JP5198213B2 - Laser microscope equipment - Google Patents

Description

  The present invention relates to a laser microscope apparatus.

  A coherent anti-Stokes Raman scattering microscope is known that uses a specific vibration of a molecule in a sample to generate coherent anti-Stokes Raman scattering light from the molecule and observes the sample by detecting this scattered light ( For example, see Patent Document 1). Since this coherent anti-Stokes Raman scattering microscope uses a specific vibration of a sample molecule, it is not necessary to label an observation target with a fluorescent probe in advance unlike a fluorescence microscope. Moreover, the molecule | numerator to observe can be changed by changing the vibration to utilize.

  By the way, in general, coherent anti-Stokes Raman scattering light includes a resonance component contributed by a specific vibration of a molecule and a non-resonance component not contributed by a specific vibration of the molecule. Since the non-resonant component corresponds to so-called noise, it is desirable to remove the non-resonant component and extract only the resonant component when observing the sample with coherent anti-Stokes Raman scattering light. On the other hand, conventionally, there has been known a method of generating non-resonant components by generating two coherent anti-Stokes Raman scattering lights using two pump lights having different frequencies and one Stokes light and taking the difference between them. (For example, refer to Patent Document 2).

Japanese translation of PCT publication No. 2002-520612 International Publication No. 2007/050596

  However, the use of pulse laser beams having three different frequencies requires a laser light source or a frequency conversion device for generating pulse laser beams of the respective frequencies, which complicates the device configuration and increases the device cost. In addition, there is an inconvenience that the operation becomes complicated.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a laser microscope apparatus that can improve image quality by removing non-resonant components while suppressing complication of the apparatus configuration. .

In order to achieve the above object, the present invention employs the following means.
The present invention includes two optical paths for guiding pulsed laser light having two different frequency bands distributed in the time axis direction and having a frequency difference substantially equal to a specific vibration frequency of a molecule in a sample, A pulse timing adjusting means provided on at least one of the optical paths for adjusting the temporal timing of the pulsed laser light on the sample surface of the pulsed laser light guided to the two optical paths, and guided through the two optical paths; A combining means for combining the pulse laser light, an irradiation means for generating a coherent anti-Stokes Raman scattered light by irradiating the sample with the pulse laser light combined by the combining means, and generated from the sample. By detecting the coherent anti-Stokes Raman scattering light and changing the temporal timing by the pulse timing adjusting means. Image generating means for generating an image based on the difference between two coherent anti-Stokes Raman scattering light detected by the light detecting means Te, a laser light source for generating a femtosecond pulsed laser beam, emitted from the laser light source Branch means for branching the femtosecond pulsed laser light into the two optical paths, and a specific vibration frequency of the molecules in the sample in the femtosecond pulsed laser light provided in one of the optical paths and guided through the two optical paths The frequency conversion means made of a photonic crystal fiber that gives a frequency difference substantially equal to and the frequency dispersion amount of the pulsed laser light that is provided in at least one of the two optical paths and guided through the two optical paths is substantially equivalent There is provided a laser microscope apparatus comprising: a frequency dispersion adjusting unit that adjusts in such a manner .

  According to the laser microscope apparatus of the present invention, each pulse laser beam guided through the two optical paths is combined by the combining unit, and the combined pulse laser beam is irradiated to the sample by the irradiation unit. Here, each pulse laser beam guided through the two optical paths has a frequency difference substantially equal to the specific vibration frequency of the molecule in the sample, and therefore, coherent resonance with the specific vibration of the molecule in the sample. Anti-Stokes Raman scattered light is generated and detected by the light detection means.

  At this time, the frequency difference between the two pulse laser beams can be changed by adjusting the temporal timing of the two pulse laser beams on the sample surface by the pulse timing adjusting means. Thus, coherent anti-Stokes Raman scattering light can be generated in a state where an arbitrary frequency difference is given in addition to a frequency difference that resonates with a specific vibration of a molecule in the sample. That is, it is possible to generate coherent anti-Stokes Raman scattered light having different frequencies according to the adjustment of the temporal timing of the two pulse laser beams.

