JP2005180932A - Light source unit and fluorescence measuring apparatus using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a light source unit for easily obtaining illumination light comprising arbitrary wavelength components, while obtaining high accuracy measuring fluorescence from fluorescent dye using the illumination light, and to provide a fluorescence-measuring instrument which uses the same. <P>SOLUTION: After illumination light 11 from a light source 13 is spectrally diffracted by a pre-spectroscope 19, a wavelength light 17, corresponding to the fluorescent dye, is selected by an opening part formed in an aperture member 35. Then. the wavelength light 17 is multiplexed into an excitation light 14 by a post-spectroscope 21. The fluorescent dye used to dye a measuring object outputs fluorescence, by being excited by an excitation light 14 comprising wavelength components corresponding to the fluorescent dye and the fluorescence can be measured with high accuracy. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a light source unit that outputs illumination light having a predetermined wavelength component and a fluorescence measurement device that measures fluorescence obtained using the illumination light as excitation light.

  For example, in the biochemical field, a biological tissue such as a cancer cell is stained with a fluorescent dye, and the fluorescence obtained by irradiating a measurement target containing the fluorescent dye with excitation light is measured to thereby change the state of the biological tissue. Fluorometric photometry is known.

  In this case, there are many types of fluorescent dyes having different absorbances (absorption characteristics) with respect to the wavelength of the excitation light and fluorescence intensities (luminescence characteristics) with respect to the wavelength of the output fluorescence. 9 and 10 show the relative absorbances and fluorescence intensities of the five types of fluorescent dyes a1 to a5 by standardizing the absorbance and fluorescent intensity of the fluorescent dye a1 to 100. FIG. For example, since the fluorescent dye a1 exhibits the maximum absorbance near 380 nm, the fluorescence can be most efficiently obtained by irradiating the fluorescent dye a1 with excitation light in the vicinity of this wavelength.

  On the other hand, it is possible to examine the state of the biological tissue in more detail by dyeing the biological tissue using a plurality of fluorescent dyes having different characteristics and measuring the fluorescence having different wavelengths obtained from the fluorescent dyes. In this case, as shown in FIG. 10, the fluorescence obtained from each fluorescent dye has a portion in which the wavelength ranges are superimposed on each other. It is necessary to separate the fluorescence.

  Therefore, as a general method for separating fluorescence, paying attention to the fact that the fluorescence intensity with respect to absorbance differs depending on the fluorescent dye, an optical filter that transmits only excitation light having a wavelength near the maximum absorbance of the desired fluorescent dye is used. There is a method in which the optical filter is arranged on the light source side and switched according to the fluorescent dye (see Patent Document 1).

JP 2003-98439 A

  However, it is extremely difficult to manufacture an optical filter having transmission characteristics corresponding to the absorbance characteristics of each fluorescent dye. In particular, when a new fluorescent dye is used, much labor is required to develop an optical filter corresponding to the new fluorescent dye. Further, when the type of fluorescent dye is changed, there is a possibility that an optical filter corresponding to the characteristic cannot be immediately provided.

  Furthermore, since it is practically impossible to provide one optical filter that satisfies the absorbance characteristics of all the fluorescent dyes used, measurement is performed by switching the optical filter according to each fluorescent dye. As a result, the trouble that the work becomes complicated occurs.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a light source unit capable of supplying illumination light having an arbitrary wavelength component very easily.

  In addition, the present invention provides a fluorescence measurement that can easily generate excitation light having a wavelength corresponding to the characteristics of the fluorescent dye, irradiate the measurement object, and measure the fluorescence obtained from the fluorescent dye with extremely high accuracy. An object is to provide an apparatus.

The light source unit of the present invention includes a light source that outputs illumination light,
A spectroscopic means for splitting the illumination light according to a wavelength;
A selection means for selecting wavelength light of a desired wavelength from the illumination light that has been split;
A multiplexing means for multiplexing the selected wavelength light;
(Invention of Claim 1).

