JP2006162418A - Cars three-dimensional image system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a CARS three-dimensional image system capable of speeding up scanning in the direction of optical axis of irradiation, while suppressing complication of a system constitution. <P>SOLUTION: Two-dimensional scanning in a plane (X-Y plane) vertical to the incident optical axis is mechanically performed by an x-y scanning part 18. Scanning in the direction of an optical axis (Z) is performed by simultaneously changing the energies of first and second lights and changing the focal distance of an objective lens 10. More specifically, by changing the wavelengths of the first and second lights by a wavelength conversion control part 19, without changing a wavelength difference, the energy of the first and second lights is changed. The focal distance of the objective lens 10 is thereby changed, and the depth of a measuring plane is changed. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  In the medical biology field, the present invention is capable of non-invasively and highly accurately measuring a living tissue and a fine structure of a cell constituting the same, such as a CARS (coherent anti-Stokes Raman scattering) microscope or a biological measuring device using a CARS microscope. The present invention relates to a CARS three-dimensional image device for three-dimensional measurement.

  In a conventional CARS microscope, a sample is irradiated with light of two wavelengths, and light generated from resonance of energy ranks of frequency differences between the two wavelengths of light is measured. Thereby, the specific frequency component in the molecular vibration of the substance specific to the sample is selectively measured and imaged. For this reason, it is not necessary to irradiate excitation light for high energy fluorescence, and it is not necessary to label the measurement substance with a phosphor, and the fine structure of the cell can be imaged (for example, see Patent Document 1).

JP 2002-107301 A

  In order to collect three-dimensional image data with the conventional CARS microscope as described above, not only two-dimensional scanning in a plane perpendicular to the irradiation optical axis but also scanning in the irradiation optical axis direction (so-called slice depth direction). Also need to do. For such scanning in the irradiation optical axis direction, a scanning method (mechanical scanning) by mechanical movement of the objective lens or the subject is used. For this reason, there was a limit in shortening measurement time. In addition, since it is necessary to incorporate a highly accurate moving mechanism in the objective lens portion that is closest to the sample, the apparatus configuration is complicated and expensive. Furthermore, the CARS microscope has been reduced in size and has become an obstacle to incorporation into an endoscope.

  The present invention has been made in order to solve the above-described problems. It is an object of the present invention to obtain a CARS three-dimensional image apparatus capable of speeding up scanning in the irradiation optical axis direction while suppressing the complexity of the apparatus configuration. Objective.

  The CARS three-dimensional image device according to the present invention synthesizes the first and second light beams having different wavelengths and irradiates the measured object, and measures the generated CARS light to image the three-dimensional state of the measured object. In the CARS three-dimensional image device to be converted, scanning in the direction of the irradiation optical axis is performed by changing the energy of the first and second light by the same circumference amount.

  Since the CARS three-dimensional image device of the present invention performs scanning in the direction of the irradiation optical axis by changing the energy of the first and second light by the same amount of circumference, a mechanical scanning mechanism is used in the direction of the irradiation optical axis. Scanning in the direction of the irradiation optical axis can be accelerated while suppressing the complexity of the apparatus configuration.

The best mode for carrying out the present invention will be described below with reference to the drawings.
Embodiment 1 FIG.
1 is a block diagram showing a CARS three-dimensional image apparatus according to Embodiment 1 of the present invention. In this example, a YAG laser is used as the light source unit 1. The light generated by the light source unit 1 is converted by the pulsed light generation unit 2 into ultrashort pulsed light on the order of picoseconds. The light from the pulsed light generation unit 2 is incident on the first wavelength conversion unit 3. The first wavelength conversion unit 3 is configured by a parametric amplification device using a nonlinear effect, for example. The wavelength of the light from the pulsed light generating unit 2 is converted by the first wavelength converting unit 3.

  The light that has passed through the first wavelength conversion unit 3 is branched into first and second light by a first half mirror 4 as a spectroscopic means. The first light reflected by the first half mirror 4 is turned 90 degrees by the first mirror (total reflection mirror) 5 and turned again 90 degrees by the second mirror 7, so that the second half The light enters the mirror 8.

  On the other hand, the second light transmitted through the first half mirror 4 is incident on the second wavelength converter 9. In the second wavelength conversion unit 9, an energy displacement corresponding to the vibration period of the measurement substance in the sample 11 that is the measurement target is applied to the second light. The second light that has passed through the second wavelength conversion unit 9 enters the optical path adjustment unit 6. The optical path adjustment unit 6 includes a pair of fixed mirrors 6a and 6b and a pair of movable mirrors 6c and 6d that can be displaced in a direction in which the fixed mirrors 6a and 6b come into contact with and away from each other. The first light that has passed through the optical path adjustment unit 6 is incident on the second half mirror 8. In the second half mirror 8, the first light and the second light are combined so that their optical axes coincide with each other, and irradiation light is generated.