  Here, a non-resonant component not contributed by specific vibration of molecules in the sample included in the coherent anti-Stokes Raman scattering light, that is, noise is a pulse having two different frequency bands on the sample surface by the pulse timing adjusting means. In both cases where the frequency difference of the laser light is made approximately equal to the specific vibration of the molecules in the sample or not, it is included in the coherent anti-Stokes Raman scattered light. Therefore, in the two cases where the frequency difference between the two pulsed laser beams is adjusted so as to be approximately equal to the specific vibration of the molecules in the sample or not, the intensity of the coherent anti-Stokes Raman scattered light is detected by the light detection means. , And an image is generated by the image generation unit based on the difference in the intensity of the coherent anti-Stokes Raman scattering light in the two cases, so that an image from which non-resonant components are removed can be generated. Thereby, it is possible to improve the signal-to-noise ratio and perform observation with a high-quality image. That is, according to the laser microscope apparatus of the present invention, non-resonant components of coherent anti-Stokes Raman scattered light can be removed using pulsed laser light having two different frequency bands.

In addition, the frequency of the femtosecond pulsed laser light is split so that a femtosecond pulsed laser light generated from a single laser light source is branched and a frequency difference approximately equal to a specific vibration frequency of a molecule in the sample is given in one optical path. Can be obtained by the frequency conversion means to obtain pulsed laser light having two different frequency bands, so that the apparatus configuration can be simplified by sharing the light sources of the two pulsed laser lights having different frequency bands.

Furthermore, since the frequency conversion means is a photonic crystal fiber, it becomes possible to obtain pulsed laser light having a wide frequency band to which frequency dispersion is given, easily and inexpensively. Further, by selecting the type of photonic crystal fiber to be used, pulsed laser light having various frequency components and frequency bands can be obtained. Therefore, it is possible to adjust the frequency difference between the two pulsed laser beams so as to match various vibration frequencies of the molecules in the sample.

In the above-described invention, it is preferable that the irradiation unit includes a scanner that performs two-dimensional scanning with the laser beam on the specimen.

  By adjusting each pulse laser beam guided along the two optical paths so as to have a substantially equal frequency dispersion amount by the frequency dispersion adjusting means, the frequency difference between the two pulse laser beams can be obtained at each time on the time axis. Can be constant. As a result, the energy of the two pulsed laser beams can be efficiently used to generate coherent anti-Stokes Raman scattered light. Further, by adjusting the amount of frequency dispersion by the frequency dispersion adjusting means, even for pulse laser light having a relatively wide frequency band such as femtosecond laser light, at each time on the time axis, the pulse laser light The frequency component can be dispersed on the time axis.

  In the above invention, the pulse timing adjusting means adjusts the temporal timing of the two pulse laser beams so that the intensity of the coherent anti-Stokes Raman scattering light detected by the light detecting means becomes maximum or maximum and minimum or minimum. It may be adjusted.

  By taking the difference between the maximum or maximum value of the intensity of the coherent anti-Stokes Raman scattering light and the minimum or minimum value, the non-resonant component can be removed. Thereby, the signal-to-noise ratio can be improved and a high-quality image can be obtained. In addition, the temporal timing of the two pulsed laser beams is constant so that the frequency difference between the two pulsed laser beams varies within a range in which the intensity of the coherent anti-Stokes Raman scattering light is a maximum or maximum value and a minimum or minimum value. The non-resonant component is removed by extracting only the intensity component that fluctuates at the predetermined frequency from the intensity of the coherent anti-Stokes Raman scattering light obtained by periodically varying the amplitude and the predetermined frequency. An image can also be generated.

  According to the present invention, there is an effect that image quality can be improved by removing non-resonant components while suppressing complication of the apparatus configuration.

A laser microscope apparatus 1 according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, a laser microscope apparatus 1 according to the present embodiment includes a laser light source apparatus 2 and a microscope main body 3 for observing the specimen A by irradiating the specimen A with laser light from the laser light source apparatus 2. And.

  The laser light source device 2 includes a single laser light source 4 that emits femtosecond pulse laser light, a beam splitter (branching means) 5 that branches the femtosecond pulse laser light emitted from the laser light source 4 into two, a beam Two femtosecond pulsed laser beams L1 and L2 branched by the splitter 5 are respectively passed through two optical paths 6 and 7, and two pulsed laser beams L1 ′ and L2 ′ that have passed through the two optical paths 6 and 7 are transmitted. And a laser combiner 8 for multiplexing.