  In this case, since the illumination light is separated for each wavelength by the spectroscopic means, the wavelength light having a desired wavelength is selected by the selection means from the split illumination light, and then the wavelength light is multiplexed by the multiplexing means. Thus, illumination light consisting only of a desired wavelength component can be obtained.

  An arbitrary wavelength component can be obtained by disposing an aperture member as a selection means in the optical path of the dispersed wavelength light and allowing the wavelength light to pass through an opening formed at a desired wavelength position of the aperture member. (2nd aspect of the invention). Moreover, wavelength light containing a plurality of wavelength components can be obtained by forming a plurality of openings corresponding to the wavelength positions (the invention according to claim 3).

  A spectroscope can be used as the spectroscopic means for splitting the illumination light and the multiplexing means for multiplexing the wavelength light (the invention according to claim 4). Note that the spectroscope constituting the multiplexing means can have the same configuration as the spectroscope constituting the spectroscopic means by setting the traveling direction of the light passing therethrough opposite to the spectroscope of the multiplexing means.

Further, the fluorescence measurement device of the present invention is a fluorescence measurement device that irradiates a measurement object stained with a predetermined fluorescent dye with excitation light and measures the fluorescence obtained from the fluorescent dye.
A light source that outputs illumination light;
A spectroscopic means for splitting the illumination light according to a wavelength;
A selection means for selecting wavelength light of a desired wavelength from the illumination light that has been split;
A multiplexing means for multiplexing the selected wavelength light;
An optical system for guiding the combined wavelength light to the measurement object as the excitation light;
Fluorescence measuring means for measuring the fluorescence obtained from the fluorescent dye by being irradiated with the excitation light;
(Invention of Claim 5).

  In this case, the illumination light is separated by the spectroscopic means, the wavelength component corresponding to the fluorescent dye is selected by the selection means, and then combined by the multiplexing means, and the measurement object as excitation light via the optical system. Is irradiated. And the state of the measuring object based on a desired fluorescent pigment | dye can be measured by measuring the fluorescence output from the fluorescent pigment | dye irradiated with excitation light with a fluorescence measurement means.

  In addition, by passing illumination light through an opening formed at a wavelength position corresponding to the characteristics of the fluorescent dye, excitation light composed of a wavelength component corresponding to the fluorescent dye can be obtained (claim 6). invention). In addition, by forming a plurality of openings, excitation light corresponding to a plurality of fluorescent dyes having different characteristics can be obtained simultaneously (the invention according to claim 7).

  A spectroscope can be used as the spectroscopic means for splitting the illumination light and the multiplexing means for multiplexing the wavelength light (the invention according to claim 8).

  The combined excitation light is converted into slit light by a slit member and guided to a measurement object through an optical system, and then fluorescence obtained from the fluorescent dye of the measurement object is spectroscopically dispersed by a spectroscope. To obtain the spectral information of the measurement object (the invention according to claim 9). In this case, two-dimensional image information and two-dimensional spectroscopic information of the measurement object can be obtained by scanning the slit light along the measurement object by the scanning unit.

  By moving the imaging lens constituting the optical system as scanning means, it is possible to acquire two-dimensional image information and two-dimensional spectroscopic information with the measurement object fixed (the invention according to claim 10). .

  According to the light source unit of the present invention, illumination light having an arbitrary wavelength component can be generated and supplied very easily from illumination light output from a light source.

  Moreover, according to the fluorescence measuring apparatus of the present invention, excitation light composed of wavelength components corresponding to the characteristics of the fluorescent dye can be generated very easily and supplied to the measurement object. And based on the fluorescence obtained from the fluorescent dye, the state of the measurement object can be measured with extremely high accuracy.

  FIG. 1 is a configuration block diagram of a spectral processing apparatus 10 to which a light source unit of the present invention and a fluorescence measuring apparatus using the light source unit are applied.