  The irradiation light generated by the second half mirror 8 is converged inside the sample 11 by the objective lens 9 having a focal point adjusted to a desired depth. When the irradiation light is converged in the sample 11, coherent anti-Stokes Raman scattering light (hereinafter abbreviated as CARS light) is generated due to the nonlinear effect in the sample 11. This CARS light has a wavelength different from that of the irradiation light, travels in the same optical path as the irradiation light in the opposite direction, is separated by the dichroic mirror 12, and is detected by the first detector 13. Further, the backscattered light from the sample 11 passes through the dichroic mirror 12, is separated by the third half mirror 14, and is detected by the second detector 15.

  Measurement signals from the first and second detectors 13 and 15 are transferred to the computer 17 via the signal collecting unit 16.

  Two-dimensional scanning of the sample 11 is performed by the xy scanning unit 18. The scanning in the irradiation optical axis direction is performed by sequentially converting the shift energy in the first wavelength conversion unit 3 by the wavelength conversion control unit 19 which is an energy conversion control unit. The measurement signal is transferred from the signal collecting unit 16 to the computer 17 in synchronization with the operations of the xy scanning unit 18 and the wavelength conversion control unit 19. The computer 17 images and displays the spatial distribution of molecules having a vibration order synchronized with the displacement frequency of the second wavelength conversion unit 9 using the measurement signals obtained by the spatial scanning in the three directions as described above. .

  Next, details of the three-dimensional imaging will be described. When the irradiation light is converged in the sample 11, CARS light corresponding to the irradiation wavelength is generated along the incident optical axis direction in the vicinity of the focal position. The CARS light is generated as resonance light from a specific molecule having a vibration energy rank of a frequency difference between the incident first and second light. Therefore, if the vibration frequency difference between the first and second light is set to the vibration order of lipids specifically contained in the cell membrane, for example, the membrane structure is imaged in detail without staining the cells. be able to.

  Here, since the CARS light is generated in proportion to the square of the irradiation light intensity, when the objective lens 10 is used as shown in FIG. 1, the resonance light can be set to be generated only from the vicinity of the focal position. it can. Further, since CARS light is generated by a coherent effect with irradiation light, the traveling direction thereof is the same direction as the incident excitation light and a direction reversed by 180 degrees (light coherent to the incident light). Among these, the former is called T-CARS and the latter is called E-CARS.

  Here, since the T-CARS light is emitted in the same direction as the incident light direction, it is necessary to measure from the back side of the sample 11. For this reason, it can be used for imaging only when the thickness of the sample 11 is thin and a focusing objective lens can be attached to the back side of the sample 11.

  Therefore, in this embodiment, an E-CARS type imaging method using backscattering is adopted. Thereby, it is not necessary to arrange an objective lens on the back side of the sample 11, and application to the thick sample 11 is also possible. Further, since only one objective lens 10 is used, strict focus adjustment is unnecessary. Furthermore, it can be used for non-invasive diagnosis that is attached to an endoscope and examines cell characteristics near the surface of the body cavity.

  Further, when the irradiation light is narrowed down by the objective lens 10, the CARS light is generated only in the vicinity of the focal position of the objective lens 10 where the light density of the irradiation light is sufficiently high. By scanning this focal region in three directions, it becomes possible to form a three-dimensional image with a spatial resolution of a minute region determined by the beam width of the irradiation light and the light distribution near the focal point.

  In the CARS three-dimensional image apparatus according to this embodiment, two-dimensional scanning in a plane (XY plane) perpendicular to the incident optical axis is mechanically performed by the xy scanning unit 18. On the other hand, scanning in the direction of the optical axis (Z) is performed by simultaneously changing the energy of the first and second lights and changing the focal length of the objective lens 10. Specifically, the wavelength conversion controller 19 changes the wavelengths of the first and second lights while the wavelength difference remains the same (without changing the difference between the frequencies of the two-wavelength light that is the resonance frequency). The energy of the first and second light is changed. Thereby, the focal distance of the objective lens 10 is changed, and the measurement surface depth is changed.

  FIG. 2 is a graph showing the relationship between the wavelength of irradiation light and the focal length of the objective lens 10 in the CARS three-dimensional image apparatus of FIG. Such a change in focal length is known as chromatic aberration of the lens, and should generally be suppressed because it causes distortion of the optical system. However, in this embodiment, the focal length is changed by positively using the change in wavelength and used for three-dimensional scanning.

  FIG. 3 is an explanatory diagram showing a change in the focal plane of the objective lens 10 due to a change in the wavelength of the irradiation light in the CARS three-dimensional image apparatus of FIG. By changing the wavelength of the irradiation light from λ1 to λ2, the position of the focal plane (measurement slice plane) of the objective lens 10 is changed from the focal plane 11a to the focal plane 11b.

  In normal spectroscopic measurement, if the irradiation light wavelength is changed, the measurement spectral characteristics change, so that imaging becomes impossible. However, in CARS measurement, if the energy difference between the first and second lights is constant, the target measurement order energy, that is, the measurement molecule does not change, so the energy of the first and second lights changes by the same amount. By doing so, the measurement target substance does not change. Moreover, the spectral absorption change in the sample 11 occurs due to the energy change of the irradiation light, and the data changes, but the backscattered light intensity is measured by the second detector 15 and detected by the first detector 13. By correcting the CARS light intensity, a change in data can be corrected.