  The first optical path 6 is provided with a frequency dispersion device (frequency dispersion adjusting means) 9 capable of adjusting the amount of frequency dispersion given to the femtosecond pulsed laser light L1. The second optical path 7 includes a photonic crystal fiber (frequency conversion means) 10 that allows the femtosecond pulsed laser light L2 to pass through, and an optical path adjustment that adjusts the optical path length of the pulsed laser light L2 ′ that has passed through the photonic crystal fiber 10. A device (pulse timing adjusting means) 11 is provided.

  The frequency dispersion device 9 includes, for example, a pair of prisms (not shown) that can adjust the distance between them and a mirror (not shown). The femtosecond pulsed laser light L1 that has passed through the pair of prisms is returned by the mirror after passing through the prism pair again after being folded by the mirror. In this case, the amount of frequency dispersion given to the pulsed laser light L1 'passing through the frequency dispersion device 9 can be adjusted by adjusting the interval between the prisms. A diffraction grating pair (not shown) may be used instead of the prism pair.

  The photonic crystal fiber 10 changes or expands the frequency band of the femtosecond pulsed laser light L2 to be transmitted to generate the pulsed laser light L2 ′, and the pulsed laser light L1 ′ and L2 ′ guided through the optical paths 6 and 7. Is given a frequency difference substantially equal to a specific vibration frequency of the molecules in the sample A.

  The optical path adjusting device 11 is configured by a mirror (reflector), for example (not shown). The optical path length of the pulsed laser light L2 'can be changed by turning back the optical path of the pulsed laser light L2' using at least two or more sets of reflectors and adjusting the interval between the at least two sets of reflectors. Thereby, the temporal timing of the pulse of the pulsed laser beam L2 'can be adjusted.

  The laser combiner 8 includes a multiplexing unit (not shown) that multiplexes two pulsed laser beams L1 ′ and L2 ′ that have passed through the two optical paths 6 and 7, and a component in a desired frequency band from the pulsed laser beam L2 ′. And a filter (not shown). By passing the pulse laser beam L2 'through this filter, it is possible to remove unnecessary components for generating coherent anti-Stokes Raman scattering light.

  The microscope main body 3 is, for example, a laser scanning microscope, and includes a scanner 12 and a lens group 20 that two-dimensionally scans the pulsed laser light L3 emitted from the laser light source device 2, and pulses scanned by the scanner 12. A condensing lens (irradiating means) 13 that condenses the laser light L3 on the surface of the specimen A, a condensing lens 15 that condenses coherent anti-Stokes Raman scattered light generated in a direction that transmits the specimen A, and a condensing lens 15 A second photo detector (light detection means) 16 for detecting the coherent anti-Stokes Raman scattered light collected by the second optical detector 16 and an image using the coherent anti-Stokes Raman scattered light detected by the second photo detector 16. A control unit (image generating means) 21 that generates and controls the optical path adjusting device 11 is provided.

  In the figure, reference numeral 17 is a dichroic mirror, reference numeral 18 is a stage, and reference numeral 19 is a mirror. The coherent anti-Stokes Raman scattering light generated in the sample A may be collected by the condenser lens 13, branched by the dichroic mirror 17, and detected by the first photodetector 14.

The operation of the laser microscope apparatus 1 configured as described above will be described below.
In order to observe the specimen A with coherent anti-Stokes Raman scattering light using the laser microscope apparatus 1 according to the present embodiment, the laser light source 4 is operated to emit femtosecond pulsed laser light. The femtosecond pulsed laser light emitted from the laser light source 4 is branched into two optical paths 6 and 7 by the beam splitter 5.

  The femtosecond pulsed laser light L1 branched to the first optical path 6 is given an initial amount of frequency dispersion by passing through the frequency dispersion device 9 disposed on the first optical path 6. On the other hand, the femtosecond pulsed laser light L2 branched to the second optical path 7 is deflected by the mirror 19 and then passed through the photonic crystal fiber 10, whereby the femtosecond pulsed laser light in the first optical path 6 is obtained. This is broadband light (pulse laser light L2 ′) whose frequency is changed or expanded compared to L1. At the same time, the pulsed laser light L2 'is given a predetermined frequency dispersion by adding the photonic crystal fiber 10.