  The spectral processing device 10 guides the excitation light 14 to the measurement target 12 and the light source unit 16 that outputs the excitation light 14 for exciting the fluorescent dye that stains the measurement target 12 such as a biological tissue. A microscope optical system 20 for obtaining an enlarged fluorescence image of the object 12, a polychromator 22 (spectrometer) for dispersing the fluorescence 18 constituting the fluorescence image in a desired wavelength range, and the excitation light 14 for the measurement object 12 On the other hand, an optical system 24 that guides the fluorescence 18 to the polychromator 22 and a plurality of photoelectric conversion units arranged in a two-dimensional manner are received, and the fluorescence 18 dispersed by the polychromator 22 is received and converted into an electrical signal. And an information processing unit 28 that controls the polychromator 22 and the optical system 24 and processes spectral information in accordance with an electrical signal from the CCD sensor 26.

  FIG. 2 shows the configuration of the light source unit 16. The light source unit 16 includes a light source unit 15 including a light source 13 including a xenon lamp that outputs illumination light 11, and a pre-spectrometer that splits the illumination light 11 from the light source unit 15 to obtain wavelength-specific wavelength light 17. 19 (spectral means), a post-spectrometer 21 (multiplexing means) for obtaining excitation light 14 by combining the wavelength light 17 from the front spectroscope 19, and an excitation light 14 from the post-spectrometer 21 And a light guide cable 23 led to the optical system 24.

  The light source unit 15 and the front spectroscope 19 are connected via a slit member 25. The front spectroscope 19 includes a reflection mirror 27 that reflects the illumination light 11 supplied from the light source unit 15 through the slit member 25, a first concave mirror 29 that collimates the illumination light 11 reflected by the reflection mirror 27, and A diffraction grating 31 that splits the illumination light 11 reflected by the first concave mirror 29 into wavelength light 17 for each wavelength and a second concave mirror 33 that condenses the wavelength light 17 are provided.

  The front spectroscope 19 and the rear spectroscope 21 are connected via an aperture member 35 (selecting means). As shown in FIG. 3, openings 35 a to 35 e are formed in the aperture member 35 at predetermined positions in the spectral direction (arrow Z direction) of the wavelength light 17 from the front spectroscope 19. That is, the openings 35a to 35e are formed at the wavelength positions of the wavelength light 17 centered on the wavelength (see FIG. 9) at which the absorbance of each fluorescent dye staining the measurement object 12 is maximum.

  The post-spectrometer 21 combines the split wavelength light 17 into the excitation light 14, and has the same configuration as the pre-spectrometer 19 although the function is opposite. That is, the post-spectrometer 21 receives the first concave mirror 37 that collimates the wavelength light 17 supplied from the front spectroscope 19 through the openings 35a to 35e, and the wavelength light 17 reflected by the first concave mirror 37. A diffraction grating 39 that is combined to form the excitation light 14, a second concave mirror 41 that collects the excitation light 14, and a reflection mirror that reflects the excitation light 14 collected by the second concave mirror 41 and guides it to the light guide cable 23. 43.

  Note that the diffraction grating 31 constituting the pre-spectrometer 19 and the diffraction grating 39 constituting the post-spectrometer 21 may be any one that splits the illumination light 11 or multiplexes the wavelength light 17. Instead of the diffraction gratings 31 and 39, prisms may be used.

  The post-spectrometer 21 and the light guide cable 23 are connected via a slit member 45. The light guide cable 23 is composed of a flexible cable containing a plurality of optical fibers. In the connector portions 23a and 23b at both ends of the light guide cable 23, as shown in FIG. 4, the incident end 47a and the output end 47b of the optical fiber are slit. Corresponding to the member 45 and a slit member 34 to be described later, it is configured linearly. In this case, the excitation light 14 can be efficiently transmitted from the rear spectroscope 21 to the optical system 24 via the light guide cable 23. A condensing lens may be disposed between the emission end 47 b and the slit member 34.

