon lamp or a mercury lamp, and even if it can be introduced, 0% or 1% cannot be transmitted. In addition, the single mode fiber can only be manufactured with a wavelength in the visible region and a wavelength width of about 200 nm. On the other hand, when the multimode fiber 48 is used, the core diameter can be increased to 2 to 2000 μm as described above, and the light transmittance can be secured well over a wide wavelength region of 300 to 2000 nm. A light source having a wide wavelength range such as a xenon lamp or a mercury lamp can be used.

  In this case, when a mercury lamp or a xenon lamp is used as the light source, a wavelength selection element for selecting an excitation wavelength (a liquid crystal shutter can be used for high-speed switching on the optical path from the light source to the objective lens. ) Is desirable. When an LED is used as the light source, the excitation wavelength can be selected by electrically turning on / off the LED. In this case, since the wavelength characteristic of the LED is broader than that of the laser light source, it is desirable to provide a filter in accordance with the excitation wavelength on the optical path of the light from the LED.

  Further, when the multimode fiber 48 is used, an LED can be used as a light source. Therefore, in a laser scanning microscope using such an LED, the amount of light from the LED is switched according to the scanning of the scanning optical unit 52. Thus, efficient image acquisition can be performed. The LED has a small amount of light compared to other light sources such as a mercury lamp and a xenon lamp, but the amount of light can be switched at a high speed. Therefore, for example, at the timing of capturing an image by two-dimensional scanning of the scanning optical unit 52. The average power can be reduced by increasing the amount of light and controlling the amount of light to be reduced at the timing when no image is captured. In other words, the rated power is determined for the LED, but this rated power is determined in units of time, so if the ratio of the amount of light when capturing an image and the amount of light when not capturing an image is 2: 1, It is possible to increase the current value when increasing the amount of light to twice the current value at the time of average power. By doing so, it is possible to capture an image in a state where a current twice as large as the rated current of the LED is supplied and the amount of light is sufficiently increased, so that efficient image acquisition can be performed.

  In addition, this invention is not limited to the said embodiment, In the implementation stage, it can change variously in the range which does not change the summary.

  Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent requirements are deleted from all the constituent requirements shown in the embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and is described in the column of the effect of the invention. If the above effect is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.

The figure which shows schematic structure of the optical scanning confocal microscope concerning the 1st thru | or 4th reference example of this invention. The figure which shows schematic structure of the laser microscope concerning one Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Laser light source unit, 2.3, 4 ... Laser light source 5 ... Reflection mirror, 6 ... Dichroic mirror 7 ... Dichroic mirror, 8 ... AOTF
DESCRIPTION OF SYMBOLS 9 ... Single mode fiber, 10 ... Scanning unit 11 ... Collimating lens, 12 ... Acousto-optic element 13 ... Condensing lens, 14 ... Pinhole 15 ... Confocal lens, 16 ... Scanning optical unit 17 ... Relay lens, 18 ... Imaging Lens 19 ... Objective lens, 20 ... Sample, 22.23 ... Detector 24 ... Reflection mirror, 25 ... Control unit 26 ... PC, 31 ... Multimode fiber 41 ... Laser light source unit, 42.43, 44 ... Laser light source 45 ... Reflection mirror, 46.47 ... Dichroic mirror 48 ... Multimode fiber, 49.50 ... Dichroic mirror 51 ... Laser light source, 52 ... Scanning optical unit 52a. 52b ... mirror, 53 ... pupil projection lens 54 ... reflecting mirror, 55 ... imaging lens, 56 ... objective lens 57 ... sample, 58 ... barrier filter, 59 ... confocal lens 60 ... pinhole, 61 ... dichroic mirror 62,64 ... band pass filter, 63, 65 ... detector 201.202 ... polarization component

Claims (1)

An LED light source;
A multimode optical fiber for transmitting light from the LED light source;
Optical scanning means for two-dimensionally scanning light from the optical fiber on the specimen;
An objective lens for converging the light scanned by the light scanning means on the sample and collecting the detection light emitted from the sample;
Branching means for branching detection light collected by the objective lens toward the detector before the optical fiber;
A pinhole for confocal detection of the detection light branched by the branching means ;
During the two-dimensional scanning by the light scanning means, the LED light source is controlled to increase the light amount of the LED light source at the timing when the detection light is captured, and the light amount of the LED light source is decreased at the timing when the detection light is not captured. Control means for controlling the LED light source ;
A scanning confocal microscope characterized by comprising:

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