  When the initial frequency dispersion amount given to the pulse laser beam L1 ′ by the frequency dispersion device 9 and the frequency dispersion amount given to the pulse laser beam L2 ′ by passing through the photonic crystal fiber 10 are different, FIG. ), The slopes of the frequency distributions of the pulse laser beams L1 ′ and L2 ′ are different on the time axis. In this case, the frequency difference Ω ′ between the pulsed laser beams L1 ′ and L2 ′ passing through the two optical paths 6 and 7 is different at each time on the time axis and cannot be kept constant. In this state, the energy of the pulsed laser beams L1 'and L2' cannot be efficiently used for generating coherent anti-Stokes Raman scattered light from specific vibrations of molecules in the sample A.

  Therefore, by operating the frequency dispersion device 9, the amount of dispersion given to the pulse laser light L 1 ′ passing through the first optical path 6 is changed to the pulse laser light L 2 ′ passing through the photonic crystal fiber 10 in the second optical path 7. Adjustment is made so that the amount of dispersion given is substantially equal on the specimen A surface. That is, as shown by the arrow P1 in FIG. 2A, the slope of the frequency distribution in the time axis direction is changed.

  As a result, as shown in FIG. 2B, the slopes of the frequency distributions in the time axis direction of the pulse laser beams L1 ′ and L2 ′ of the two optical paths 6 and 7 reaching the laser combiner 8 become substantially the same. Adjusted to. Thereafter, the pulse laser beams L1 'and L2' are combined by the laser combiner 8 to become the pulse laser beam L3.

  The pulse laser beam L3 synthesized in this way is incident on the microscope body 3 and scanned two-dimensionally by the scanner 12, and then condensed on the specimen A surface via the lens group 20 and the condenser lens 13. Is done. Thereby, coherent anti-Stokes Raman scattering light can be generated from the specific vibration frequency of the molecules in the sample A at each position where the pulse laser beam L3 is collected.

  The coherent anti-Stokes Raman scattered light generated in the specimen A is collected by the condenser lens 15 disposed on the opposite side of the condenser lens 13 across the specimen A, and detected by the second photodetector 16. . Then, by storing the coordinates of the condensing position of the pulsed laser light L3 on the specimen A surface and the light intensity of the coherent anti-Stokes Raman scattered light detected by the second photodetector 16 in association with each other, The control unit 21 generates a two-dimensional coherent anti-Stokes Raman scattering light image.

Here, the coherent anti-Stokes Raman scattering light image obtained as described above includes a resonance component contributed by a specific vibration of the molecule and a non-resonance component not contributed by the specific vibration of the molecule. Yes.
When the specific molecular vibration frequency in the specimen and Omega, the strength I _c of the coherent anti-Stokes Raman scattering light generated from the molecule, the following using the physical quantity of the third-order nonlinear susceptibility of the molecule chi ⁽³⁾ It is expressed as (1).
_{I c (Ω) α | χ} (3) R (Ω) + χ (3) NR | 2 = | χ (3) R (Ω) | 2 + (χ (3) NR) 2 + 2Reχ (3) R (Ω ) Χ ⁽³⁾ _NR (1)

In the above equation (1), χ ⁽³⁾ _R represents the third-order nonlinear susceptibility corresponding to the resonance component, and χ ⁽³⁾ _NR represents the third-order nonlinear susceptibility corresponding to the non-resonance component. Also, the first term | χ ⁽³⁾ _R (Ω) | ² is the contribution of the resonance component, the second term (χ ⁽³⁾ _NR ) ² is the contribution of the non-resonance component, and the second term 2Reχ ⁽³⁾ _R (Ω) χ ⁽³⁾ _NR represents the contribution of interference between the resonance component and the non-resonance component. Here, the coherent anti-Stokes Raman scattering light obtained by superposing the sections thereof, and the frequency Omega _d frequency Omega _p and intensity I _c which the intensity I _c becomes maximum (peak) is minimum (dip) Exists.

The non-resonant component can be removed by using the change in the spectrum of the third term of the above formula (1). That is, the second photodetector 16 detects the coherent anti-Stokes Raman scattering light for each of the frequency Omega _d the strength I _c of the coherent anti-Stokes Raman scattered light is frequency Omega _p and minima as a maximum, By taking the difference, the non-resonant component can be removed.