  The optical system 24 reflects the excitation light 14 having passed through the slit 36 of the slit member 34 by using the excitation light 14 supplied from the light source unit 16 through the light guide cable 23 as slit light, and the microscope optical system. A beam splitter 42 that guides the fluorescence 18 introduced from the microscope optical system 20 to the polychromator 22 and an imaging lens 44 that collimates the excitation light 14 reflected by the beam splitter 42.

  As shown in FIG. 5, the slit 36 of the slit member 34 is set so as to form a slit image 38 of the excitation light 14 that is long in the arrow Y direction on the measurement object 12. The beam splitter 42 is preferably a dichroic mirror that reflects the excitation light 14 and transmits the fluorescence 18.

  The imaging lens 44 (scanning means) is placed on the moving stage 46. The moving stage 46 is configured to be movable in the direction of arrow N via a ball screw rotated by a motor 48. It is assumed that the arrow N direction is set corresponding to the direction orthogonal to the arrow Y direction of the measurement object 12 shown in FIG.

  The microscope optical system 20 includes a prism 50 that reflects the excitation light 14 guided from the optical system 24, an objective lens 52 that condenses the excitation light 14 reflected by the prism 50 onto the measurement object 12, and the prism 50. 1 is provided with an eyepiece 56 that guides the fluorescence 18 from the measurement object 12 to the observer 54 in a state of being retracted to the position of the dotted line shown in FIG.

  The polychromator 22 includes a slit member 58 on which a slit image 38 of the fluorescence 18 guided from the optical system 24 is formed, a reflection mirror 62 that reflects the fluorescence 18 that has passed through the slit 60 of the slit member 58, and a reflection mirror 62. A first concave mirror 64 that collimates the fluorescent light 18 reflected by the first concave mirror 64, a diffraction grating 66 that splits the fluorescent light 18 reflected by the first concave mirror 64, and a second concave mirror 68 that collects the spectrally fluorescent light 18 on the CCD sensor 26. With. As shown in FIG. 5, it is assumed that the slit 60 of the slit member 58 and the grating 65 of the diffraction grating 66 are set to be long in the same direction as the slit image 38.

  The diffraction grating 66 is mounted on a rotary stage 72 that is rotated by a motor 70. By adjusting the incident angle of the fluorescent light 18 with respect to the diffraction grating 66, the CCD sensor 26 receives zero-order diffracted light (regularly reflected light) or n next time. It is comprised so that folding light (n: 1 or more integers) can be guide | induced.

  The information processing unit 28 (fluorescence measurement means) controls the spectral processing apparatus 10 by the control unit 74, and can be configured by, for example, a personal computer. The control unit 74 performs processing of spectral information and fluorescence image information of the measurement target 12 based on the fluorescence 18 obtained from the measurement target 12.

  The control unit 74 displays an input unit 76 for inputting setting conditions for measurement and an instruction for displaying the measurement result, a setting screen, and spectral information and fluorescence image information of the measurement object 12. Display unit 78, imaging lens driving unit 80 for moving imaging lens 44 constituting optical system 24, diffraction grating driving unit 82 for rotating diffraction grating 66 constituting polychromator 22, CCD A signal processing unit 84 that processes an electrical signal supplied from the sensor 26 is connected to a spectral image information storage unit 86 that stores spectral information and fluorescence image information of the measurement object 12.

  The spectral processing apparatus 10 of the present embodiment is basically configured as described above. Next, a spectral processing method for the measurement object 12 using the spectral processing apparatus 10 will be described.

  First, the preparation process will be described with reference to the flowchart shown in FIG.

  The measurement object 12 made of a living tissue or the like is stained with a predetermined fluorescent dye (step S1) and placed at the focal position of the objective lens 52 constituting the microscope optical system 20, while the fluorescent dye used for the measurement object 12 is The aperture member 35 corresponding to the characteristic is selected (step S2) and installed between the front spectroscope 19 and the rear spectroscope 21 of the light source unit 16.