  In the three-dimensional scanning procedure, first, the shift energy by the wavelength conversion control unit 19 is fixed to a minimum, and then the two-dimensional scanning by the xy scanning unit 18 is performed to measure the CARS signal distribution on the first measurement slice plane. At this time, the signal of the second detector 15 is also taken at the same time, and the light intensity detected by the first detector 13 is corrected. Next, the shift energy by the wavelength conversion control unit 19 is increased and fixed by an appropriate amount, similarly, two-dimensional scanning is performed, and the CARS signal distribution on the second measurement slice plane is measured. By sequentially repeating such operations, three-dimensional imaging can be performed.

  Here, the distance between each measurement slice plane is defined from the relationship between the focal length of the objective lens 10 and the wavelength. Conversely, the step value of the energy shift can be determined from the distance between each measurement slice plane. FIG. 2 shows an example of the wavelength dependency of the focal length. When the focal length is 5 mm, it is possible to move the focal point by about 2 μm if a wavelength change of 10 nm is applied in the vicinity of 800 nm.

As described above, in the CARS three-dimensional image apparatus according to this embodiment, scanning in the irradiation optical axis direction is performed by changing the energy of the first and second lights by the same amount of circumference, so Mechanical scanning is unnecessary, and the scanning in the irradiation optical axis direction can be speeded up while suppressing the complexity of the apparatus configuration.
Moreover, after changing the wavelength of the light from the common light source unit 1 by the first wavelength conversion unit 3, the light is divided into the first and second light by the first half mirror 4, and the second light Since the wavelength is changed by the second wavelength conversion unit 9, the wavelength of the first and second lights can be easily made equal by controlling the shift energy in the first wavelength conversion unit 3 by the wavelength conversion control unit 19. The circumference can be changed.

  Furthermore, since the scanning in the irradiation optical axis direction is not performed by the mechanical scanning, the application of the CARS three-dimensional image device to the endoscope becomes easy. That is, the CARS three-dimensional endoscope is an operation for operating the insertion portion connected to the insertion portion inserted into the body cavity of the subject, the head portion provided at the distal end portion of the insertion portion, and the proximal end portion of the insertion portion. Part. The head unit is provided with an objective lens 10 and an xy scanning unit 18 which are an emitting unit for irradiation light and a light receiving unit for CARS light. Moreover, the optical fiber which guides irradiation light and CARS light is incorporated in the insertion part. In such an endoscope, since it is not necessary to perform mechanical scanning in the irradiation optical axis direction, the head portion can be reduced in size. As for the displacement of the head portion in the direction of the irradiation optical axis, the scanning is performed at a sufficiently high speed with respect to the displacement speed so that the displacement can be ignored, or an interval for maintaining the distance between the measured portion and the head portion. What is necessary is just to mount a holder in a head part.

  In the above example, two-dimensional scanning in a plane perpendicular to the irradiation optical axis is performed by mechanical optical axis movement. However, for example, if a multi-micro lens used in a confocal microscope is used, mechanical scanning is performed. Multipoint focus measurement is possible without this. Therefore, if the axial scanning by energy change and the microlens array method are shared, a very small CARS microscope that does not require mechanical operation can be realized.

It is a block diagram which shows the CARS three-dimensional image apparatus by Embodiment 1 of this invention. It is a graph which shows the relationship between the wavelength of irradiation light in the CARS three-dimensional image apparatus of FIG. 1, and the focal distance of an objective lens. It is explanatory drawing which shows the change of the focal plane of the objective lens by the wavelength change of the irradiation light in the CARS three-dimensional image apparatus of FIG.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 Light source part, 3 1st wavelength converter, 4 1st half mirror (spectral means), 9 2nd wavelength converter, 19 Wavelength conversion control part

Claims (4)

  In the CARS three-dimensional image device that combines the first and second lights having different wavelengths and irradiates the measurement object, and measures the generated CARS light, thereby imaging the three-dimensional state of the measurement object. A CARS three-dimensional image device that scans in the direction of the irradiation optical axis by changing the energy of the first and second light by the same amount of circumference.   A wavelength conversion controller that changes the energy of the first and second lights by changing the wavelengths of the first and second lights while the wavelength difference between the first and second lights remains the same. The CARS three-dimensional image apparatus according to claim 1, further comprising:   A light source unit, a first wavelength conversion unit that changes a wavelength of light from the light source unit, a spectroscopic unit that splits light from the first wavelength conversion unit into the first and second light, and The CARS three-dimensional image apparatus according to claim 2, further comprising a second wavelength conversion unit that changes a wavelength of the second light.   An insertion portion to be inserted into the body cavity of the subject; a head portion provided at a distal end portion of the insertion portion, and provided with an emission portion of the irradiation light to the measurement object and a light receiving portion of the CARS light; The CARS three-dimensional image apparatus according to claim 1, wherein the CARS three-dimensional image apparatus is provided.

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