  Therefore, the temporal timings of the two pulsed laser beams L1 'and L2' are changed so that the intensity of the coherent anti-Stokes Raman scattering light detected by the second photodetector 16 becomes maximum and minimum. That is, the control unit 21 operates the optical path adjusting device 11 to delay the pulsed laser light L2 ′ passing through the second optical path 7 in the time axis direction as indicated by an arrow P2 in FIG. .

The time distribution of the frequency of the coherent anti-Stokes Raman scattering light thus obtained is shown in FIGS. 3 (a) and 3 (b). FIG. 3A shows a state where the intensity of the coherent anti-Stokes Raman scattering light is maximized. At this time, the pulsed laser light L1 of the two optical paths 6, 7 ', L2' frequency difference is Omega _p.
FIG. 3B shows that the intensity of the coherent anti-Stokes Raman scattering light is minimized by delaying the pulsed laser light L2 ′ passing through the second optical path 7 by Δt in the time axis direction from the state of FIG. This shows the state. At this time, the pulsed laser light L1 of the two optical paths 6, 7 ', L2' frequency difference is Ω + ΔΩ = Ω _d.

Then, taking the difference between the maximum value and the minimum value of the intensity I _c of the thus coherent anti-Stokes Raman scattering light obtained by, for generating a two-dimensional image by the control unit 21 based on said difference. Thereby, it is possible to generate a high-quality coherent anti-Stokes Raman scattering light image in which noise is removed and the signal-to-noise ratio is improved.

As described above, according to the laser microscope apparatus 1 according to the present embodiment, the pulse laser beams L1 ′ and L2 ′ are adjusted by adjusting the time timing of the pulse laser beam L2 ′ by the optical path adjusting device 11. Can be generated, and two coherent anti-Stokes Raman scattering lights having different frequencies can be generated. In addition, by taking the difference between the intensity of the two coherent anti-Stokes Raman scattering light generated in this way and generating an image by the image generation means based on the difference, an image from which non-resonant components have been removed is generated. Is possible.
In particular, by taking the difference between the maximum or minimum value and the minimum or minimum value of the intensity of the coherent anti-Stokes Raman scattering light, it is possible to perform observation with an image in which the non-resonant component is removed and the signal-to-noise ratio is further improved. .

  Further, the femtosecond pulsed laser light generated from the single laser light source 4 is branched by the beam splitter 5 so as to give a frequency difference substantially equal to the specific vibration frequency of the molecule in the sample in the second optical path 7. By converting the frequency of the femtosecond pulsed laser beam by the photonic crystal fiber 10, the light source of the two pulsed laser beams L1 ′ and L2 ′ having different frequency bands can be shared to simplify the apparatus configuration.

  Further, the frequency dispersion device 9 adjusts the amount of frequency dispersion so that the frequency difference Ω ′ between the pulse laser beams L1 ′ and L2 ′ of the two optical paths 6 and 7 becomes constant at each time on the time axis. The energy of the pulse laser beams L1 ′ and L2 ′ can be efficiently used for generating coherent anti-Stokes Raman scattering light. Further, by adjusting the amount of frequency dispersion by the frequency dispersion device 9, even for pulse laser light having a relatively wide frequency band such as femtosecond laser light, at each time on the time axis, the pulse laser light The frequency component can be dispersed on the time axis.

  Further, by using the photonic crystal fiber 10 as the frequency conversion means, the apparatus can be configured more simply and inexpensively. Further, since the optical path adjustment device 11 is also configured by a mirror (reflector), the device can be configured more simply and inexpensively. As the frequency conversion means, for example, any one of a bulk, a thin film, a film, and a photonic crystal structure having the same function and action may be used instead of the photonic crystal fiber 10.

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, in the present embodiment, the control unit 21 has been described as taking the difference between the maximum or minimum value and the minimum or minimum value of the intensity of the coherent anti-Stokes Raman scattering light. Instead, two pulse lasers are used. The temporal timing of the two pulsed laser beams is set to a predetermined amplitude and a predetermined value so that the frequency difference of the light changes in a range in which the intensity of the coherent anti-Stokes Raman scattering light is maximum or maximum and minimum or minimum. It is also possible to generate an image from which non-resonant components are removed by extracting only the intensity component that fluctuates at the predetermined frequency from the intensity of the coherent anti-Stokes Raman scattering light obtained by periodically varying the frequency. .