  For example, when the measurement object 12 is stained with five types of fluorescent dyes a1 to a5 having the characteristics shown in FIGS. 9 and 10, the position corresponding to the wavelength at which the absorbance of each of the fluorescent dyes a1 to a5 is maximum is centered. The aperture member 35 in which the openings 35 a to 35 e are formed is selected and installed in the light source unit 16. In this case, the width in the spectral direction (arrow Z direction) of each of the openings 35a to 35e can be set according to the necessary range of the wavelength component to be selected.

  Next, the observer 54 moves the prism 50 to the position indicated by the dotted line in FIG. 1, and then adjusts the position of the microscope optical system 20 while observing the measurement object 12 via the eyepiece lens 56. The object 12 is aligned (step S3).

  Next, the prism 50 is moved to the position indicated by the solid line, the slit member 34 of the optical system 24 and the slit member 58 of the polychromator 22 are set to the fully open state (step S4), and the motor 70 is driven by the diffraction grating driving unit 82. The rotary stage 72 is driven to rotate, and the diffraction grating 66 is set to a state in which the zero-order diffracted light of the fluorescence 18 is guided to the CCD sensor 26 (step S5). Instead of setting the slit members 34 and 58 to the fully open state, they may be removed.

  After setting the spectral processing apparatus 10 as described above, the light source unit 16 is driven to irradiate the measurement object 12 with the excitation light 14 (step S6).

  In FIG. 2, the illumination light 11 output from the light source 13 of the light source unit 15 enters the front spectroscope 19 through the slit member 25, is reflected by the reflection mirror 27, and then is collimated by the first concave mirror 29. To the diffraction grating 31. The diffraction grating 31 splits the illumination light 11 into the wavelength light 17, and the wavelength light 17 is reflected by the second concave mirror 33 and guided to the aperture member 35.

  As shown in FIG. 3, the aperture member 35 has a spectral direction (in the direction of arrow Z) of the wavelength light 17 corresponding to the vicinity of the wavelength at which the absorbance of each of the fluorescent dyes a1 to a5 staining the measurement object 12 is maximum. Openings 35a to 35e are formed at predetermined positions. Accordingly, the wavelength light 17 guided to the aperture member 35 is only introduced into the post-spectrometer 21 through the apertures 35a to 35e through the wavelength light 17 in the vicinity of the wavelength at which the absorbance of the fluorescent dyes a1 to a5 is maximum. The Moreover, unlike the optical filter whose transmittance is controlled, the apertures 35a to 35e of the aperture member 35 allow the wavelength light 17 to be efficiently introduced into the post-spectrometer 21 in order to allow the desired wavelength light 17 to pass completely. be able to.

  The wavelength light 17 introduced into the post-spectrometer 21 passes through the first concave mirror 37, the diffraction grating 39, and the second concave mirror 41, and is multiplexed by the reverse action of the front spectroscope 19, Excitation light 14 having only wavelengths near the wavelength at which the absorbances of the fluorescent dyes a1 to a5 are maximum is generated. The excitation light 14 is introduced from the connector portion 23 a to the optical fiber of the light guide cable 23 through the reflection mirror 43 and the slit member 45.

  The excitation light 14 introduced into the light guide cable 23 passes through the slit member 34 in the fully opened state of the optical system 24 from the connector portion 23 b and is guided to the beam splitter 42. The excitation light 14 reflected by the beam splitter 42 is irradiated onto the measurement object 12 through the imaging lens 44, the prism 50 and the objective lens 52.

  The fluorescent dyes a1 to a5 of the measurement object 12 irradiated with the excitation light 14 are excited by the excitation light 14 and output fluorescence 18. In this case, since the excitation light 14 is composed only of wavelengths in the vicinity of the wavelength at which the absorbance of each of the fluorescent dyes a1 to a5 is maximum, the fluorescence 18 that is suitably separated for each of the fluorescent dyes a1 to a5 is obtained.