  Further, in the present embodiment, the single laser light source 4 that generates femtosecond pulsed laser light and the beam splitter 5 that branches into the optical paths 6 and 7 are used. The laser light sources 4 may be attached one by one. In this case, the laser light sources 4 need to be synchronized with each other in pulse timing.

  Further, the frequency dispersion device 9 may be provided in the second optical path 7 or may be provided in both the optical paths 6 and 7. Even when a frequency dispersion device is provided between the laser light source 4 and the beam splitter 5, or when the laser light source 4 and the frequency dispersion device are integrated, the same configuration as that of the present embodiment. By doing so, the non-resonant component of the coherent anti-Stokes Raman scattering light can be removed.

  A light amount adjusting means (not shown) such as a neutral density filter may be disposed in at least one of the optical paths 6 and 7. Thereby, the light quantity balance of the pulse laser beams L1 'and L2' passing through the two optical paths 6 and 7 can be achieved.

1 is a block diagram illustrating an overall configuration of a laser microscope apparatus according to an embodiment of the present invention. It is a graph which shows the time distribution of the frequency of the pulse laser beam transmitted through the two optical paths of the laser microscope apparatus of FIG. 1, and shows (a) before adjustment and (b) after frequency dispersion amount adjustment, respectively. It is a graph which shows the intensity | strength of the coherent anti-Stokes Raman scattering light at the time timing adjustment of the pulse of the pulse laser beam transmitted through two optical paths of the laser microscope apparatus of FIG. b) Each state is minimized.

Explanation of symbols

A specimen L1, L2 femtosecond pulse laser beam L1 ', L2' pulsed laser beam _{Ω, Ω ', Ω p,} Ω d frequency (difference)
DESCRIPTION OF SYMBOLS 1 Laser microscope apparatus 4 Laser light source 5 Beam splitter (branching means)
6,7 Optical path 8 Laser combiner
9 Frequency dispersion device (Frequency dispersion adjusting means)
10 Photonic crystal fiber (frequency conversion means)
DESCRIPTION OF SYMBOLS 11 Optical path adjustment device 13 Condensing lens 14 1st photodetector 16 2nd photodetector 21 Control part

Claims (3)

Two optical paths for guiding pulsed laser light having two different frequency bands distributed in the time axis direction and having a frequency difference substantially equal to a specific vibration frequency of a molecule in the sample;
A pulse timing adjusting unit that is provided in at least one of the two optical paths and adjusts the temporal timing of the pulsed laser light on the sample surface of the pulsed laser light guided to the two optical paths;
A multiplexing means for multiplexing the pulsed laser light guided through the two optical paths;
Irradiating means for irradiating the sample with pulsed laser light combined by the combining means to generate coherent anti-Stokes Raman scattering light;
Photodetection means for detecting coherent anti-Stokes Raman scattering light generated from the specimen;
Image generating means for generating an image based on a difference between two coherent anti-Stokes Raman scattered lights detected by the light detecting means by changing a temporal timing by the pulse timing adjusting means ;
A laser light source that generates femtosecond pulsed laser light;
Branching means for branching the femtosecond pulsed laser light emitted from the laser light source into the two optical paths;
A frequency conversion means comprising a photonic crystal fiber that is provided in any one of the optical paths and gives the femtosecond pulsed laser light guided through the two optical paths a frequency difference substantially equal to a specific vibration frequency of molecules in the sample; ,
A frequency dispersion adjusting means that is provided in at least one of the two optical paths and adjusts so that the amount of frequency dispersion of the pulse laser light guided through the two optical paths is substantially equal;
A laser microscope apparatus comprising: The pulse timing adjusting means, claim strength of the coherent anti-Stokes Raman scattering light detected by the light detecting means so that a maximum or maximum and minimum or minimum, to adjust the temporal timing of the two pulse laser beams laser microscope apparatus according to 1. The laser microscope apparatus according to claim 1, wherein the irradiation unit includes a scanner that performs two-dimensional scanning with the laser light on the specimen .

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