  The fluorescence 18 output from the measurement object 12 is guided to the polychromator 22 via the objective lens 52, the prism 50, the imaging lens 44 and the beam splitter 42. Next, after passing through the slit member 58 in the fully opened state, the fluorescence 18 is condensed on the CCD sensor 26 via the reflection mirror 62, the first concave mirror 64, the diffraction grating 66, and the second concave mirror 68. In this case, since the diffraction grating 66 is set so as to totally reflect the fluorescence 18, a fluorescence image of the measurement object 12 made of the fluorescence 18 is formed on the CCD sensor 26.

  Therefore, the control unit 74 converts the fluorescence 18 guided to the CCD sensor 26 into an electrical signal by the signal processing unit 84, and then displays the fluorescence image of the measurement object 12 on the display unit 78 (step S7). The operator adjusts the microscope optical system 20 while confirming the fluorescence image of the measurement object 12 displayed on the display unit 78, and focuses the measurement object 12 on the CCD sensor 26 (step S8).

  Here, during the focusing, the entire measurement object 12 is irradiated with the excitation light 14, but since the time required for the focusing is a short time, the fluorescent light is hardly faded by the excitation light 14. It doesn't matter. It is more preferable to adjust the excitation light 14 output from the light source unit 16 to the minimum necessary light amount necessary for focusing.

  After the focusing is completed, the slit member 34 of the optical system 24 and the slit member 58 of the polychromator 22 are set to a predetermined slit width (step S9). At this time, the excitation light 14 output from the light source unit 16 is narrowed down by the slit 36 of the slit member 34, and a slit image 38 including the excitation light 14 is formed on the measurement object 12 (see FIG. 5).

  The fluorescent image obtained from the measurement object 12 corresponding to the formation position of the slit image 38 is formed at the position of the slit 60 of the slit member 58 provided at the entrance of the polychromator 22. Next, the fluorescence 18 that has passed through the slit member 58 forms an image on the CCD sensor 26 via the reflection mirror 62, the first concave mirror 64, the diffraction grating 66, and the second concave mirror 68. The control unit 74 displays the slit-like fluorescent image (slit image) on the display unit 78 (step S10).

  In this state, when the control unit 74 drives the motor 48 by the imaging lens driving unit 80 and moves the imaging lens 44 in the arrow N direction via the moving stage 46, the slit formed on the measurement object 12. Since the image 38 moves in the arrow N direction, the slit image displayed on the display unit 78 also moves.

  Therefore, the operator sets the measurement range in the direction of the arrow N of the measurement object 12 while confirming the position of the slit image displayed on the display unit 78 (step S11). If the entire fluorescence image of the measurement object 12 displayed for focusing in step S7 and the slit image in step S10 are displayed in an overlapping manner, the measurement range can be set more easily.

  After the above preparation process is completed, the spectroscopic measurement process of the measurement object 12 is started. The process will be described with reference to the flowchart shown in FIG.

  First, the motor 70 is driven by the diffraction grating driving unit 82 to rotate the rotary stage 72, and the fluorescence 18 incident on the diffraction grating 66 is dispersed to set the first-order diffracted light to the second concave mirror 68 (step). S12).

  Next, the imaging lens driving unit 80 drives the motor 48 to move the moving stage 46, and the imaging lens 44 is positioned at the initial position of the measurement range set in step S11 (step S13).

  In the state described above, the excitation light 14 output from the light source unit 16 is guided to the measurement object 12 through the slit member 34 of the optical system 24, so that the measurement object 12 corresponding to the initial position of the imaging lens 44 can be obtained. A slit image 38 made of the excitation light 14 is formed at the site. The fluorescence 18 output from the fluorescent dyes a1 to a5 of the measurement object 12 by the excitation light 14 is guided to the polychromator 22 through the microscope optical system 20 and the optical system 24, and after being separated by the diffraction grating 66, A spectral image is formed on the CCD sensor 26 via the second concave mirror 68 (step S14). This spectral image represents the wavelength information of the fluorescence 18 obtained from the slit image 38 of the measurement object 12 in the direction of the arrow X shown in FIG. 5, and from the slit image 38 in the direction of the arrow Y. The image information of the obtained fluorescence 18 is represented.

  The spectrally divided fluorescence 18 including the wavelength information and the image information is converted into an electrical signal by each photoelectric conversion unit constituting the CCD sensor 26, subjected to signal processing by the signal processing unit 84, and then stored as spectral image information. Stored in the unit 86 (step S15).

  Next, the imaging lens driving unit 80 drives the motor 48 to move the moving stage 46 in the direction of arrow N by a minute distance (step S16). Then, steps S14 and S15 are repeated until the position of the imaging lens 44 reaches the end position of the measurement range set in step S11 (step S17). Thereby, the spectral image information of the measurement object 12 is acquired and stored in the spectral image information storage unit 86.

  Furthermore, it is necessary to measure the time-dependent state of the measurement object 12, and when the next spectral image information is acquired (step S18), the processing from step S13 is repeated. That is, after the imaging lens 44 is returned to the initial position again, the spectral image information of each slit image 38 is acquired and moved in the spectral image information storage unit 86 while being moved by a minute distance in the direction of arrow N.

  As described above, the spectral image information storage unit 86 stores the entire spectral image information of the measurement object 12 for each predetermined measurement time.

  In the present embodiment, the aperture member 35 in the light source unit 16 is used to generate the excitation light 14 composed only of wavelength components in the vicinity of the wavelength at which the absorbance of the fluorescent dyes a1 to a5 staining the measurement object 12 is maximum. Since the measurement object 12 is irradiated, the fluorescence 18 obtained from each of the fluorescent dyes a1 to a5 can be well separated and measured. Therefore, without changing the aperture member 35, the fluorescence 18 obtained from the plurality of fluorescent dyes a1 to a5 can be simultaneously measured to perform an efficient analysis.

  Further, the change of the fluorescent dyes a1 to a5 that stain the measurement object 12 can be easily dealt with by replacing the aperture member 35. Furthermore, since the wavelength component of the excitation light 14 can be adjusted very easily by changing the positions of the openings 35a to 35e of the aperture member 35, it is very easy to cope with new fluorescent dyes. Can do. Note that the aperture member 35 may have only one opening 35a to 35e, and the aperture member 35 may be replaced for each fluorescent dye a1 to a5.

  Furthermore, in this embodiment, when acquiring the spectral image information of the entire measurement object 12, the excitation light 14 as the slit image 38 is measured instead of irradiating the entire measurement object 12 with the excitation light 14. Since the fluorescent light 18 is obtained by irradiating the object 12, the excitation light 14 is not irradiated to the part not involved in the measurement, and the fading of the fluorescent dyes a <b> 1 to a <b> 5 by the excitation light 14 is suppressed accordingly. Is done. Therefore, even when the measurement object 12 is irradiated with the excitation light 14 every predetermined measurement time and spectral information is obtained, fading of the fluorescent dyes a1 to a5 is greatly reduced. It can be acquired with high accuracy.

  Moreover, since the imaging lens 44 is moved instead of moving the measuring object 12 in order to irradiate the measuring object 12 with the excitation light 14 as the slit image 38, for example, an external force is applied. Even if it is the measuring object 12 like a biological tissue in which the state may change, spectral image information can be acquired without affecting the state.

  In this case, for example, when the excitation light 14 irradiates the position N in the arrow N direction of the measurement object 12 at time T, the spectral image acquired by the photoelectric conversion unit in the X column and the Y row of the CCD sensor 26. If the information is P (X, Y, N, T) (see FIG. 8), the spectral image information P has a wavelength corresponding to the coordinate X at the coordinates (N, Y) of the measurement object 12 at time T. It represents the fluorescence output value. Therefore, on the basis of the spectral image information P, the display unit 78 displays spectral information at an arbitrary time T at an arbitrary coordinate (N, Y) of the measurement object 12, and the arbitrary measurement object 12 is displayed. The entire image information at time T can be displayed.

It is a block diagram of the configuration of the spectral processing apparatus of the present embodiment. It is a block diagram of the light source unit shown in FIG. FIG. 3 is a configuration explanatory diagram of an aperture member in the light source unit shown in FIG. 2. FIG. 2 is a configuration explanatory diagram of a connector portion in a light guide cable that connects a light source unit and an optical system shown in FIG. 1. FIG. 2 is an explanatory diagram illustrating an arrangement relationship of main elements in an optical system constituting the spectral processing apparatus shown in FIG. 1. It is a flowchart of the preparation process in the spectral processing apparatus shown in FIG. It is a flowchart of the spectral measurement process in the spectral processing apparatus shown in FIG. It is explanatory drawing of the spectral image information acquired by the spectral processing apparatus shown in FIG. It is explanatory drawing of the light absorbency characteristic of fluorescent dye. It is explanatory drawing of the fluorescence intensity characteristic of fluorescent dye.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Spectral processing apparatus 12 ... Measurement object 13 ... Light source 14 ... Excitation light 15 ... Light source part 16 ... Light source unit 19 ... Pre-spectrometer 20 ... Microscope optical system 21 ... Post-spectrometer 22 ... Polychromator 23 ... Light guide cable 24 ... Optical system 26 ... CCD sensor 28 ... Information processing unit 35 ... Aperture members 35a to 35e ... Opening 44 ... Imaging lens 46 ... Moving stage

Claims (10)

A light source that outputs illumination light;
A spectroscopic means for splitting the illumination light according to a wavelength;
A selection means for selecting wavelength light of a desired wavelength from the illumination light that has been split;
A multiplexing means for multiplexing the selected wavelength light;
A light source unit comprising: The unit of claim 1, wherein
The light source unit according to claim 1, wherein the selection unit includes an aperture member having an opening formed at a position corresponding to the desired wavelength light. The unit of claim 2, wherein
The aperture member has a plurality of openings formed at positions corresponding to the plurality of light beams having different wavelengths. The unit of claim 1, wherein
The light source unit, wherein the spectroscopic means and the multiplexing means are constituted by a spectroscope. In a fluorescence measuring apparatus that irradiates a measurement object stained with a predetermined fluorescent dye with excitation light and measures the fluorescence obtained from the fluorescent dye,
A light source that outputs illumination light;
A spectroscopic means for splitting the illumination light according to a wavelength;
A selection means for selecting wavelength light of a desired wavelength from the illumination light that has been split;
A multiplexing means for multiplexing the selected wavelength light;
An optical system for guiding the combined wavelength light to the measurement object as the excitation light;
Fluorescence measuring means for measuring the fluorescence obtained from the fluorescent dye by being irradiated with the excitation light;
A fluorescence measuring apparatus comprising: The apparatus of claim 5.
The fluorescence measuring apparatus according to claim 1, wherein the selection unit includes an aperture member having an opening formed at a position corresponding to the desired wavelength light. The apparatus of claim 6.
The fluorescence measurement apparatus, wherein the aperture member has a plurality of openings formed at positions corresponding to the plurality of light beams having different wavelengths. The apparatus of claim 5.
The fluorescence measuring apparatus, wherein the spectroscopic means and the multiplexing means are constituted by a spectroscope. The apparatus of claim 5.
A slit member in which an opening for converting the combined excitation light into slit light is formed;
Scanning means for scanning the measurement object by moving the slit light in a direction perpendicular to the longitudinal direction of the opening formed in the slit member;
A spectroscope that splits the fluorescence obtained from the fluorescent dye by being irradiated with the slit light, and guides the fluorescence to the fluorescence measuring means;
A fluorescence measuring apparatus comprising: The apparatus of claim 9.
The fluorescence measurement apparatus according to claim 1, wherein the scanning unit includes an imaging lens that constitutes the optical system and moves the slit light along the measurement object by moving in a direction orthogonal to the optical axis.

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